FI20245658A1

FI20245658A1 - Method of amplification and storage of circular nucleic acid molecules

Info

Publication number: FI20245658A1
Application number: FI20245658A
Authority: FI
Inventors: Simona Adamusová; Tatu Hirvonen; Anttoni Korkiakoski; Nea Laine; Anna Musku; Juha-Pekka Pursiheimo; Tuula Rantasalo; Manu Tamminen
Original assignee: Genomill Health Oy
Priority date: 2024-05-23
Filing date: 2024-05-23
Publication date: 2025-11-24
Also published as: WO2025242958A1

Abstract

Disclosed is method for amplification, prolonged storage and/or sequencing, of circular nucleic acid molecules (cNA). Method comprises providing cNA comprising two single-stranded loop sequences (ssLS) connected by two complementary sequences (CS) that are capable of annealing such that a double-stranded portion (dsP) is formed, wherein dsP comprises recognition site for nicking endonuclease (rsNE); contacting cNA with oligonucleotide primer (OP) complementary to a ssLS and allowing annealing thereof; amplifying cNA starting from OP using rolling circle amplification with strand-displacing polymerase and allowing annealing of CS, thereby obtaining concatemeric molecules comprising single-stranded portions (ssP) and hairpin loop structures having dsP; nicking dsP with NE; performing denaturation step such that nicked concatemeric molecules disintegrate into separate segments having complementary ends (CE); and allowing intramolecular annealing of CE and ligating separate segments intramolecularly, thereby obtaining cNA. Method further comprises performing: storing cNA for predefined duration; and/or subjecting cNA to high-throughput sequencing technology.

Description

METHOD OF AMPLIFICATION AND STORAGE OF CIRCULAR NUCLEIC

ACID MOLECULES

TECHNICAL FIELD

The present disclosure relates to methods for amplification, and subsequent prolonged storage and/or sequencing, of circular nucleic acid molecules. Moreover, the present disclosure relates to methods for providing single oligonucleotides comprising hairpin loops. Moreover, the present disclosure relates to methods for removal of specific subpopulations of circular nucleic acid molecules from populations of circular nucleic acid molecules. Furthermore, the present disclosure relates to methods for reduction of specific subpopulations of circular nucleic acid molecules from populations of circular nucleic acid molecules.

BACKGROUND

Improving storage capacity of a data storage medium may enhance efficient data storage and safeguard such data storage medium from a potential damage. Currently, a single chip of dimension ranging up to few millimeters (mm) can store data up to several terabytes (TB). However, such data storage medium is susceptible to damage from external factors like radiation, moisture, dust, ionic pollution, or mishandling. Moreover, + 20 such data storage medium has a low shelf-life, thus risking the data

S stored therein. Additionally, the storage capacity of such data storage 3 medium is insufficient for accommodating the exponential growth of data

N generated daily.

E Recently, utilizing nucleic acids, such as deoxyribonucleic acid (DNA), are 3 25 emerging as data storage medium. Typically, DNA storage molecules

X have been demonstrated as PCR-amplifiable molecules & (https://www.science.org/doi/10.1126/science.1226355), yeast artificial chromosomes (https://academic.oup.com/nsr/article/8/5/nwab028/6134071),

bacterial plasmids (https://www.nature.com/articles/s41563-021- 01021-3), bacterial genomes (https://www.nature.com/articles/s41467- 023-38876-w) or thermoresponsive capsules (https: //www.nature.com/articles/s41565-023-01377-4). Such DNA storage molecules have been demonstrated to include encoding of entire books, Universal Declaration of Human Rights in multiple languages, a music video, and an entire Netflix series episode into DNA. Notably, the utilization of DNA as data storage medium involves complex synthesis technologies (writing data onto DNA) and sequencing technologies (reading or extracting data from DNA).

However, such technologies are underdeveloped, prone to error, resource intensive, time-consuming and lack economic prospects. Additionally, conventional DNA storage technologies are associated with high cost of synthesis and sequencing, and relatively slow read and write speeds.

Moreover, reading of the stored data from the DNA storage molecules leads to destruction and therefore depletion of the storage data thus raising concern over stability, sustainability and durability of such storage molecules. Furthermore, storing data within living organisms raises ethical questions and regulatory issues that need to be addressed as the technology develops.

Therefore, in light of the foregoing discussion, there exists a need to

S overcome the aforementioned drawbacks. 3 SUMMARY &

I The aim of the present disclosure is to provide a method for amplification, > 25 and subsequent prolonged storage and/or sequencing, of one or more

O circular nucleic acid molecules, methods for removal and reduction of a

X specific subpopulation of circular nucleic acid molecules from a population of circular nucleic acid molecules, and a method for providing single oligonucleotide comprising a hairpin loop, to produce circular nucleic acids from linear nucleic acids, using single oligonucleotides, for high- density, long-term data storage by performing standard database operations (such as index, copy and query) on such storage molecules using enzymatic reactions as well as reading the storage molecules using next-generation DNA sequencing. The aim of the present disclosure is achieved by a method for amplification, and subsequent prolonged storage and/or sequencing, of one or more circular nucleic acid molecules, methods for removal and reduction of a specific subpopulation of circular nucleic acid molecules from a population of circular nucleic acid molecules, and a method for providing single oligonucleotide comprising a hairpin loop, as defined in the appended independent claims to which reference is made to. Advantageous features are set out in the appended dependent claims.

Throughout the description and claims of this specification, the words "comprise", "include", "have", and "contain" and variations of these words, for example "comprising" and "comprises", mean "including but not limited to", and do not exclude other components, items, integers or steps not explicitly disclosed also to be present. Moreover, the singular encompasses the plural unless the context otherwise reguires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the x context reguires otherwise. &

O BRIEF DESCRIPTION OF THE DRAWINGS

N FIG. 1 is a schematic illustration of a flow diagram depicting steps of a

E 25 method for amplification, and subseguent prolonged storage and/or 3 sequencing, of one or more circular nuclear acid molecules, in accordance 3 with an embodiment of the present disclosure;

S FIG. 2 is a schematic illustration of a flow diagram depicting conversion of linear nucleic acid molecules into one or more circular nucleic acid molecules using single oligonucleotide or oligonucleotide complex annealing and a combination of gap fill and ligation, in accordance with an embodiment of the present disclosure;

FIG. 3 is a schematic illustration of a flow diagram depicting conversion of linear nucleic acid molecules into one or more circular nucleic acid molecules using single oligonucleotide or oligonucleotide complex annealing and a combination of targeted binding, gap fill and ligation, in accordance with an embodiment of the present disclosure;

FIG. 4 is a schematic illustration of a flow diagram depicting conversion of linear nucleic acid molecules into one or more circular nucleic acid molecules using single oligonucleotide or oligonucleotide complex annealing and ligation, in accordance with an embodiment of the present disclosure;

FIGs. 5A and 5B are schematic illustrations of a flow diagram depicting conversion of linear nucleic acid molecules into one or more circular nucleic acid molecules using folding of the nucleic acid molecules by the interaction between first and second pair of complementary single stranded sequences and subsequent ligation or combination of gap fill and ligation, in accordance with an embodiment of the present disclosure;

FIG. 5C is a schematic illustration of a flow diagram depicting synthetic generation of the linear nucleic acid molecules and conversion of the said < synthetic linear nucleic acid molecules into one or more circular nucleic

S acid molecules using folding of the nucleic acid molecules by the ro interaction between first and second pair of complementary single stranded sequences and subsequent ligation or combination of gap fill

E 25 and ligation, in accordance with an embodiment of the present disclosure; > FIGs. 6A, 6B, 6C, 6D, 6E and 6F are schematic illustrations of a flow 3 diagram of producing one or more circular nucleic acid molecules from a

N single-stranded or at least partially double-stranded nucleic acid molecules using ligation of hairpin loop, in accordance with an embodiment of the present disclosure;

FIG. 7 is a schematic illustration of a flow diagram depicting conversion of linear nucleic acid molecules into one or more circular nucleic acid molecules using oligonucleotide pair by annealing and a combination of targeted binding, gap fill and ligation, folding of the nucleic acid molecules 5 by the interaction between first and second pair of complementary single stranded sequences and subsequent ligation with a looped oligonucleotide, in accordance with an embodiment of the present disclosure;

FIG. 8 is a schematic illustration of a flow diagram producing triple- hairpin-looped molecules which are subsequently used in rolling circle amplification to produce hairpin-looped concatemeric molecules which are finally cleaved into monomers using nicking endonucleases, in accordance with an embodiment of the present disclosure;

FIG. 9A is a schematic illustration of a flow diagram for removal of a specific subpopulation of circular nucleic acid molecules from a population of circular nucleic acid molecules, in accordance with an embodiment of the present disclosure; and

FIG. 9B is a schematic illustration of a flow diagram for reduction of a specific subpopulation of circular nucleic acid molecules from a population of circular nucleic acid molecules, in accordance with an embodiment of the present disclosure.

N

N DETAILED DESCRIPTION OF EMBODIMENTS

5 @ The following detailed description illustrates embodiments of the present

I disclosure and ways in which they can be implemented. Although some > 25 modes of carrying out the present disclosure have been disclosed, those

O skilled in the art would recognize that other embodiments for carrying

X out or practicing the present disclosure are also possible.

In a first aspect, the present disclosure provides a method for amplification, and subseguent prolonged storage and/or seguencing, of one or more circular nucleic acid molecules, the method comprising the steps of: (i) providing the one or more circular nucleic acid molecules each comprising two single-stranded loop sequences connected by two complementary sequences that are capable of annealing to each other such that a double-stranded portion is formed, wherein the double- stranded portion comprises a recognition site for a nicking endonuclease; (ii) contacting said circular nucleic acid molecules with an oligonucleotide primer complementary to one of the two single-stranded loop sequences and allowing annealing to take place; (iii) amplifying said circular nucleic acid molecules starting from said oligonucleotide primer using a rolling circle amplification with a strand- displacing polymerase and allowing annealing of the two complementary sequences that are capable of annealing to each other, thereby obtaining concatemeric molecules comprising single-stranded portions and hairpin loop structures having double-stranded portions comprising said recognition site for the nicking endonuclease; (iv) nicking the double-stranded portions of the concatemeric molecules with the nicking endonuclease having specificity for said recognition site; (v) performing a denaturation step such that the nicked concatemeric = molecules disintegrate into separate segments having complementary

S

N ends; and 2 n (vi) allowing an intramolecular annealing of the complementary ends of

Al - the separate segments and ligating the separate segments a > 25 intramolecularly, thereby obtaining said circular nucleic acid molecules, 00

LO

N

N (vii) storing the said circular nucleic acid molecules for a predefined duration; and

(viii) subjecting said circular nucleic acid molecules obtained in step (vi) or in step (vii) to a high-throughput sequencing technology.

The aforementioned method for amplification, and subsequent prolonged storage and/or sequencing, of one or more circular nucleic acid molecules produces circular nucleic acid molecules, converted from different types of DNA starting material, including targeted DNA libraries, non-specific

DNA libraries and completely synthetic DNA molecules, having high- density, long-term data storage properties. Moreover, the circular nucleic acid molecules is configured to store up to 215 petabytes (215 million gigabytes) of data per gram, which is orders of magnitude greater than traditional storage media. The circular nucleic acid molecules can last hundreds to thousands of years when properly stored, far outstripping the lifespan of current digital storage media that may degrade within decades. The circular nucleic acid molecules is an energy-efficient long- term storage solution as it works with an electricity supply to maintain the data once it's synthesized. Additionally, the circular nucleic acid molecules are copied enzymatically, eliminating the possibility of external interference, such as cyber-attacks, electronic disturbances, and so on.

In a second aspect, the present disclosure provides a method for providing single oligonucleotide comprising a hairpin loop, said method comprising the steps of:

N (ia) providing linear nucleic acid molecules comprising:

N ro a 5' portion comprising a linear sequence flanked on each side by one e of a pair of complementary single-stranded seguences, followed by

E 25 a single-stranded center portion comprising seguences 3 complementary to a loop adaptor oligonucleotide, followed by 3 a 3' portion comprising a linear sequence flanked on each side by one

N of a pair of complementary single-stranded seguences,

wherein both the pairs of complementary single-stranded sequences contain a recognition site for a nicking endonuclease; (ib) allowing both the pairs of complementary single-stranded sequence to anneal to form hairpin loops and contacting with a loop adaptor oligonucleotide comprising a hairpin loop flanked by single-stranded sequences complementary to the center portion defined in step (ia); (ic) allowing the loop adaptor oligonucleotide to anneal to the center portion to generate annealed complexes; (id) ligating the annealed complexes to obtain closed molecules; (ie) annealing the closed molecules with an oligonucleotide primer complementary to loop derived from the loop adaptor oligonucleotide; (if) amplifying said closed molecules starting from said oligonucleotide primer using a rolling circle amplification with a strand-displacing polymerase and allowing annealing of complementary sequences, thereby obtaining concatemeric molecules comprising single-stranded portions and hairpin loop structures; (ig) nicking the double-stranded portions with a nicking endonuclease having specificity for said recognition sites; and (ih) performing a denaturation step to obtain single oligonucleotides comprising a hairpin loop. <

N

< Beneficially, using the single oligonucleotide with the hairpin loop 3 structure simplifies the design and synthesis process compared to using

Q multiple oligonucleotides. Moreover, the single oligonucleotide reduces

E the complexity and cost associated with oligonucleotide design and 2 25 synthesis, making the approach more accessible and efficient. Moreover,

O

0 the hairpin loop structure of the single oligonucleotides facilitates the

N

N circularization of linear nucleic acid molecules by providing complementary seguences that anneal to the ends of the linear nucleic acid molecule. Additionally, the hairpin loop structure promotes efficient intramolecular ligation, leading to the formation of circular nucleic acid molecules, which are often desirable for certain applications such as cloning or library preparation. Furthermore, the hairpin loop structure facilitates primer annealing and extension, leading to robust amplification of the circular nucleic acid molecule with high efficiency and yield.

In a third aspect, the present disclosure provides a method for removal of a specific subpopulation of circular nucleic acid molecules from a population of circular nucleic acid molecules, the method comprising steps of: (i) providing a population of at least partially single-stranded circular nucleic acid molecules comprising an endonuclease restriction recognition sequence, wherein said circular nucleic acid molecules optionally comprise two loop sequences separated by two sequences that are capable of annealing to each other such that a double-stranded portion is formed; (ii) contacting said circular nucleic acid molecules with an oligonucleotide primer and allowing annealing to take place, wherein the oligonucleotide primer is complementary to a single-stranded sequence or a part of the single-stranded sequence that is to be removed from the population of circular nucleic acid molecules; < (iii) generating double-stranded molecules by extending the annealed

S oligonucleotide primer using a non-strand displacing polymerase; = (iv) cleaving said double-stranded molecules with an endonuclease 0

N capable of cleaving the endonuclease restriction recognition seguence to

I

= 25 generate linearized molecules; and 00 2 (v) remove linearized molecules from the population of nucleic acid

LO

N molecules, optionally by exonuclease degradation.

N

Beneficially, the circular nucleic acid molecules, such as circular DNA, are distinct from linear DNA, and have their own replication machinery.

Notably, the circular nucleic acid molecules have inherent advantages over the linearized (or linear) nucleic acid molecules. For example, the circular DNA are highly stable as they are less prone to degradation compared to linear DNA over long periods of time, thereby potentially enhance the longevity of stored data. Moreover, the circular DNA is easy to handle and manipulate in the laboratory compared to linear DNA. The closed-loop structure of circular DNA molecules generally simplify certain aspects of DNA synthesis, storage, and retrieval processes. Furthermore, the circular DNA can be more compact than linear DNA, as it lacks free ends, thereby allowing for more efficient use of space when storing large amounts of data. Additionally, the circular DNA can be efficiently replicated with high fidelity, thereby enabling scalable data storage solutions.

In a fourth aspect, the present disclosure provides a method for reduction of a specific subpopulation of circular nucleic acid molecules from a population of circular nucleic acid molecules, the method comprising the steps of: (i) providing a population of at least partially single-stranded circular nucleic acid molecules, wherein said circular nucleic acid molecules optionally comprise two loop sequences separated by two sequences that are capable of annealing to each other such that a double-stranded

N portion is formed; a (ii) contacting said circular nucleic acid molecules and allowing a annealing with

N

E 25 (a) a first complementary oligonucleotide primer, which is © complementary to a target sequence or a part of the target sequence that

O is to be reduced in freguency in the population of circular nucleic acid

O molecules, wherein the first complementary oligonucleotide primer is modified to prevent extension, and wherein the first complementary oligonucleotide primer comprises oligonucleotides that cannot be displaced by a strand-displacing polymerase; and (b) a second complementary oligonucleotide primer, which is complementary to a universal sequence; (iii) amplifying said circular nucleic acid molecules starting from said second complementary oligonucleotide primer using a rolling circle amplification with a strand-displacing polymerase, thus obtaining a modified population of circular nucleic acid molecules wherein molecules containing the target sequence have not been amplified.

Beneficially, by limiting or reducing a specific subpopulation of circular nucleic acid molecules, such as those containing the target sequence, from a population of circular nucleic acid molecules, reduces the amount of non-relevant target material in a circular DNA pool, thereby focusing the sequencing read-out effort to target DNA pools of interest.

Throughout the present disclosure, the terms "circular nucleic acid molecules" and "linear nucleic acid molecules" as used herein refer to different structural forms of nucleic acids, such as DNA molecules (hereafter, nucleic acid or nucleic acid molecule may be interchangeably used as DNA), found in various organisms, prokaryotic and eukaryotic.

Typically, the circular nucleic acid molecules form a closed loop without < any free ends, hence circular. Examples of circular nucleic acid molecules

S are found in prokaryotic cells (such as bacteria in form of plasmids, and ro archaea) as well as in some organelles of eukaryotic cells (such as mitochondria and chloroplasts). The circular nucleic acid molecules are

E 25 independently replicating genetic elements separate from the © chromosomal or nuclear nucleic acid or DNA. The circular DNA molecules

O are replicated by a mechanism called rolling-circle replication in bacteria.

O Moreover, the circular DNA molecules replicate semi-conservatively in organelles of eukaryotic cells. Such circular DNA molecules in living cells are typically double-stranded, whereas in the present disclosure, the described circular DNA molecules are, for the most part, single-stranded.

The linear nucleic acid molecules refers to DNA molecules that have two distinct ends. In eukaryotic cells, examples of linear nucleic acid molecules include chromosomes, having repetitive sequences, namely, telomeres, that help protect the ends of the DNA from degradation and fusion with other DNA molecules. These linear nucleic acid molecules, found predominantly in eukaryotic cells, are double-stranded, and found for example in animals and plants. Notably, during cell division, linear

DNA is replicated, and the two resulting DNA molecules are separated into daughter cells. The linear nucleic acid molecules may be processed to form the circular nucleic acid molecules, as discussed in the following paragraphs.

Optionally, the linear nucleic acid molecules may comprise at least one of a double-stranded region, a single-stranded region. Throughout the present disclosure, the term "linear at least partially double-stranded nucleic acid molecules" refers to linear strands of DNA molecule comprising a linear arrangement of nucleotides with some regions of both double-stranded and single-stranded DNA along their length. In other words, the double-stranded regions may be continuous along the length of the molecule or interspersed with single-stranded regions. Beneficially,

S the combination of double-stranded and single-stranded regions provides

N flexibility and versatility in the function of the linear at least partially 3 double-stranded nucleic acid molecules. : 25 Optionally, the linear nucleic acid molecules are provided as a sample, = © wherein the sample pertains to a patient sample, clinical sample, food

O sample, environmental sample, forensic sample or archeological sample.

N In this regard, the linear nucleic acid molecules may be obtained from * patient samples, forensic samples or clinical samples provided for diagnostic or clinical purposes. Such patient samples, forensic samples or clinical samples may include samples such as blood, saliva, urine, hair, semen, tissues, tissue biopsies, or swabs from various anatomical sites.

The linear nucleic acid molecules may be obtained from food samples consisting of food products or ingredients collected from agricultural, industrial, or commercial sources, such as raw materials, processed foods, beverages, or food matrices such as soil, water, and animal feed.

The linear nucleic acid molecules may be obtained from environmental samples comprising various biological, chemical, and physical components obtained from natural or human-made environments, including soil, water, air, sediments, and microbial communities. The linear nucleic acid molecules may be obtained from archaeological samples consisting of organic or inorganic materials recovered from archaeological sites, artifacts, human remains, or ancient specimens preserved in sedimentary layers, ice cores, or fossil deposits.

Notably, both the linear DNA and the circular DNA find application as a potential DNA storage medium. The linear DNA, such as synthetic DNA strands, may offer advantages in terms of sequence design flexibility, compatibility with existing DNA sequencing technologies, and potential integration with other DNA-based applications. However, the circular DNA is highly stable thus less prone to degradation compared to linear DNA over long periods of time. Moreover, the circular DNA permits certain < laboratory manipulations which are not available to linear DNA. The

S closed-loop structure of circular DNA molecules may simplify certain 3 aspects of DNA synthesis, storage, and retrieval processes. Furthermore, & 25 the circular DNA can be more compact than linear DNA, as it lacks free z ends, thus allows for more stability when storing large amounts of data. 8 Furthermore, the circular DNA can be efficiently replicated with high ? fidelity and minimal distortions, thus potentially facilitate keeping safe

N the stored data.

Pursuant to the present disclosure, the aforementioned method provides amplification, and subseguent prolonged storage and/or seguencing of the circular nucleic acid molecules, such as circular DNA, to prepare the circular nucleic acid molecules as the DNA storage molecules (or DNA storage medium). It may be appreciated that amplification of the circular nucleic acid molecules, such as circular DNA, is a common technique for generating multiple copies of target DNA sequences. The amplification of the circular nucleic acid molecules is achieved by rolling circle amplification using a strand-displacing DNA polymerase, known to a person of ordinary skill in the art. The amplified circular nucleic acid molecules may be subsequently stored for prolonged durations and read using existing DNA-seguencing technologies or similar future technologies while ensuring preservation of their integrity and the data stored thereon over time.

Notably, the linear DNA molecules have two distinct ends: a 5' end having a phosphate group attached to the 5' carbon of the sugar, and a 3' end having a hydroxyl (-OH) group attached to the 3' carbon in the deoxyribose sugar of the nucleotides. The 5' end is typically the "beginning" or "start" of the nucleic acid sequence and the 3' end is typically the "end" or "termination" of the nucleic acid sequence. The circular DNA forms a closed loop without distinct 5' or 3' ends like linear

DNA, and the sequence continues uninterrupted in a circular fashion.

Optionally, the linear DNA may exists as double-stranded DNA consisting

N of two antiparallel (i.e., they run in opposite directions), complementary

N strands arranged in a double helix with defined 5' and 3' ends on each 3 strand, wherein each strand consists of a specific sequence of nucleotides

N 25 that is complementary to the other. In the context of DNA, specifically a & double-stranded DNA (dsDNA), the term "complementary" refers to the 3 specific pairing between nucleotide bases on two separate DNA strands

X that form hydrogen bonds with each other. The four nucleotide bases

N found in DNA adenine (A), thymine (T), cytosine (C), and guanine (G) exhibit a complementary pairing pattern, such that adenine (A) pairs of one strand with thymine (T) of other strand via two hydrogen bonds, and cytosine (C) pairs of one strand with guanine (G) of other strand via three hydrogen bonds. In terms of sequence complementarity, the 5" end is partially complementary to the 3’ end, such that the nucleotide sequences in the 5’ end of a first strand can base-pair with sequences in the 3’ end of a second strand, and the nucleotide sequences in the 3’ end of the first strand can base-pair with sequences in the 5” end of the second strand.

For example, if one strand of DNA has the sequence 5'-ATCG-3', its complementary strand will have the sequence 3'-TAGC-5'.

Alternatively, the linear DNA may exist as single-stranded DNA consisting of a single, continuous strand of nucleotides with a defined 5' end and 3' end. Optionally, the circular DNA may exists as double-stranded DNA consisting of two complementary strands forming a closed loop.

Alternatively, the circular DNA may exists as single-stranded DNA forming a closed loop without any complementary strand. Beneficially, the double-stranded DNA serves as the stable genetic material in organisms, encoding the instructions for the development, growth, functioning, and reproduction of living organisms, and the single- stranded DNA serves as templates for DNA replication, transcription, genomes such as in certain viruses. It may be appreciated that in case of a single-stranded DNA (ssDNA), the term "complementary" refers to the specific pairing of a sequence of nucleotides in one strand with a < complementary sequence of nucleotides within the same strand or with

S another ssDNA molecule or with free nucleotides, namely, 3 deoxyribonucleoside triphosphates (dNTPs). dNTPs are nucleotides that & 25 are added by a DNA polymerase enzyme to a growing DNA strand to z complement the existing template strand.

Beneficially, the complementary base pairing is crucial for maintaining

S the double-stranded structure of DNA. Additionally, the complementary

N base pairing ensures accurate DNA replication and allows for the faithful transmission of genetic information during processes like replication, transcription, and DNA repair.

The method comprises providing the one or more circular nucleic acid molecules each comprising two single-stranded loop sequences connected by two complementary sequences that are capable of annealing to each other such that a double-stranded portion is formed, wherein the double-stranded portion comprises a recognition site for a nicking endonuclease. Throughout the present disclosure, the term "single-stranded loop sequences" (SSLS) as used herein refers to a common structural motif, namely, the stem loop sequences or hairpin loop sequences, found in ssDNA (or RNA) molecules, where the strand folds back on itself, creating a loop structure typically consisting of unpaired bases. The two single-stranded loop sequences connected by two complementary sequences that are capable of annealing to each other such that a double-stranded portion may mimic a dumbbell shaped structure, wherein the two single-stranded loop sequences are at the end and the central, stem-like double-stranded portion connects them.

The double-stranded portion comprises specific DNA sequence that is recognized and cleaved (at or near such recognition sequence or recognition site) by a given nicking endonuclease. Notably, the nicking endonuclease is a type of enzyme that cleaves only one strand of a DNA molecule, creating a single-strand break or "nick" in the DNA backbone, in contrast to restriction endonucleases which typically cleave both < strands of the DNA molecule, creating double-strand breaks (DSBs).

S Typically, the restriction site is a palindromic seguence, which reads the 3 same on both strands when read in the 5' to 3' direction. Notably, the & 25 cleavage of the DNA molecule may produce blunt ends (straight cuts z across both DNA strands with no overhanging ends) or sticky ends > (asymmetrical cuts resulting in overhanging ends with single-stranded 3 DNA sequences). Beneficially, cleavage results in precisely cutting DNA molecules at specific sequences, facilitating the manipulation of DNA fragments and the construction of recombinant DNA molecules.

Moreover, the method comprises contacting said circular nucleic acid molecules with an oligonucleotide primer complementary to one of the two single-stranded loop sequences and allowing annealing to take place.

The oligonucleotide primer is a short (18 to 25 nucleotides in length), single-stranded DNA molecule that serves as a starting point for DNA synthesis by annealing (binding) specifically to a complementary sequence within a target circular nucleic acid molecule. In this regard, during annealing process, the oligonucleotide primer provides a 3' hydroxyl (-OH) group, which serves as the starting point for DNA polymerase to add nucleotides and extend the DNA strand in the 5' to 3' direction by adding nucleotides to the 3' end of the oligonucleotide primer. Optionally, the annealing is performed at a temperature ranging from 50-65°C. Beneficially, the oligonucleotide forms high specificity, stable base pairs with the target sequence of the circular nucleic acid molecules.

Optionally, the oligonucleotide primer provided for contacting said circular nucleic acid molecules is a universal primer. Typically, the universal primer is designed to anneal to a conserved region of DNA that is common to multiple target sequences, for amplifying a broad range of sequences, such as conserved genes, regions, or motifs across different species or variants. Beneficially, the universal primer is more flexible, < cost-effective and time-efficient as designing and optimizing primers for

R each target individually is not reguired in this case. 3 Optionally, the oligonucleotide primer provided for contacting said

N 25 circular nucleic acid molecules is a target-specific primer. The target- & specific primer is designed to anneal specifically to a unigue seguence

E within the target circular DNA molecule, to amplify a specific gene, region,

S or seguence of interest, allowing for the selective amplification of the

N target from a complex mixture of nucleic acids. Beneficially, the target- specific primer ensures highly specific amplification of the target DNA molecule, reducing the risk of false-positive results and improving the accuracy of downstream analyses.

Furthermore, the method comprises amplifying said circular nucleic acid molecules starting from said oligonucleotide primer using a rolling circle amplification with a strand-displacing polymerase and allowing annealing of the two complementary sequences that are capable of annealing to each other, thereby obtaining concatemeric molecules comprising single- stranded portions and hairpin loop structures having double-stranded portions comprising said recognition site for the nicking endonuclease.

Rolling circle amplification (RCA) is an isothermal amplification technique used to amplify circular DNA molecules through repetitive cycles of primer extension mediated by a strand-displacing polymerase, such as a DNA polymerase. In RCA, a single oligonucleotide primer anneals to a circular

DNA molecule, initiating DNA synthesis. As the strand-displacing polymerase extends the oligonucleotide primer and displaces the downstream DNA strand, generating a long single-stranded DNA (ssDNA) concatemer that coils around the circular DNA molecule. The displaced strand acts as a template for continuous oligonucleotide primer extension, resulting in the generation of multiple copies of the circular

DNA molecule. Herein, the two complementary sequences that are capable of annealing to each other belong to the circular DNA molecule < and the strand-displacing polymerase. Beneficially, the strand-displacing

S polymerase in RCA allows for efficient amplification of circular DNA 3 molecules without the need for thermal denaturation of the DNA strands. & 25 The strand-displacing polymerase enables continuous DNA synthesis z along the circular template, thereby resulting in exponential amplification > of the circular DNA sequence with high efficiency and specificity. 3 The concatemeric molecules are long, circular DNA molecules that contain

N repeated copies of the original circular DNA molecule arranged in tandem (i.e., linked in series), with single-stranded portions and hairpin loop structures containing double-stranded portions. For example, if the sequences in the circular DNA molecule are arranged as ATGC, then in a corresponding concatemeric molecule the sequences may be

ATGCATGCATGCATGC and so on (assuming amplification (or replication of DNA synthesis) was initiated between genes C and A). Moreover, concatemeric molecules initiate to form from the site of nicking of the circular DNA molecules. Notably, the hairpin loop structures are formed within the concatemeric molecules when single-stranded regions, beyond the site of nicking, fold back on themselves due to complementary base pairing, resulting in stem-loop structures with double-stranded portions that result from complementary base pairing between the two single strands. The double-stranded portions of the hairpin loop structures contain recognition sites for a nicking endonuclease, similar to the circular nucleic acid molecules, to enable preparation of multiple copies of the circular nucleic acid molecules using subsequent enzymatic reactions.

Furthermore, the method comprises nicking the double-stranded portions of the concatemeric molecules with the nicking endonuclease having specificity for said recognition site. The nicking endonuclease nicks at the specific recognition site within the double-stranded portion of the concatemeric molecules to create single-stranded nicks within the double-stranded regions, effectively breaking the concatemeric molecules to yield separate segments.

S Furthermore, the method comprises performing a denaturation step such

N that the nicked concatemeric molecules disintegrate into separate 3 segments having complementary ends. Denaturation is a process that

N 25 disrupts the hydrogen bonds between complementary base pairs, causing & the double-stranded DNA molecules to separate into single-stranded 3 segments. In this regard, the nicked concatemeric molecules are 3 subjected to heating at a high temperature, typically around 90-95°C

N (which is > melting temperature (Tm) of the DNA), for a short period of time, resulting in generation of separate segments from the nicked concatemeric molecules. The separated segments consist of single-

stranded DNA with nicked regions and complementary ends that can anneal to each other or other DNA molecules or oligonucleotide primers.

Furthermore, the method comprises allowing an intramolecular annealing of the complementary ends of the separate segments and ligating the separate segments intramolecularly, thereby obtaining said circular nucleic acid molecules. Intramolecular annealing refers to the process by which complementary sequences within the same DNA molecule (or segment) come together and form stable base pairs through hydrogen bonding. In this regard, the separate single-stranded DNA segments, each containing complementary ends, anneal intramolecularly bringing the separate segments close together and aligning their sequences for ligation to form a circular structure, namely, the circular DNA molecule.

Herein, a DNA ligase enzyme catalyzes the formation of phosphodiester bonds or covalent bonds between adjacent nucleotides in the DNA backbone, sealing the nicked ends of the separate segments together.

Furthermore, the method comprises performing storing the said circular nucleic acid molecules for a predefined duration. The predefined duration refers to a specific period of time for which the circular nucleic acid molecules are stored. This duration is determined based on factors such as the stability of the molecules, the intended downstream applications, and practical considerations such as storage space and cost. Moreover, it

N may be appreciated that the storage conditions (such as temperature,

N pH, humidity, and so on) are optimized to prevent degradation, 3 denaturation, or other detrimental effects that could compromise the

N 25 quality or functionality of the circular nucleic acid molecules. The duration & may range from hours to days, weeks, or even years, depending on the

E reguirements of the experiment or application. In an example, the

S predefined duration is at least one month. Alternatively, optionally, the

N predefined duration is a longer storage times, including very long ones, such as for example, years or decades. Beneficially, storing the circular nucleic acid molecules for a predefined duration provides preservation of the circular DNA molecules, allowing flexibility in experimental timelines, to perform downstream application or analyses at a later time without the need to repeat the entire process of generating the circular DNA molecules.

In an example, the downstream application of the circular DNA molecules may include using DNA as a storage medium due to DNA's potential for high-density, long-term data storage. In this regard, different types of

DNA starting material, including targeted DNA libraries, non-specific DNA libraries and completely synthetic DNA molecules, may be converted into

DNA storage molecules; and subsequently, enzymatic operations on the

DNA storage molecules corresponding to standard database operations such as index, copy and query may be performed.

Optionally, the circular DNA molecules containing the encoded data can be stored in a variety of forms, such as in solution, dried onto a substrate, or encapsulated in microscopic beads, as long as such storage methods ensure the stability and longevity of the DNA molecules.

Furthermore, the method comprises performing subjecting said circular nucleic acid molecules obtained during steps of intramolecular annealing or storing for the predefined time, to a high-throughput sequencing technology. High-throughput sequencing technology, also known as next- generation sequencing (NGS), refers to a set of powerful methods for

S massively parallel (simultaneous) sequencing of millions to billions of

LÖ nucleic acid molecules in a single sequencing run. This allows for the e rapid, accurate and cost-effective analysis of large numbers of DNA or z 25 RNA seguences. In other words, the high-throughput seguencing = © technology or next-generation sequencing (NGS) enables retrieving and

O reading the DNA storage molecules to determine the seguence of

N nucleotides in the DNA. The seguencing data is then decoded back into * binary format using the reverse of the encoding scheme, thus recovering the original digital data. Optionally, the high-throughput seguencing platforms include Illumina, Ion Torrent, Pacific Biosciences (PacBio), and

Oxford Nanopore Technologies, each platform utilizes distinct sequencing chemistries and detection methods, such as sequencing-by-synthesis, sequencing-by-ligation, single-molecule real-time (SMRT) sequencing, and nanopore sequencing.

Optionally, the step of providing one or more circular nucleic acid molecules comprises the steps of: (ia) providing linear at least partially double-stranded nucleic acid molecules and optionally performing end-repair of said linear nucleic acid molecules such that blunt-ended or 3” A-tailed nucleic acid molecules are generated; (ib) contacting said nucleic acid molecules with adaptor nucleic acids comprising double-stranded portions and two single-stranded portions, wherein the ends of the double-stranded portions of the adaptor nucleic acids are compatible with the ends of said nucleic acid molecules such that they can be joined by ligation; (ic) ligating the adaptor nucleic acids to said linear nucleic acid molecules; (id) performing a denaturation step such that strands of said nucleic acid molecules separate; (ie) contacting the separated strands with a single oligonucleotide

N comprising a hairpin loop, or with a plurality of oligonucleotides capable a of annealing to form an oligonucleotide complex comprising a hairpin = loop, said hairpin loop having said double-stranded portions comprising

N

- the recognition site for a nicking endonuclease, a a © 25 wherein said single oligonucleotide or the oligonucleotide complex

LO

O

O stranded portions derived from the adaptor nucleic acids; (if) allowing annealing to take place to form annealed complexes;

(ig) rendering the annealed complexes circular by filling gaps using a polymerase and nucleotides and/or performing a ligation such that circular nucleic acid molecules are generated.

In this regard, the linear DNA comprising at least partially double- stranded regions, is subjected to end-repair process to generate blunt- ended or 3' A-tailed nucleic acid molecules from linear DNA molecules with damaged or uneven ends, for downstream applications, such as library construction or DNA storage molecule preparation. Typically, 3' A- tailed nucleic acid molecules have ends where one strand terminates in an overhanging single-stranded region consisting of a stretch of adenine (A) nucleotides at the 3' ends of the DNA molecules. The 3' A-tailed nucleic acid end-repair is achieved by treating the linear DNA molecule with a terminal transferase enzyme or non-proofreading DNA polymerase and dATP (deoxyadenosine triphosphate). It may be appreciated that after the end-repair, the nucleic acid molecules are typically purified to remove enzymes, nucleotides, and other reaction components, ensuring high-quality DNA for downstream applications. Beneficially, the generation of blunt-ended or 3' A-tailed nucleic acid molecules improves the efficiency and accuracy of subsequent enzymatic reactions, enabling reliable and reproducible results.

The end-repair blunt-ended or 3' A-tailed nucleic acid molecules are

S contacted with adaptor nucleic acids (or adaptors) that are short DNA

N molecules or synthetic oligonucleotides designed with specific seguences 3 and structural features for a desired downstream application. Optionally,

N 25 the adaptor nucleic acids may be used for adding priming sites for & amplification or seguencing, ligating DNA fragments together or with 3 other surfaces or components, and so on. In this regard, the double- x stranded portions of the adaptors form a stable duplex through

N complementary base pairing, while the single-stranded overhangs or regions at each end of the double-stranded portion provide sites for interactions with target nucleic acid molecules. It may be appreciated that the two single-stranded portions of the adaptor nucleic acids do not hybridize with each other. The compatible ends of the double-stranded portions of the adaptor nucleic acids are ligated to the ends of said linear nucleic acid molecules using a ligase enzyme. The ligase enzyme results in the formation of a phosphodiester bond between the compatible ends, thereby, joining the adaptor and target molecules. The ligated molecules are subjected to denaturation to separate the double-stranded molecule into single-stranded molecules.

The single-stranded portions of the adaptor nucleic acids are subsequently contacted with oligonucleotides, selected from single oligonucleotide or plurality of oligonucleotides that anneal together to form oligonucleotide complex, both having hairpin loop, to anneal and form the annealed complex comprising the annealed separated strands of the adaptors and the single oligonucleotide or the plurality of oligonucleotides. The term "single oligonucleotide" refers to a single- stranded DNA molecule, of 10-100 nucleotides in length. The term "plurality of oligonucleotides" refers to multiple copies of said single- stranded DNA molecule. In this regard, the amount of the single oligonucleotide or plurality of oligonucleotides is more compared to the target DNA molecule. Herein, the annealed complexes are rendered circular to form the circular nucleic acid molecules using at least one of: x filling gaps using a polymerase and nucleotides and/or performing a

N ligation. The process of filling gaps or nicks employs for example a DNA 3 polymerase enzyme and free nucleotides (dNTPs). Alternatively, the & 25 annealed complexes can be circularized through ligation, where the ends z of the annealed molecules are covalently joined together by a ligase enzyme.

N Optionally, said single oligonucleotide comprises 5” and 3' ends that are

N complementary to the two single-stranded portions derived from the adaptor nucleic acids. For example, a single-stranded portions of the adaptor nucleic acid is 5'-AGCTGATC-3', the complementary single oligonucleotide sequence is designed such that the 5' end of the single oligonucleotide has the sequence 3'-TACGACTA-5', which is complementary to the 5' end of the single-stranded portion of the adaptor nucleic acid (5'-AGCTGATC-3'), and the 3' end of the single oligonucleotide has the sequence 5'-CATGACT-3', which is complementary to the 3' end of the single-stranded portion of the adaptor nucleic acid (3'-TCGACTAG-5'). When the single oligonucleotide is annealed to the adaptor nucleic acid, the complementary seguences at the 5' and 3' ends of the single oligonucleotide form base-pairs with the corresponding sequences on the single-stranded portion of the adaptor nucleic acid, forming a stable duplex structure, namely, the annealed complex. Beneficially, the complementarity between the single oligonucleotide and the two single-stranded portions derived from the adaptor nucleic acids allows the single oligonucleotide to anneal or hybridize with the adaptor nucleic acids.

Optionally, the plurality of oligonucleotides comprises a first oligonucleotide and a second oligonucleotide, and wherein the first oligonucleotide comprises: a 5’ end that consists of a hairpin loop, a single-stranded portion, and a 3' end that is complementary to the two single-stranded portions derived from the adaptor nucleic acids, and

N wherein the second oligonucleotide comprises a 5' end that is a complementary to the two single-stranded portions derived from the > adaptor nucleic acids and comprises a 3’ end that is complementary to : 25 the single-stranded portion of the first oligonucleotide that is adjacent to = © the hairpin loop of the first oligonucleotide. 8 In this regard, the terms "first oligonucleotide" and "second

N

S oligonucleotide" as used herein refer to different single oligonucleotides, each of which has one end complementary to the two single-stranded portions derived from the adaptor nucleic acids, and the other end complementary to each other. Herein, the first oligonucleotide is different from the second oligonucleotide, wherein the second oligonucleotide lacks a hairpin loop. Herein, both the first and second oligonucleotides are designed to anneal to specific regions of the adaptor nucleic acid. The first oligonucleotide forms a hairpin loop structure while binding to the adaptor nucleic acid, and the second oligonucleotide complements the adjacent region of the first oligonucleotide. Together, they facilitate the formation of specific structures required for subsequent steps in the experimental procedure. In an example, the adaptor nucleic acid sequence is 5'-AGCTGATC-3', the first oligonucleotide sequence is 5'-

TACGACTAGCTGATCGTAG-3', and the second oligonucleotide sequence is 5'-GCTAGCATCGACTAGC-3".

Optionally, the plurality of oligonucleotides comprises: a first oligonucleotide, a second oligonucleotide and a bridge oligonucleotide, wherein the first oligonucleotide comprises: a 5’ end that is complementary to a portion of the bridge oligonucleotide, a portion that is complementary to a single-stranded portion of the second oligonucleotide, and a 3' end that is complementary to the single- stranded portions derived from the adaptor nucleic acids, and wherein the second oligonucleotide comprises: a 5° end that is complementary to the single-stranded portions derived from the adaptor

N nucleic acids, a portion that is complementary to a single-stranded a portion of the first oligonucleotide and a 3' end that is complementary to e a portion of the bridge oligonucleotide, and

E 25 wherein the bridge oligonucleotide comprises seguences complementary © to the 5’ end of the first oligonucleotide and the 3' end of the second

O oligonucleotide.

S In this regard, the term "bridge oligonucleotide" as used herein refers to a short DNA molecule that is configured to connect or bridge two complementary nucleic acid seguences on two separate nucleic acid molecules or regions. Herein, the bridge oligonucleotide serves to connect the first and second oligonucleotides by complementing both their 5' and 3' ends. This arrangement allows for the formation of a complex structure involving multiple oligonucleotides. In an example, the sequences of the first oligonucleotide is 5'-AATTCCGG-3', the second oligonucleotide is 5'-

GCTAAGATC-3', and the bridge oligonucleotide is 5'-CGGAATT-3'.

Moreover, the portion of the bridge oligonucleotide may be selected from a 5' end sequence, a 3' end sequence, or a central sequence thereof.

Optionally, the plurality of oligonucleotides comprises a first oligonucleotide, a second oligonucleotide, a hairpin loop oligonucleotide and a bridge oligonucleotide or a plurality of bridge oligonucleotides capable of annealing to the hairpin loop oligonucleotide to form a bridge oligo complex, wherein the first oligonucleotide comprises: a 5° end that is complementary to the single-stranded portions derived from the adaptor nucleic acids and a 3’ end that is complementary to a portion of the bridge oligonucleotide or the bridge oligo complex; wherein the second oligonucleotide comprises: a 5° end that is complementary to a portion of the bridge oligonucleotide or the bridge oligo complex and a 3’ end that is complementary to the single-stranded portions derived from the adaptor nucleic acids at the 3’ end,

S wherein the hairpin loop oligonucleotide comprises, starting from the 5” ro end of the molecule, a left bridge oligo-specific sequence, a hairpin loop e and a right bridge oligo-specific sequence, and

E 25 wherein the bridge oligonucleotide or plurality of bridge oligonucleotides 3 contains: a sequence complementary to the 3’ end of the first 3 oligonucleotide, sequences complementary to the left bridge oligo-

N specific seguence and the right bridge oligo-specific seguence in the hairpin loop oligonucleotide and a sequence complementary to the 5’ end of the first oligonucleotide and the 3’ end of the second oligonucleotide.

In this regard, the term "hairpin loop oligonucleotide" refers to a short single-stranded DNA molecule that folds back on itself to form a characteristic hairpin loop structure, as discussed above. The hairpin loop oligonucleotide comprises three main parts, the left bridge oligo-specific sequence at the 5' end, a right bridge oligo-specific sequence at the 3' end, and a hairpin loop structure therebetween. Herein, the left bridge oligo-specific sequence and the right bridge oligo-specific sequence refer to the sequences of the hairpin loop oligonucleotide that are complementary to the bridge oligonucleotide or the plurality of bridge oligonucleotides. Specifically, the left bridge oligo-specific sequence and the right bridge oligo-specific sequence refer to the portions of the hairpin loop oligonucleotide that are specifically complementary to the sequence on the left side and the right side of the bridge oligonucleotide, respectively.

The hairpin loop oligonucleotide anneals with the bridge oligonucleotides or the plurality of bridge oligonucleotides to form a bridge oligo complex due to complementary pairing between specific regions thereof.

Beneficially, the bridge oligo complex serves as a molecular scaffold or connector, bringing together different components or molecules in a molecular assembly. In an example, the sequences of the first oligonucleotide is 5'-AGCTGATC-3', the second oligonucleotide is 5'- < TAGCGTCA-3', the hairpin loop oligonucleotide is 5'-ATCGGATC-

S HairpinLoop-TAGCTAGC-3', and the bridge oligonucleotide is 5'- 3 GATCGATC-HairpinLoop-AGCTGATC-3'. Moreover, the portion of the & 25 bridge oligonucleotide or plurality of bridge oligonucleotides may be z selected from a 5' end sequence, a 3' end sequence, or a central sequence thereof.

N Optionally, the plurality of oligonucleotides comprises a hairpin loop

N oligonucleotide or a plurality of oligonucleotides capable of annealing to form a hairpin loop oligo complex, and a bridge oligonucleotide capable of annealing to the hairpin loop oligonucleotide or a hairpin loop oligo complex to form a bridge oligo complex, wherein the hairpin loop oligonucleotide or the hairpin loop oligo complex comprises, starting from the 5’ end of the molecule, a left bridge oligo- specific sequence, a hairpin loop sequence and a right bridge oligo- specific sequence, and wherein the bridge oligonucleotide contains a 5 end that is complementary to the single-stranded portions derived from the adaptor nucleic acids at the 3’ end, and a 3' end that is complementary to the single-stranded portions derived from the adaptor nucleic acids at the 5’ end, and sequences complementary to the left bridge oligo-specific sequence and the right bridge oligo-specific sequence in the hairpin loop oligonucleotide.

In this regard, the plurality of hairpin loop oligonucleotides comprises multiple hairpin loop oligonucleotides, such as a first hairpin loop oligonucleotides, a second hairpin loop oligonucleotides, and so on. The hairpin loop oligonucleotides or the plurality of hairpin loop oligonucleotides anneal with the bridge oligonucleotide to form the bridge oligo complex. Herein the 5' and 3' ends of the bridge oligo complex are complementary to the 3' and 5' ends of the single-stranded portions derived from the adaptor nucleic acids, respectively, and a central

N seguence thereof is complementary to the left bridge oligo-specific a seguence and the right bridge oligo-specific seguence. In an example, > the right bridge oligo-specific sequence of the hairpin loop oligonucleotide : 25 5'-ATCGGATC-HairpinLoop-TAGCTAGC-3' is complementary to the bridge = © oligonucleotide 5'-GCTAGCTA-3". 8 Optionally, the plurality of oligonucleotides are capable of annealing to

N

S form a hairpin loop oligo complex and a bridge oligonucleotide, wherein said plurality of oligonucleotides comprises:

(a) a first oligonucleotide, a second oligonucleotide and a loop bridging oligonucleotide, wherein the first oligonucleotide comprises: a 5° end that is complementary to a portion of the bridge oligonucleotide, a portion that is complementary to a single-stranded portion of the second oligonucleotide, and a 3’ end that is complementary to the loop bridging oligonucleotide, and wherein the second oligonucleotide comprises: a 5° end that is complementary to the loop bridging oligonucleotide, a portion that is complementary to a single-stranded portion of the first oligonucleotide and a 3' end that is complementary to a portion of the bridge oligonucleotide, and wherein the loop bridging oligonucleotide comprises sequences complementary to the first 5’ end of the first oligonucleotide and the 3’ end of the second oligonucleotide, or (b) a first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide comprises: a 5’ end that consists of a hairpin loop, a single-stranded portion and a 3° end that is complementary to the bridge oligonucleotide, and +

N wherein the second oligonucleotide comprises a 5' end that is

N ro complementary to bridge oligonucleotide and a 3' end that is e complementary to a single-stranded portion of the first oligonucleotide

I that is adjacent to the hairpin loop of the first oligonucleotide. a 2 25 In this regard, the term "/oop bridging oligonucleotide" as used herein

O

0 refer to a short seguence of DNA that acts as a bridge between the first

N

N and second oligonucleotides. In an example, seguences of the first oligonucleotide is 5'--ATCGTA-AGCT-3', the second oligonucleotide is 5'-

CGCTT-CTAG-3’, and the loop bridging oligonucleotide is 5-AGCTT-3".

Herein, the first oligonucleotide may or may not contain a hairpin loop.

Optionally, the step of providing one or more circular nucleic acid molecules comprises the steps of: (ia) providing linear at least partially double-stranded nucleic acid molecules; (ib) performing a denaturation step such that strands of said linear nucleic acid molecules separate; (ic) contacting the separated strands with a single oligonucleotide comprising a hairpin loop, said hairpin loop comprising a recognition site for a nicking endonuclease, wherein said single oligonucleotide comprises 5" and 3' ends that are complementary to specific sequences in the separated strands; (id) allowing annealing to take place to form annealed complexes; (ie) rendering the annealed complexes circular by filling gaps using a polymerase and nucleotides; and performing a ligation such that circular nucleic acid molecules are generated.

Herein, the optional steps of end-repair of the linear at least partially double-stranded nucleic acid molecules and adaptor nucleic acids ligation to said linear nucleic acid molecules are eliminated. Moreover, herein, the <

N term "specific sequences in the separated strands" refers to any of the 5'

N e end seguence, 3' end seguence or a central seguence of the linear nucleic e acid molecules. Beneficially, eliminating such optional steps enables

I faster turnaround times, cost-effective and time-efficient process. a © 25 Notably, end-repair and ligation can potentially introduce biases or

LO

O degraded or damaged nucleic acids. By skipping these steps, the integrity of the nucleic acids may be better preserved, leading to more accurate downstream application analysis results. Moreover, by eliminating these steps, the overall yield of circular nucleic acid molecules may be increased, allowing for greater amounts of product to be generated from the same starting material.

Optionally, the step of providing one or more circular nucleic acid molecules comprises the steps of: (ia) providing linear at least partially double-stranded nucleic acid molecules; (ib) performing a denaturation step such that strands of said linear nucleic acid molecules separate; (ic) contacting the separated strands with a plurality of oligonucleotides capable of annealing to form an oligonucleotide complex comprising a hairpin loop, said hairpin loop comprising a recognition site for a nicking endonuclease, wherein the plurality of oligonucleotides comprises a first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide comprises: a 5' end that consists of a hairpin loop, a single-stranded portion, and a 3' end that is complementary to a specific sequence in the separated strands, and wherein the second oligonucleotide comprises a 5’ end that is complementary to a specific sequence in the separated strands and comprises a 3’ end that is complementary to a single-stranded portion of

N the first oligonucleotide that is adjacent to the hairpin loop of the first & . . i oligonucleotide;

S e (id) allowing annealing to take place to form annealed complexes;

E (ie) rendering the annealed complexes circular by filling gaps using a 2 25 polymerase and nucleotides; and performing a ligation such that circular

O o nucleic acid molecules are generated.

N

O

N Herein, using the plurality of oligonucleotides, while eliminating the optional steps of end-repair of the linear at least partially double-stranded nucleic acid molecules and adaptor nucleic acids ligation to said linear nucleic acid molecules, enables amplification or sequencing of specific

DNA regions, introducing molecular labels or tags into DNA sequences for visualization, purification, or downstream analysis, constructing diverse libraries for high-throughput sequencing or screening, and so on.

Moreover, as discussed above, benefits of annealing plurality of oligonucleotides to the separated strands of a denatured linear DNA molecule, while eliminating the optional steps of end-repair of the linear at least partially double-stranded nucleic acid molecules and adaptor nucleic acids ligation to said linear nucleic acid molecules, makes the process more time-efficient, cost-effective, less prone to biases, and so forth.

Optionally, the step of providing one or more circular nucleic acid molecules comprises the steps of: (ia) providing linear at least partially double-stranded nucleic acid molecules; (ib) performing a denaturation step such that the strands of said linear nucleic acid molecules separate; (ic) contacting the separated strands with a plurality of oligonucleotides capable of annealing to form an oligonucleotide complex comprising a hairpin loop, said hairpin loop comprising a recognition site for a nicking < endonuclease, wherein the plurality of oligonucleotides comprises: a first

S oligonucleotide, a second oligonucleotide and a bridge oligonucleotide, g wherein the first oligonucleotide comprises: a 5' end that is

N complementary to a portion of the bridge oligonucleotide, a portion that

E 25 is complementary to a single-stranded portion of the second 3 oligonucleotide, and a 3' end that is complementary to a specific 3 sequence in the separated strands, and

S wherein the second oligonucleotide comprises: a 5' end that is complementary to a specific seguence in the separated strands, a portion that is complementary to a single-stranded portion of the first oligonucleotide and a 3’ end that is complementary to a portion of the bridge oligonucleotide, and wherein the bridge oligonucleotide comprises sequences complementary to the 5’ end of the first oligonucleotide and the 3' end of the second oligonucleotide; (id) allowing annealing to take place to form annealed complexes; (ie) rendering the annealed complexes circular by filling gaps using a polymerase and nucleotides; and performing a ligation such that circular nucleic acid molecules are generated.

Herein, by bridging the first and second oligonucleotides with the bridge oligonucleotide, the plurality of oligonucleotides are annealed to denatured DNA strands with high specificity, in a single step. This allows for multiplexed annealing, facilitating the simultaneous incorporation of multiple sequences into the linear DNA molecule, besides the other benefits of removing such optional steps.

Optionally, the step of providing one or more circular nucleic acid molecules comprises the steps of: (ia) providing one or more linear nucleic acid molecules, each comprising (a) a sequence of interest, and + 20 (b) a first and second pair of complementary single-stranded

N

< sequences, wherein each pair of complementary single-stranded 3 sequences is capable of annealing to each other such that a double-

S stranded stem portion is formed,

I

E- wherein the seguence of interest is flanked on both ends by a single- 3 25 stranded sequence of the first pair, and

LO

N wherein the two single-stranded seguences of the second pair are

N separated by a loop seguence and wherein a single-stranded seguence of the second pair is linked to a single-stranded sequence of the first pair, optionally via a nucleotide linker; (ib) optionally denaturing the molecules; (ic) allowing the pairs of the complementary sequences to anneal; and (id) joining the 5" and 3’ prime ends of the linear nucleic acid molecule using a ligase or a combination of a polymerase, nucleotides and a ligase to form a circular nucleic acid molecule comprising a double-stranded portion comprising a recognition site for a nicking endonuclease.

In this regard, the sequence of interest is the specific DNA sequence targeted for manipulation or analysis. The two pairs of complementary single-stranded sequences, namely the first and the second pairs of complementary single-stranded sequences, comprise the sequence of interest flanked on both ends of the first pair, while the second pair consists of two single-stranded sequences separated by a loop sequence, optionally a hairpin loop. The first and second pairs are configured to form double-stranded stem portion by self-annealing and ligate together via the nucleotide linker. The nucleotide linker is a short strand of nucleotides that serves as a bridge between two single-stranded DNA sequences, namely the single-stranded sequence of the second pair and the single- stranded sequence of the first pair, by facilitating covalent linkage < therebetween. Moreover, nucleotide linkers facilitate connecting the ends

S of single-stranded DNA seguences, forming a loop structure, such as in ro the first pair. In this regard, the nucleotide linkers are designed to have e complementary seguences to the DNA fragments they are linking,

E 25 ensuring efficient annealing and ligation. > Optionally, the linear nucleic acid molecules are denatured to separate 3 the complementary single-stranded sequences from each other. It may

N be appreciated that if the one or more linear nucleic acid molecules are double-stranded, a denaturation step may be performed to separate the two strands. Denaturation is typically achieved by heating the sample to high temperatures or treating it with denaturing agents like urea or formamide. This step is optional and depends on the application. For example, if the molecules are already single-stranded or if double- stranded molecules are desired, denaturation may be skipped.

The denatured linear nucleic acid molecules are subsequently allowed to anneal, enabling the pairs of complementary single-stranded sequences to reassociate with each other. Beneficially, the annealing process reforms the double-stranded stem portion, bringing the sequence of interest and the loop sequence into proximity. The 5’ and 3’ ends of the first and second pairs of the linear nucleic acid molecules are joined together to form a circular nucleic acid molecule containing a sequence of interest, by using a ligase enzyme or a combination of a polymerase, nucleotides, and a ligase. Notably, when using a ligase, such as DNA ligase, to join the 5” and 3’ ends of two linear nucleic acid sequences, the ligase enzyme catalyzes the formation of a phosphodiester bond between the 3’ hydroxyl group of one nucleotide and the 5’ phosphate group of the adjacent nucleotides, to form the desired circular nucleic acid molecule. However, combination of enzymes and nucleotides is used to fill in the gap between the 5’ and 3’ ends of the linear nucleic acid molecule before sealing the ends with the ligase enzyme. In this regard, polymerase, such as DNA polymerase enzyme, adds nucleotides, such as < dNTPs, to fill in the gap between the 5” and 3’ ends of the linear DNA

S molecule, using the complementary strand as a template. Subseguently, 3 the ligase enzyme catalyzes the sealing of the nick between the newly & 25 synthesized strand and the original linear DNA molecule, forming a z continuous circular DNA molecule. Beneficially, using such combination 8 ensures that the DNA ends are properly filled in before ligation occurs, ? resulting in a more efficient and precise circularization process.

N Optionally, the one or more linear nucleic acid molecules provided are generated synthetically. The synthetically generated linear nucleic acid molecules refer to DNA molecules that are produced in-vitro using chemical synthesis methods rather than being isolated from natural sources. The synthetically generated linear nucleic acid molecules have specific sequences, lengths, and modifications tailored to desired needs for the downstream application thereof.

Optionally, the step of providing one or more circular nucleic acid molecules comprises the steps of: (ia) providing linear at least partially double-stranded nucleic acid molecules and optionally performing end-repair of said linear nucleic acid molecules such that blunt-ended or 3’ A-tailed nucleic acid molecules are generated; (ib) contacting said nucleic acid molecules with adaptor nucleic acids comprising a double-stranded portion and said first and second pair of complementary single-stranded sequences, and wherein ends of the double-stranded portions of the adaptor nucleic acids are compatible with ends of said nucleic acid molecules such that they can be joined by ligation; and (ic) ligating the adaptor nucleic acids to said nucleic acid molecules.

Herein, the first and second pair of complementary single-stranded sequences of the adaptor nucleic acids bond (anneal or hybridize) with the linear at least partially double-stranded nucleic acid molecules or the

N end-repaired linear at least partially double-stranded nucleic acid a molecules, to form stable base-paired complexes. The ends of the double- = stranded portions of the adaptor nucleic acids are ligated to said linear at

N least partially double-stranded nucleic acid molecules or the end-repaired a 25 linear at least partially double-stranded nucleic acid molecules using the

O ligase enzyme. Beneficially, adaptor nucleic acids designed with specific 3 sequences that are complementary to the ends of the nucleic acid

N molecules, allow for precise and sequence-specific ligation, ensuring that the correct molecules are joined together.

Optionally, the step of providing one or more circular nucleic acid molecules comprises the steps of: (ia) providing one or more linear nucleic acid molecules, optionally performing a denaturation step, and phosphorylating 5’ ends thereof; (ib) contacting and annealing the 5’ phosphorylated linear nucleic acid molecules with first and second adaptor nucleic acids, wherein each of the first adaptor nucleic acids comprise a first strand comprising first pair of complementary single-stranded sequences, which are capable of annealing to each other such that a hairpin loop comprising a double-stranded stem portion is formed, followed by a single-stranded half-pair sequence complementary to the 3’ end of a first strand of the corresponding second adaptor nucleic acid, followed by a double- stranded portion and a single-stranded portion attached to a second strand, wherein the single-stranded portion contains random N nucleotides to permit binding to non-universal ends, and wherein the second adaptor nucleic acids comprise a 5' phosphorylated first strand forming a double-stranded portion with a second strand, said first strand further comprising a single-stranded sequence complementary to the half-pair of the first adaptor nucleic acid, and wherein the second strand contains random N nucleotides to permit < binding to non-universal ends

R (ic) allowing the linear nucleic acid molecules and the first and second 3 adaptor nucleic acids to anneal;

N (id) joining the 5' and 3’ prime ends of the annealed nucleic acid molecule a 25 using a ligase.

O

O Herein, phosphorylation of the 5’ ends of the one or more linear nucleic

X acid molecules involves adding phosphate groups to the terminal 5’ hydroxyl groups of the DNA molecules. This step is typically carried out using a kinase enzyme (e.g., T4 polynucleotide kinase) in the presence of ATP or another phosphate donor. Notably, phosphate group provides a reactive site for the formation of phosphodiester bonds with other nucleic acid molecules or adapters, thus the phosphorylation of the 5’ ends is essential for subsequent enzymatic reactions, such as ligation or polymerization. The 5’ phosphorylated linear nucleic acid molecules is contacted and annealed with first and second adaptor nucleic acids, at least one of which comprises a 5' phosphorylated strand, herein the second adaptor nucleic acid. In this regard, the complementary sequences of the 5' phosphorylated linear nucleic acid molecules and the first and second adaptor nucleic acids come together and form stable base-paired complexes through hydrogen bonding. The 5’ and 3’ ends of the annealed nucleic acid molecules are joined together using a ligase enzyme that catalyzes the formation of phosphodiester bonds between the adjacent nucleotides, resulting in the circularization of the linear nucleic acid molecules.

The term "half-pair" refers to a partial complementary sequence present on the first adaptor nucleic acid that corresponds to the single-stranded sequence on the second adaptor nucleic acid, facilitating their annealing and subsequent ligation to linear nucleic acid molecules. The term "non- universal ends" refers to DNA sequences that do not have predefined or specific sequences recognized by the first or second adaptor nucleic acids. x The non-universal ends may typically arise from various sources, such as

R fragmented DNA molecules resulting from random shearing or enzymatic 3 digestion, PCR amplified DNA fragments, or randomly generated & 25 sequences comprising sequences with random nucleotides (denoted as z N) to the ends of DNA molecules. Beneficially, the single-stranded portion 8 of the adaptor nucleic acid containing random N nucleotides provides ? flexibility in binding to these non-universal ends. In other words, by

N incorporating random nucleotides, the adaptor nucleic acid can anneal to a broader range of sequences, including those with non-specific or varied ends, thus enhancing the versatility and adaptability of the adaptor nucleic acids for use with diverse nucleic acid molecules with non- universal ends. Optionally, the two annealing steps may occur simultaneously in the same reaction mixture.

Optionally, the step of providing one or more circular nucleic acid molecules comprises the steps of: (ia) providing linear sticky-ended at least partially double-stranded nucleic acid molecules; (ib) contacting said linear sticky-ended nucleic acid molecules with adaptor nucleic acids such that the linear sticky-ended nucleic acid molecules and the adaptor nucleic acids are joined by sticky ends, wherein the adaptor nucleic acids comprise a hairpin loop and at least one end compatible with sticky-ends of a corresponding linear sticky- ended nucleic acid molecule; (ic) ligating the adaptor nucleic acids to the linear sticky-ended nucleic acid molecules; (id) performing a denaturation step to generate single-stranded molecules; and (ie) ligating the 5” and 3’ prime ends of the single-stranded molecules to form a circular nucleic acid molecule using a ligase capable of single- stranded-molecule circularization. +

N

S The term "sticky ends" refer to DNA ends that have short single-stranded 3 overhangs complementary to each other. The adaptor nucleic acids serve

S as bridge molecules, facilitating the circularization of the linear DNA

E fragments by providing complementary seguences that anneal to the 2 25 sticky ends and allowing for the subseguent ligation reaction. Herein, the

O o linear sticky-ended nucleic acid molecules are converted into the circular

N

N nucleic acid molecules by ligating adaptor nucleic acids to their ends.

Notably, denaturation is essential to separate the double-stranded linear sticky-ended nucleic acid molecules.

Optionally, the step of providing one or more circular nucleic acid molecules comprises the steps of: (ia) providing linear sticky-ended at least partially double-stranded nucleic acid molecules; (ib) contacting said linear sticky-ended nucleic acid molecules with adaptor nucleic acids such that the linear sticky-ended nucleic acid molecules and the adaptor nucleic acids are joined by sticky ends; wherein the adaptor nucleic acids comprise a hairpin loop flanked by two double-stranded portions formed via annealing with a linear oligonucleotide, wherein at least one end of the double-stranded portions has sticky ends compatible with sticky-ends of a corresponding linear sticky-ended nucleic acid molecule, (ic) ligating the adaptor nucleic acids to the linear sticky-ended nucleic acid molecules; (id) performing a denaturation step to generate single-stranded molecules; and (ie) allowing the single-stranded molecules to self-anneal and ligating the 5" and 3’ prime ends of the single-stranded molecules to form a circular nucleic acid molecule using a ligase capable of single-stranded-molecule circularization. <

S The linear oligonucleotide is typically a short, single-stranded DNA ro molecule composed of a sequence of nucleotides connected by e phosphodiester bonds. The term "linear" as used herein refers to a lack

I of any secondary structures such as hairpin loops or duplex regions in the a © 25 linear oligonucleotide, unlike longer nucleic acid molecules that can form

LO

O molecules with the combination of the adaptor nucleic acids and a linear oligonucleotide provide additional seguence specificity during annealing and enhance the efficiency of annealing reactions. In this regard, the adaptor nucleic acids can be designed with specific sequences that complement the sticky ends of the nucleic acid molecules, ensuring precise and directional binding, while the linear oligonucleotide can further increase specificity by providing additional hybridization sites or structural features. Optionally, the double-stranded portions have a 3' end or a 5' end comprising the sticky ends, either or both of which may be compatible with sticky-ends of a corresponding linear sticky-ended nucleic acid molecule.

The term "self-anneal" refers to the process by which single-stranded nucleic acid molecules spontaneously form double-stranded structures through complementary base pairing. In this regard, the single-stranded regions of one molecule can hydrogen bond with complementary sequences on another molecule, forming double-stranded regions. After self-annealing occurs and double-stranded regions are formed, the 5” and 3' ends of the single-stranded molecules are ligated together using the ligase enzyme. The ligase enzyme catalyzes the formation of phosphodiester bonds between the adjacent nucleotides at the ends of the molecules, resulting in the circularization of the nucleic acid molecules to form circular nucleic acid molecules.

Optionally, the step of providing one or more circular nucleic acid molecules comprises the steps of:

N (ia) providing linear sticky-ended at least partially double-stranded 5 nucleic acid molecules; e (ib) contacting such linear sticky-ended nucleic acid molecules with

E 25 adaptor nucleic acids such that the linear sticky-ended nucleic acid © molecules and the adaptor nucleic acids are joined by sticky ends; ? wherein the adaptor nucleic acids comprise a hairpin loop flanked by a

N double-stranded portion formed via annealing with a linear oligonucleotide, wherein the double-stranded portion has a sticky end compatible with sticky-ends of a corresponding linear sticky-ended nucleic acid molecule, wherein the hairpin loop is flanked on the other side by a single-stranded sequence complementary to a portion of a splint oligonucleotide and wherein the linear oligonucleotide further comprises a single-stranded sequence complementary to an adjacent portion of said splint oligonucleotide; (ic) ligating the adaptor nucleic acids to the linear sticky-ended nucleic acid molecules; (id) performing a denaturation step to generate single-stranded molecules; and (ie) adding the splint oligonucleotide and allowing it to anneal with the single-stranded molecules and ligating the 5’ and 3’ prime ends of the single-stranded molecules to form a circular nucleic acid molecule.

The term "splint oligonucleotide" refers to a short, single-stranded DNA molecule that serves as a bridge or template for the ligation of two other nucleic acid molecules. In this regard, the splint oligonucleotide facilitates the joining of the two nucleic acid molecules, herein the linear sticky- ended at least partially double-stranded nucleic acid and the single- stranded sequence of the hairpin loop, by bringing them into close proximity and providing a template for the ligation reaction. Beneficially, the splint oligonucleotide helps to stabilize the interaction between the < two nucleic acid molecules and promotes the formation of a stable

S complex, thereby facilitating the ligation reaction catalyzed by the ligase 3 enzyme, as discussed above.

N Optionally, the step of providing one or more circular nucleic acid

E: 25 molecules comprises the steps of:

E (ia) providing linear single-stranded nucleic acid molecules;

X (ib) contacting the linear single-stranded nucleic acid molecules with adaptor nucleic acids and a splint oligonucleotide such that the linear single-stranded nucleic acid molecules and the adaptor nucleic acids are joined via the splint oligonucleotide; wherein the adaptor nucleic acids comprise a hairpin loop flanked by two single-stranded portions, wherein one single-stranded portion is complementary to the splint oligonucleotide and wherein the other single- stranded portion is complementary to an end of a corresponding linear single-stranded nucleic acid molecule, and wherein the splint oligonucleotide comprises a portion that is complementary to a single-stranded portion of the adaptor nucleic acid and a portion that is complementary to other end of the corresponding linear single-stranded nucleic acid molecule, (ic) ligating the adaptor nucleic acids to the linear single-stranded nucleic acid molecules; (id) optionally performing a denaturation step to dissociate the splint oligonucleotide and generate single-stranded molecules; and (ie) allowing the single-stranded molecules to self-anneal and ligating the 5" and 3' prime ends of the resulting single-stranded molecules to form circular nucleic acid molecules using a ligase capable of single-stranded- molecule circularization.

Herein, the splint oligonucleotide is dissociated from the linear single-

N stranded nucleic acid molecules by subjecting the reaction mixture a containing the splint oligonucleotide and the ligated nucleic acid = molecules to conditions that disrupt the hydrogen bonds holding the

N

- double-stranded structure together, to generate single-stranded a > 25 molecules for easy manipulation later. 00

LO

O

O molecules comprises the steps of: (ia) providing one or more linear nucleic acid molecules, optionally performing a denaturation step, and phosphorylating 5' ends thereof;

(ib) contacting the 5’ phosphorylated linear nucleic acid molecules with adaptor nucleic acids, wherein the adaptor nucleic acids comprise an annealed first and second strand, wherein the first strand comprises a 5’ phosphorylated portion complementary to the second strand, followed by a hairpin loop followed by a 3’ portion complementary to the second strand, wherein the hairpin loop comprises a recognition site for a nicking endonuclease, and wherein the second strand comprises a 5’ portion comprising random N nucleotides to permit binding to non-universal sequences, followed by a portion that is complementary to the first strand, followed by a 3’ portion comprising random N nucleotides to permit binding to non-universal sequences; (ic) allowing the 5” phosphorylated nucleic acid molecules and the adaptor nucleic acids to anneal to generate annealed complexes; (id) closing the nicks in the annealed complexes using a DNA ligase, thereby joining the 5’ phosphorylated nucleic acid molecules to the adaptor nucleic acids, to generate ligated complexes; (ie) nicking double-stranded portions of the ligated complexes with a nicking endonuclease having specificity for said recognition site to generate nicked structures;

N (if) performing a denaturation step such that the nicked structures a disintegrate into separate segments having complementary ends; and

O

@ (ig) allowing intramolecular annealing of the complementary ends of the

I separate segments and ligating the separate segments intramolecularly, a © 25 thereby obtaining circular nucleic acid molecules.

LO

O

0 Herein, the annealed first and second strands of the adaptor nucleic acids

N

N refer to a configuration where at least one of the first and second strands has specific seguences that are complementary to each other, which allow the first and second strands to fold back on itself and form a stable double-stranded structure. Optionally, the first strand of the adaptor nucleic acid is typically designed to contain such specific sequences that are complementary to each other, which allow the first strand to fold back on itself and form a stable double-stranded structure. While the second strand is complementary to the first strand, allowing it to hybridize with the first strand and stabilize the double-stranded structure.

Moreover, the terms "annealed complex" and "ligated complex" refer to the combinations of the 5’ phosphorylated nucleic acid molecules to the adaptor nucleic acids with and without nicks or gaps, respectively. In this regard, DNA ligase enzyme is used to close or fill the nicks in the annealed complexes, such as by employing dNTPs. The term "intramolecular annealing" refers to the process by which complementary single-stranded segments within the same molecule come together and base pair with each other, forming stable duplex regions within the same molecule.

Optionally, DNA ligase enzyme is used for the intramolecular annealing to obtain the circular nucleic acid molecules.

Optionally, the step of providing one or more circular nucleic acid molecules comprises the steps of: (ia) synthesizing nucleic acid molecules containing a hairpin loop flanked by single-stranded sequences having 5° and 3' ends that are complementary to a splint oligonucleotide; and

S (ib) annealing said nucleic acid molecules with the splint oligonucleotide ro comprising sequences that are complementary to the 5” and 3’ ends of e the nucleic acid molecules and ligating the ends thereof.

E 25 In this regard, the nucleic acid molecules, such as a linear double- 3 stranded or single-stranded nucleic acid molecule, or a circular double- 3 stranded or single-stranded nucleic acid molecule, containing a specific

N structure are synthesized. Optionally, the hairpin loop is flanked by single-stranded seguences at both ends that are complementary to the splint oligonucleotide and anneal therewith using a DNA ligase enzyme, forming circular nucleic acid molecules.

Optionally, the step of providing one or more circular nucleic acid molecules comprises the steps of: (ia) synthesizing linear single-stranded nucleic acid molecules containing a hairpin loop; and (ib) self-ligating said linear nucleic acid molecules to form a circular nucleic acid molecule using a ligase capable of single-stranded-molecule circularization.

Herein, the linear single-stranded nucleic acid molecule is synthesized using the methods known to a person skilled in the art. Typically, oligonucleotide primers that encode for the hairpin loop may be employed. The hairpin loop containing linear single-stranded nucleic acid molecules are circularized by self-ligation, by bringing the 5' and 3' ends thereof into proximity by the folding of the hairpin loop, and allowing them to be ligated together intramolecularly using a DNA ligase enzyme as discussed above.

Optionally, the step of providing one or more circular nucleic acid molecules comprises the steps of: (ia) providing one or more linear nucleic acid molecules and optionally

N performing a denaturation step,

N ro (ib) contacting and annealing the linear nucleic acid molecules with first e and second adaptor nucleic acids, generating annealed complexes,

E wherein the first adaptor nucleic acids comprise a 5’ portion and a 3’ 2 25 portion, and wherein the 3’ portion is complementary to a portion of the

O

0 linear nucleic acid molecules, and wherein the 5’ portion is partially

N

N complementary to the 3' portion of the second adaptor nucleic acids, and wherein the second adaptor nucleic acids comprise a 5’ portion and a 3’ portion, and wherein the 5' portion is phosphorylated and is complementary to a portion of the linear nucleic acid molecules and wherein the 3’ portion is partially complementary to the 5’ portion of the second adaptor nucleic acids; wherein the complementary portions of the first and second adaptor nucleic acids differ in lengths such that upon annealing of these complementary portions to each other sticky ends are generated; (ic) rendering the annealed complexes double-stranded by filling gaps using a polymerase and nucleotides and performing a ligation; (id) performing a denaturation to provide separated strands; (ie) allowing annealing of the complementary portions derived from the first and second adaptor nucleic acids to provide a self-annealed nucleic acid; (if) contacting and annealing the self-annealed nucleic acid with a third adaptor nucleic acid comprising a loop and a double-stranded portion, wherein the double-stranded portion comprises a sticky end that matches with the sticky end of the self-annealed nucleic acid; and (ig) ligating the annealed third adaptor nucleic acid to the self-annealed nucleic acid.

Herein, the term "portion" refers to a defined segment or fragment of the nucleic acid chain, characterized by its sequence or position within the +

N molecule. The portion can be delineated based on its position within the

N ro linear nucleic acid molecule, such as starting from a particular nucleotide e position and extending to another position downstream or upstream

I along the sequence. The terms "5" portion" and "3' portion" refer to a © 25 specific regions of first and second adaptor nucleic acids relative to their

LO

O

O refers to the end of the molecule where the phosphate group is attached to the 5’ carbon of the sugar moiety of the nucleotide. Similarly, the 3’ portion of the first adaptor nucleic acids refers to the end of the molecule where the hydroxyl group is attached to the 3’ carbon of the sugar moiety of the nucleotide. These two complementary strands anneal to each other via complementary base-pair to form a double-stranded structure. The complementary portions of the first and second adaptor nucleic acids differ in lengths such that the nucleotide sequences on one adaptor molecule are longer or shorter than the corresponding sequences on the other adaptor molecule. Upon annealing, the difference in length leads to the generation of sticky ends due to unpaired nucleotides. Beneficially, the sticky ends provide specificity for the joining of DNA molecules with complementary sticky ends, facilitating the assembly of DNA molecules in a desired orientation.

The present disclosure also relates to the method for providing single oligonucleotide as described above. Various embodiments and variants disclosed above, with respect to the aforementioned method for amplification, apply mutatis mutandis to the method for removal.

The method for providing single oligonucleotide comprising a hairpin loop, said method comprising the steps of: (ia) providing linear nucleic acid molecules comprising: a 5' portion comprising a linear sequence flanked on each side by one of a pair of complementary single-stranded sequences, followed by x a single-stranded center portion comprising seguences complementary to & a loop adaptor oligonucleotide, followed by

LO

= a 3' portion comprising a linear seguence flanked on each side by one of

N

- a pair of complementary single-stranded seguences, a a © 25 wherein both the pairs of complementary single-stranded sequences

LO

N

N (ib) allowing both the pairs of complementary single-stranded seguence to anneal to form hairpin loops and contacting with a loop adaptor oligonucleotide comprising a hairpin loop flanked by single-stranded sequences complementary to the center portion defined in step (ia); (ic) allowing the loop adaptor oligonucleotide to anneal to the center portion to generate annealed complexes; (id) ligating the annealed complexes to obtain closed molecules; (ie) annealing the closed molecules with an oligonucleotide primer complementary to loop derived from the loop adaptor oligonucleotide; (if) amplifying said closed molecules starting from said oligonucleotide primer using a rolling circle amplification with a strand-displacing polymerase and allowing annealing of complementary sequences, thereby obtaining concatemeric molecules comprising single-stranded portions and hairpin loop structures; (ig) nicking the double-stranded portions with a nicking endonuclease having specificity for said recognition sites; and (ih) performing a denaturation step to obtain single oligonucleotides comprising a hairpin loop.

The term "single-stranded center portion" refers to a segment of an oligonucleotide molecule between the 5’ portion and the 3” portion. The term "loop adaptor oligonucleotide" refers to a short nucleic acid molecule that typically contains a loop structure. This loop structure may arise from <

N complementary seguences within the oligonucleotide that form a stem-

N ro loop structure when annealed together. The loop adaptor oligonucleotide e may also contain additional seguences at its ends. Herein, the loop

I adaptor oligonucleotide serves as a complementary binding partner for a © 25 the single-stranded center portion of another oligonucleotide molecule.

LO

O Following the annealing step, the annealed complexes are ligated

O together using the ligase enzyme. The ligation seals the ends of the annealed complexes, creating continuous circular (or closed) or linear molecules. The closed molecules are subseguently annealed to the oligonucleotide primers that binds to the loop region of the closed molecules, providing a starting point for subsequent amplification steps, resulting in long concatemeric molecules with single-stranded portions and hairpin loop structures. The nicking step creates breaks in the double-stranded DNA, specifically at the recognition sites, resulting in linearization of the concatemeric molecules. The final denaturation step separates the strands of the linearized concatemeric molecules, resulting in single oligonucleotides containing a hairpin loop structure, which was originally derived from the loop region of the loop adaptor oligonucleotide.

Optionally, the single oligonucleotide provided has been generated via the aforementioned method of the second aspect.

The present disclosure also relates to the method for removal as described above. Various embodiments and variants disclosed above, with respect to the aforementioned method for amplification, apply mutatis mutandis to the method for removal.

The method for removal of a specific subpopulation of circular nucleic acid molecules from a population of circular nucleic acid molecules, the method comprising steps of: (i) providing a population of at least partially single-stranded circular < nucleic acid molecules comprising an endonuclease restriction recognition

S seguence, wherein said circular nucleic acid molecules optionally ro comprise two loop sequences separated by two sequences that are e capable of annealing to each other such that a double-stranded portion

E 25 is formed; 3 (ii) contacting said circular nucleic acid molecules with an oligonucleotide 3 primer and allowing annealing to take place, wherein the oligonucleotide

N primer is complementary to a single-stranded seguence or a part of the single-stranded seguence that is to be removed from the population of circular nucleic acid molecules;

(iii) generating double-stranded molecules by extending the annealed oligonucleotide primer using a non-strand displacing polymerase; (iv) cleaving said double-stranded molecules with an endonuclease capable of cleaving the endonuclease restriction recognition sequence to generate linearized molecules; and (v) remove linearized molecules from the population of nucleic acid molecules, optionally by exonuclease degradation.

Herein, the specific subpopulation of circular nucleic acid molecules is the linearized molecules that are generated by extending the annealed oligonucleotide primer, using a non-strand displacing polymerase, over the part of the single-stranded sequence that is to be removed. Notably, such extension results in the synthesis of complementary DNA strands, generating double-stranded molecules from the circular nucleic acid molecule. The non-strand displacing polymerase is an enzyme that catalyzes the synthesis of a complementary DNA strand based on a template DNA strand. In this regard, the non-strand displacing polymerase require a primer annealed to the template strand and extend the primer by adding nucleotides to the 3' end, generating a complementary DNA strand.

Beneficially, removing specific subpopulations of linearized molecules < allows for the selective purification of the desired circular molecules, as

S certain linearized molecules may interfere (for example contribute to ro background noise or non-specific signals) with downstream applications or analyses. The selective purification of the desired circular molecules

E 25 leads to increased specificity, sensitivity, accuracy and reproducibility in © subsequent downstream applications or analyses. ? The present disclosure also relates to the method for reduction as

N described above. Various embodiments and variants disclosed above, with respect to the aforementioned method for amplification and the aforementioned method for removal, apply mutatis mutandis to the method for reduction.

The method for reduction of a specific subpopulation of circular nucleic acid molecules from a population of circular nucleic acid molecules, the method comprising the steps of: (i) providing a population of at least partially single-stranded circular nucleic acid molecules, wherein said circular nucleic acid molecules optionally comprise two loop sequences separated by two sequences that are capable of annealing to each other such that a double-stranded portion is formed; (ii) contacting said circular nucleic acid molecules and allowing annealing with (a) a first complementary oligonucleotide primer, which is complementary to a target sequence or a part of the target sequence that is to be reduced in frequency in the population of circular nucleic acid molecules, wherein the first complementary oligonucleotide primer is modified to prevent extension, and wherein the first complementary oligonucleotide primer comprises oligonucleotides that cannot be displaced by a strand-displacing polymerase; and (b) a second complementary oligonucleotide primer, which is x complementary to a universal seguence;

O a (iii) amplifying said circular nucleic acid molecules starting from said = second complementary oligonucleotide primer using a rolling circle

N

- amplification with a strand-displacing polymerase, thus obtaining a a > 25 modified population of circular nucleic acid molecules wherein molecules 00

O containing the target sequence have not been amplified.

LO s

O In this regard, the reduction of a specific subpopulation of circular nucleic acid molecules refers to reduction of amplification of certain specific seguences of the circular nucleic acid molecules. The first complementary oligonucleotide primer is designed to bind to a target sequence or a part of the target sequence within the circular nucleic acid molecules that is intended to be reduced in frequency, at a 5' end or portion, a 3' end or portion, or a central portion of said target sequence. It may be appreciated that the first complementary oligonucleotide primer is modified to prevent extension by DNA polymerase. Additionally, the first complementary oligonucleotide primer comprises oligonucleotides that cannot be displaced by a strand-displacing polymerase. Herein, the strand-displacing polymerase refers to a type of DNA polymerase enzyme that possesses the ability to displace or "push aside" the existing DNA strand while synthesizing a new complementary DNA strand. Examples of strand-displacing polymerases include Phi29 DNA polymerase, Bst DNA polymerase, and Bsm DNA polymerase.

The second complementary oligonucleotide primer is designed to bind to a universal sequence present in all circular nucleic acid molecules in the population. The universal sequence refers to a segment of DNA that is highly conserved and commonly found across different species or organisms, and often used as a primer-binding site or a recognition site due to its widespread presence and conservation.

The rolling circle amplification (RCA) using a strand-displacing polymerase process selectively amplifies circular molecules starting from 5 the second complementary oligonucleotide primer. However, the first

N complementary oligonucleotide primer, being modified to prevent 3 extension, prevents the amplification of molecules containing the target

N 25 sequence. Consequently, the abundance of these molecules in the & population is reduced compared to other circular molecules that have

E been amplified from the second complementary oligonucleotide primer.

X Optionally, the specific subpopulation of circular nucleic acid molecules comprises data stored on the circular nucleic acid molecules. In an example, the data is a video of 1 GB. In another example, the data is a folder of images. It may be appreciated that by reduction of such specific subpopulation of circular nucleic acid molecules from a population of circular nucleic acid molecules, the data is prevented from being amplified, as amplification of data may introduce biases or errors in the original data thus compromising the integrity of the data as well as the

DNA storage molecule.

Beneficially, the method of the fourth aspect allows for the selective reduction of a specific subpopulation of circular nucleic acid molecules by preventing their amplification during rolling circle amplification, thereby modifying the composition of the nucleic acid population.

Optionally, the method comprises step of subjecting said circular nucleic acid molecules to a high-throughput sequencing technology, wherein after step of allowing an intramolecular annealing of the complementary ends of the separate segments and ligating the separate segments intramolecularly, thereby obtaining said circular nucleic acid molecules, but before step of subjecting said circular nucleic acid molecules to a high-throughput sequencing technology, the methods for removal and reduction of a specific subpopulation of circular nucleic acid molecules from a population of circular nucleic acid molecules are performed on the population of circular nucleic acid molecules obtained. In this regard, the methods for removal and reduction of a specific subpopulation of circular

S nucleic acid molecules from a population of circular nucleic acid molecules

N may be performed before storing the circular nucleic acid molecules for a 3 predefined duration. : 25 Optionally, the method comprises steps of storing the circular nucleic acid = © molecules for a predefined duration and subjecting said circular nucleic

O acid molecules to a high-throughput seguencing technology, and wherein

N after step of storing the circular nucleic acid molecules for a predefined * duration, but before step of subjecting said circular nucleic acid molecules to a high-throughput sequencing technology, the method for removal and reduction of a specific subpopulation of circular nucleic acid molecules from a population of circular nucleic acid molecules are performed on the population of circular nucleic acid molecules that was stored for a predefined duration. In this regard, the methods for removal and reduction of a specific subpopulation of circular nucleic acid molecules from a population of circular nucleic acid molecules may be performed before subjecting said circular nucleic acid molecules to a high- throughput sequencing technology.

DETAILED DESCRIPTION OF THE DRAWINGS

Referring to FIG. 1, illustrated is a flow diagram 100 depicting steps of a method for amplification, and subsequent prolonged storage and/or sequencing, of one or more circular nuclear acid molecules 102, in accordance with an embodiment of the present disclosure. The method utilizes one or more circular nucleic acid molecules 102 each comprising two single-stranded loop sequences 102A connected by two complementary sequences that are capable of annealing to each other such that a double-stranded portion 102B is formed, wherein the double- stranded portion 102B comprises a recognition site for a nicking endonuclease. At step S1, said circular nucleic acid molecules 102 were brought in contact with an oligonucleotide primer 104 complementary to < one of the two single-stranded loop sequences and allowing annealing to

S take place. At step S2, said circular nucleic acid molecules 102 are ro amplified from said oligonucleotide primer 104 using a rolling circle e amplification with a strand-displacing polymerase 106 and allowed

E 25 annealing of the two complementary seguences that are capable of © annealing to each other, thereby obtaining concatemeric molecules 108

O comprising single-stranded portions 108A and hairpin loop structures

N

S 108B having double-stranded portions 108C comprising said recognition site for the nicking endonuclease. At step S3, the double-stranded portions 108C of the concatemeric molecules 108 are nicked with the nicking endonuclease having specificity for said recognition site 110. At step S4, a denaturation step is performed such that the nicked concatemeric molecules disintegrate into separate segments 112 having complementary ends 114, 116. At step S5, an intramolecular annealing of the complementary ends of the separate segments is allowed and at step S6, the separate segments are ligated intramolecularly, thereby obtaining said circular nucleic acid molecules 102.

Referring to FIG. 2, illustrated is a schematic flow diagram 200 depicting conversion of linear nucleic acid molecules into one or more circular nucleic acid molecules 220 (or circular molecules) using single oligonucleotide or oligonucleotide complex by annealing and a combination of gap fill and ligation, in accordance with an embodiment of the present disclosure. At step S1, linear at least partially double- stranded nucleic acid molecules 202 are provided and optionally end- repair of said linear nucleic acid molecules is performed such that blunt- ended or 3’ A-tailed nucleic acid molecules 204 are generated. At step

S2, said nucleic acid molecules 204 are brought into contact with adaptor nucleic acids 206 comprising two single-stranded portions 206A and double-stranded portions 206B, wherein the ends of the double-stranded portions 206B of the adaptor nucleic acids 206 are compatible with the ends of said nucleic acid molecules 204 such that they can be joined by < ligation. At step S3, the adaptor nucleic acids 206 are ligated to said

S nucleic acid molecules 204 to produce an adaptor-nucleic acid complex 3 208. At step S4, a denaturation step is performed such that strands of & 25 said nucleic acid molecules 208 are separated and the separated strands z 210 is brought into contact with a single oligonucleotide 212 comprising > a hairpin loop, or with a plurality of oligonucleotides 214 capable of 3 annealing to form an oligonucleotide complex comprising a hairpin loop,

N said hairpin loop having said double-stranded portions comprising the recognition site for a nicking endonuclease, wherein said single oligonucleotide or the oligonucleotide complex comprises 5’ and 3' ends that are complementary to the two single-stranded portions 216 derived from the adaptor nucleic acids. At step S5, annealing is allowed to take place to form annealed complexes 218. At step $6, the annealed complexes are rendered circular by filling gaps using a polymerase and nucleotides and/or performing a ligation such that circular molecules 220 are generated.

In one embodiment, at the aforementioned step S4, the separated strands are contacted with a single oligonucleotide 212 comprising a hairpin loop comprising the recognition site for a nicking endonuclease, wherein said single oligonucleotide comprises 5’ and 3’ ends that are complementary to the two single-stranded portions derived from the adaptor nucleic acids.

In another embodiment, at the aforementioned step S4, the separated strands are contacted with plurality of oligonucleotides 214 capable of annealing to form an oligonucleotide complex comprising a hairpin loop comprising a recognition site for a nicking endonuclease, wherein the plurality of oligonucleotides comprises a first oligonucleotide and a second oligonucleotide, and wherein the first oligonucleotide comprises: a 5’ end that consists of a hairpin loop, a single-stranded portion, and a 3' end that is complementary to the two single-stranded portions derived from the adaptor nucleic acids, and wherein the second oligonucleotide

S comprises a 5' end that is complementary to the two single-stranded

N portions derived from the adaptor nucleic acids and comprises a 3' end 3 that is complementary to the single-stranded portion of the first

N 25 oligonucleotide that is adjacent to the hairpin loop of the first & oligonucleotide.

O

O In yet another embodiment, at the aforementioned step S4, the

N separated strands are contacted with plurality of oligonucleotides 214 * capable of annealing to form an oligonucleotide complex comprising a hairpin loop comprising a recognition site for a nicking endonuclease,

wherein the plurality of oligonucleotides comprises: a first oligonucleotide, a second oligonucleotide and a bridge oligonucleotide, wherein the first oligonucleotide comprises: a 5° end that is complementary to a portion of the bridge oligonucleotide, a portion that is complementary to a single-stranded portion of the second oligonucleotide, and a 3’ end that is complementary to the single- stranded portions derived from the adaptor nucleic acids, and wherein the second oligonucleotide comprises: a 5’ end that is complementary to the single-stranded portions derived from the adaptor nucleic acids, a portion that is complementary to a single-stranded portion of the first oligonucleotide and a 3’ end that is complementary to a portion of the bridge oligonucleotide, and wherein the bridge oligonucleotide comprises sequences complementary to the 5’ end of the first oligonucleotide and the 3’ end of the second oligonucleotide.

In still yet another embodiment, at the aforementioned step S4, the separated strands are contacted with plurality of oligonucleotides 214 capable of annealing to form an oligonucleotide complex comprising a hairpin loop comprising a recognition site for a nicking endonuclease, wherein the plurality of oligonucleotides comprises a first oligonucleotide, a second oligonucleotide, a hairpin loop oligonucleotide and a bridge oligonucleotide or a plurality of bridge oligonucleotides capable of < annealing to the hairpin loop oligonucleotide to form a bridge oligo

S complex, wherein the first oligonucleotide comprises: a 5’ end that is 3 complementary to the single-stranded portions derived from the adaptor & 25 nucleic acids and a 3’ end that is complementary to a portion of the bridge z oligonucleotide or the bridge oligonucleotide complex; wherein the 8 second oligonucleotide comprises: a 5’ end that is complementary to a ? portion of the bridge oligonucleotide or the bridge oligo complex and a 3’

R end that is complementary to the single-stranded portions derived from the adaptor nucleic acids at the 3' end, wherein the hairpin loop oligonucleotide comprises, starting from the 5’ end of the molecule, a left bridge oligo-specific sequence, a hairpin loop and a right bridge oligo- specific sequence, and wherein the bridge oligonucleotide or plurality of bridge oligonucleotides contains: a sequence complementary to the 3’ end of the first oligonucleotide, sequences complementary to the left bridge oligo-specific sequence and the right bridge oligo-specific sequence in the hairpin loop oligonucleotide and a sequence complementary to the 5’ end of the first oligonucleotide and the 3’ end of the second oligonucleotide.

Referring to FIG. 3 illustrated is a schematic flow diagram 300 depicting conversion of linear at least partially double-stranded nucleic acid molecules 302 into one or more circular nucleic acid molecules 312 using single oligonucleotide 306 or oligonucleotide complex 308 annealing and a combination of targeted binding, gap fill and ligation, in accordance with an embodiment of the present disclosure. At first, linear at least partially double-stranded nucleic acid molecules 302 are provided. At step S1, a denaturation step is performed such that strands 304 of linear at least partially double-stranded nucleic acid molecules 302 are separated and the separated strands 304 are contacted with a single oligonucleotide 306 comprising a hairpin loop, said hairpin loop comprising a recognition site for a nicking endonuclease, wherein said single oligonucleotide comprises 5’ and 3’ ends that are complementary to specific sequences < in the separated strands. At step S2, annealing is allowed to take place

S to form annealed complexes 310. At step S3, the annealed complexes 3 310 are rendered circular by filling gaps using a polymerase and & 25 nucleotides; and performing a ligation such that circular nucleic acid z molecules 312 are generated. > In one embodiment, conversion of linear at least partially double- 3 stranded nucleic acid molecules 302 into one or more circular nucleic acid

N molecules 312 using plurality of oligonucleotide 306 or oligonucleotide complex 308 annealing and a combination of targeted binding, gap fill and ligation. At first, linear at least partially double-stranded nucleic acid molecules 302 are provided. At step S1, a denaturation step is performed such that strands 304 of linear nucleic acid molecules separated and the separated strands 304 are contacted with a plurality of oligonucleotides 308 capable of annealing to form an oligonucleotide complex 310 comprising a hairpin loop, said hairpin loop comprising a recognition site for a nicking endonuclease, wherein the plurality of oligonucleotides 308 comprises a first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide comprises: a 5’ end that consists of a hairpin loop, a single-stranded portion, and a 3’ end that is complementary to a specific sequence in the separated strands 304, and wherein the second oligonucleotide comprises a 5’ end that is complementary to a specific sequence in the separated strands 304 and comprises a 3' end that is complementary to a single-stranded portion of the first oligonucleotide that is adjacent to the hairpin loop of the first oligonucleotide. At step S2, annealing is allowed to take place to form annealed complexes 310. At step $3, the annealed complexes 310 are rendered circular by filling gaps using a polymerase and nucleotides; and performing a ligation such that circular nucleic acid molecules 312 are generated.

In another embodiment, conversion of linear at least partially double- stranded nucleic acid molecules 302 into one or more circular nucleic acid molecules 312 using plurality of oligonucleotide 308 or oligonucleotide < complex annealing and a combination of targeted binding, gap fill and

S ligation. At first, linear at least partially double-stranded nucleic acid 3 molecules 302 are provided. At step S1, a denaturation step is performed & 25 such that strands 304 of linear at least partially double-stranded nucleic z acid molecules separated and the separated strands 304 are contacted > with a plurality of oligonucleotide 308 comprising a hairpin loop, said 3 hairpin loop comprising a recognition site for a nicking endonuclease, wherein the plurality of oligonucleotides 308 comprises: a first oligonucleotide, a second oligonucleotide and a bridge oligonucleotide, wherein the first oligonucleotide comprises: a 5° end that is complementary to a portion of the bridge oligonucleotide, a portion that is complementary to a single-stranded portion of the second oligonucleotide, and a 3' end that is complementary to a specific sequence in the separated strands 304, and wherein the second oligonucleotide comprises: a 5’ end that is complementary to a specific sequence in the separated strands, a portion that is complementary to a single-stranded portion of the first oligonucleotide and a 3’ end that is complementary to a portion of the bridge oligonucleotide, and wherein the bridge oligonucleotide comprises sequences complementary to the 5” end of the first oligonucleotide and the 3’ end of the second oligonucleotide. At step S2, annealing is allowed to take place to form annealed complexes 310. At step S3, the annealed complexes are rendered circular by filling gaps using a polymerase and nucleotides; and performing a ligation such that circular nucleic acid molecules 312 are generated.

Referring to FIG. 4 illustrated is a flow diagram 400 depicting conversion of linear at least partially double-stranded nucleic acid molecules 402 into one or more circular nucleic acid molecules 418 using single oligonucleotide 412 or oligonucleotide complex annealing and ligation, in accordance with an embodiment of the present disclosure. At step S1, linear at least partially double-stranded nucleic acid molecules 402 is < provided and optionally performed end-repair of said linear nucleic acid

S molecules such that blunt-ended or 3' A-tailed nucleic acid molecules 404 3 are generated. At step S2, said nucleic acid molecules 404 are brought & 25 into contact with adaptor nucleic acids 406 comprising double-stranded z portions and two single-stranded portions, wherein the ends of the 8 double-stranded portions of the adaptor nucleic acids 406 are compatible ? with the ends of said nucleic acid molecules 404 such that they can be

N joined by ligation. At step S3, the adaptor nucleic acids 406 are ligated to said nucleic acid molecules 404, to result in a ligated complex 408. At step S4, a denaturation step is performed such that strands 410 of said nucleic acid molecules 408 separated and the separated strands 410 are contacted with a plurality of oligonucleotides 412 capable of annealing to form an oligonucleotide complex comprising a hairpin loop, said hairpin loop having said double-stranded portions comprising the recognition site for a nicking endonuclease, wherein said plurality of oligonucleotides 412 or the oligonucleotide complex comprises 5 and 3' ends that are complementary to the two single-stranded portions 414 derived from the adaptor nucleic acids 406. At step S5, annealing is allowed to take place to form annealed complexes 416. At step S6, the annealed complexes are rendered circular by filling gaps using a polymerase and nucleotides and/or performing a ligation such that circular molecules 418 are generated.

In one embodiment, at the aforementioned step S4, the separated strands 414 are brought into contact with a plurality of oligonucleotides 412 capable of annealing to form an oligonucleotide complex comprising a hairpin loop comprising a recognition site for a nicking endonuclease, wherein the plurality of oligonucleotides 412 comprises a hairpin loop oligonucleotide or a plurality of oligonucleotides capable of annealing to form a hairpin loop oligo complex, and a bridge oligonucleotide capable of annealing to the hairpin loop oligonucleotide or a hairpin loop oligo complex to form a bridge oligo complex, wherein the hairpin loop < oligonucleotide or the hairpin loop oligo complex comprises, starting from

S the 5’ end of the molecule, a left bridge oligo-specific sequence, a hairpin 3 loop sequence and a right bridge oligo-specific sequence, and wherein & 25 the bridge oligonucleotide contains a 5’ end that is complementary to the z single-stranded portions derived from the adaptor nucleic acids 406 at 8 the 3' end, and a 3’ end that is complementary to the single-stranded ? portions derived from the adaptor nucleic acids 406 at the 5” end, and

R seguences complementary to the left bridge oligo-specific seguence and the right bridge oligo-specific sequence in the hairpin loop oligonucleotide.

In another embodiment, at the aforementioned step S4, the separated strands 414 are brought into contact with a plurality of oligonucleotides 412 capable of annealing to form an oligonucleotide complex comprising a hairpin loop oligo complex and said bridge oligonucleotide, wherein said plurality of oligonucleotides comprises: a first oligonucleotide, a second oligonucleotide and a loop bridging oligonucleotide, wherein the first oligonucleotide comprises: a 5’ end that is complementary to a portion of the bridge oligonucleotide, a portion that is complementary to a single- stranded portion of the second oligonucleotide, and a 3' end that is complementary to the loop bridging oligonucleotide, and wherein the second oligonucleotide comprises: a 5’ end that is complementary to the loop bridging oligonucleotide, a portion that is complementary to a single- stranded portion of the first oligonucleotide and a 3’ end that is complementary to a portion of the bridge oligonucleotide, and wherein the loop bridging oligonucleotide comprises sequences complementary to the first 5’ end of the first oligonucleotide and the 3’ end of the second oligonucleotide.

In yet another embodiment, at the aforementioned step S4, the separated strands 414 are brought into contact with a plurality of oligonucleotides 412 capable of annealing to form an oligonucleotide complex comprising a hairpin loop oligo complex and said bridge < oligonucleotide, wherein said plurality of oligonucleotides comprises: a

S first oligonucleotide and a second oligonucleotide, wherein the first 3 oligonucleotide comprises: a 5” end that consists of a hairpin loop, a & 25 single-stranded portion and a 3’ end that is complementary to the bridge z oligonucleotide, and wherein the second oligonucleotide comprises a 5’ 8 end that is complementary to bridge oligonucleotide and a 3’ end that is ? complementary to a single-stranded portion of the first oligonucleotide

R that is adjacent to the hairpin loop of the first oligonucleotide.

Referring to FIG. SA illustrated is a flow diagram 500 depicting conversion of linear at least partially double-stranded nucleic acid molecules 502 into one or more circular nucleic acid molecules 520 using folding of the nucleic acid molecules 502 by the interaction between first and second pair of complementary single stranded sequences and subsequent ligation or combination of gap fill and ligation, in accordance with an embodiment of the present disclosure. At step S1, linear at least partially double-stranded nucleic acid molecules 502 are provided and optionally performed end-repair of said linear nucleic acid molecules such that blunt-ended or 3’ A-tailed nucleic acid molecules 504 are generated.

At step S2, said nucleic acid molecules 504 are brought into contact with adaptor nucleic acids 506 comprising a double-stranded portion and said first and second pair of complementary single-stranded seguences, wherein ends of the double-stranded portions of the adaptor nucleic acids 506 are compatible with ends of said nucleic acid molecules 504 such that they can be joined by ligation. At step S3, the adaptor nucleic acids 506 are ligated to said nucleic acids molecules 504. At step S4, the nucleic acid molecules with the ligated adaptor ends 508 are denaturated to dissociate the strands and renaturated to allow annealing of the pairs of the complementary seguences 510 and 512 into a nicked hairpin loop 514. At step S5, the 5' and 3' prime ends of the linear nucleic acid molecule are joined using a ligase or a combination of a polymerase, nucleotides and a ligase to form a circular nucleic acid molecule 516 comprising a double-stranded portion comprising a recognition site for a

S nicking endonuclease. At step S6, the nicked hairpin loop in the ro renaturated nucleic acid molecules is ligated into a hairpin loop 518,

Q 25 resulting in circular nucleic acid molecule 520. z Referring to FIG. 5B, illustrated is a flow diagram 500 depicting a method x for conversion of linear nucleic acid molecules 502 and 504 into one or ? more circular nucleic acid molecules 526 using folding of the nucleic acid

R molecules 502 and 504 by the interaction between first and second pair of complementary single stranded seguences 508 and 510 and subseguent ligation or combination of gap fill and ligation, in accordance with an embodiment of the present disclosure. At step S1, one or more nucleic acid molecules 502 and 504 are provided, optionally a denaturation step is performed and 5’ ends are phosphorylated thereof.

At step S2, the 5’ phosphorylated linear nucleic acid molecules 506 are contacted and annealed with first adaptor nucleic acid 508 and second adaptor nucleic acid 510, wherein each of the first adaptor nucleic acid 508 comprise a first strand comprising first pair of complementary single- stranded sequences, which are capable of annealing to each other such that a hairpin loop comprising a double-stranded stem portion is formed, followed by a single-stranded half-pair sequence complementary to the 3" end of a first strand of the corresponding second adaptor nucleic acid 510, followed by a double-stranded portion and a single-stranded portion attached to a second strand, wherein the single-stranded portion contains random N nucleotides to permit binding to non-universal ends, and wherein the second adaptor nucleic acid 510 comprise a 5' phosphorylated first strand forming a double-stranded portion with a second strand, said first strand further comprising a single-stranded sequence complementary to the half-pair of the first adaptor nucleic acid 508, and wherein the second strand contains random N nucleotides to permit binding to non-universal ends. At step $3, the linear nucleic acid molecules 506 are allowed to anneal to the first adaptor nucleic acid 508 and second adaptor nucleic acid 510 through the N-containing parts 512

S and 514 of the first adaptor nucleic acid 508 and second adaptor nucleic ro acid 510. At step S4, the first and second adaptor nucleic acids are

Q 25 ligated to the linear single-stranded nucleic acid molecules 506, resulting x in nucleic acid molecules 520 containing the paired reverse- © complementary sequences 516 and 518. At step S5, the nucleic acid 8 molecules 520 with the ligated adaptor ends are denatured to dissociate

O the strands and renaturated to allow annealing of the pairs of the complementary seguences 516 and 518 into a nicked hairpin loop 522.

At step S6, the nicked hairpin loop 522 in the renaturated nucleic acid molecules is ligated into a hairpin loop 524, resulting in the circular nucleic acid molecule 526.

Referring to FIG. 5C, illustrated is a flow diagram 500 depicting synthetic generation of the linear nucleic acid molecules 502, in accordance with an embodiment of the present disclosure. At step S1, the two paired reverse complementary sequences 504 and 506 of the synthetically generated linear nucleic acid molecules 502 are allowed to anneal into a nicked hairpin loop 508. At step S2, the nicked hairpin loop 508 in the synthetically generated linear nucleic acid molecules is ligated into a hairpin loop 510, resulting into a circular nucleic acid molecule 512.

Referring to FIG. 6A, illustrated is an exemplary flow 600 of producing one or more circular nucleic acid molecules from linear sticky-ended 602 at least partially double-stranded nucleic acid molecules using adaptor ligation and subsequent circular ligation, in accordance with an embodiment of the present disclosure. At first, the linear sticky-ended at least partially double-stranded nucleic acid molecules 602 are provided.

At step S1, said linear sticky-ended nucleic acid molecules 602 are brought into contact with adaptor nucleic acids 604 such that the linear sticky-ended nucleic acid molecules 602 and the adaptor nucleic acids 604 are joined by sticky ends 606, wherein the adaptor nucleic acids 604 comprise a hairpin loop and at least one end compatible with sticky-

S ends of a corresponding linear sticky-ended nucleic acid molecule 602,

N and the adaptor nucleic acids 604 are ligated to the linear sticky-ended 3 nucleic acid molecules 602. At step S2, a denaturation step is performed

N 25 to generate single-stranded molecules 608. At step S3, the 5" and 3’ & prime ends of the single-stranded molecules 608 are ligated to form a 3 circular nucleic acid molecule 610 using a ligase capable of single- 3 stranded-molecule circularization. - Referring to FIG. 6B, illustrated is an exemplary flow 600 of producing one or more circular nucleic acid molecules 610 from at least partially double-stranded nucleic acid molecules 602 using adaptor ligation and subsequent circular ligation, in accordance with an embodiment of the present disclosure. At first, linear sticky-ended at least partially double- stranded nucleic acid molecules 602 are provided. At step S1, said linear sticky-ended nucleic acid molecules 602 are brought into contact with adaptor nucleic acids 604 such that the linear sticky-ended nucleic acid molecules 602 and the adaptor nucleic acids 604 are joined by sticky ends 606; wherein the adaptor nucleic acids 604 comprise a hairpin loop flanked by two double-stranded portions formed via annealing with a linear oligo, wherein at least one end of the double-stranded portions has sticky ends compatible with sticky-ends of a corresponding linear sticky- ended nucleic acid molecule 602, and the adaptor nucleic acids 604 are ligated to the linear sticky-ended nucleic acid molecules 602 to generate double-stranded molecules with the ligated adapter ends 606. At step

S2, a denaturation step of double-stranded molecules 606 is performed to generate single-stranded molecules 608. At step S3, the single- stranded molecules 608 are allowed to self-anneal and 5’ and 3' prime ends of the single-stranded molecules 608 are ligated to form a circular nucleic acid molecule 610 using a ligase capable of single-stranded- molecule circularization.

Referring to FIG. 6C, illustrated is an exemplary flow 600 of producing < one or more circular nucleic acid molecules 614 from single-stranded

S nucleic acid molecules 602 using splinted adaptor ligation and 3 subsequent circular ligation, in accordance with an embodiment of the & 25 present disclosure. At first, linear sticky-ended at least partially double- z stranded nucleic acid molecules 602 are provided. At step S1, such linear 8 sticky-ended nucleic acid molecules 602 are brought into contact with ? adaptor nucleic acids 604 such that the linear sticky-ended nucleic acid

R molecules 602 and the adaptor nucleic acids 604 are joined by sticky end ligation to generate molecules with the ligated adapter ends 606; wherein the adaptor nucleic acids 604 comprise a hairpin loop flanked by a double-stranded portion formed via annealing with a linear oligo, wherein the double-stranded portion has a sticky end compatible with sticky-ends of a corresponding linear sticky-ended nucleic acid molecule 602, wherein the hairpin loop 604 is flanked on the other side by a single-stranded sequence complementary to a portion of a splint oligonucleotide 610 and wherein the linear oligo further comprises a single-stranded sequence complementary to an adjacent portion of said splint oligonucleotide 610. At step S2, a denaturation step is performed to generate single-stranded molecules 608 and the splint oligonucleotide 610 is added. At step S3, the splint oligonucleotide 610 is allowed to anneal with the single-stranded molecules 608 and the 5’ and 3' prime ends of the single-stranded molecules 608 are brought to proximity by the splint oligo 610 to result in nicked circular nucleic acid molecule 612.

At step S4, the nick in the nicked circular nucleic acid molecule 612 is ligated, resulting in a circular nucleic acid molecule 614.

Referring to FIG. 6D, illustrated is an exemplary flow 600 of producing one or more circular nucleic acid molecules 614 from single-stranded synthetic nucleic acid molecules 602 using circular ligation, in accordance with an embodiment of the present disclosure. At first, linear single- stranded nucleic acid molecules 602 are provided. At step S1, such linear sticky-ended nucleic acid molecules 602 are brought into contact with < adaptor nucleic acids 604 and a splint oligonucleotide 606 such that the

S linear single-stranded nucleic acid molecules 602 and the adaptor nucleic 3 acids 604 are joined via the splint oligonucleotide 606 to produce a & 25 complex 608; wherein the adaptor nucleic acids 604 comprise a hairpin z loop flanked by two single-stranded portions, wherein one single- > stranded portion is complementary to the split oligo, and wherein the 3 splint oligonucleotide 606 comprises a portion that is complementary to a single-stranded portion of the adaptor nucleic acid 604 and a portion that is complementary to other end of the corresponding linear single- stranded nucleic acid molecule 602. At step S2, the adaptor nucleic acids

604 are ligated to the linear single-stranded nucleic acid molecules 602, resulting in splint oligonucleotide-tailed single-stranded circular nucleic acid molecule 610. At step S3, optionally a denaturation step is performed to dissociate the splint oligonucleotide and generate single- stranded molecules circular nucleic acid molecule 612. At step S4, the single-stranded molecules circular nucleic acid molecules 612 are ligated to form circular nucleic acid molecules 614 using a ligase capable of single-stranded-molecule circularization.

Referring to FIG. 6E, illustrated is another exemplary flow 600 of producing one or more circular nucleic acid molecules 618 from single- stranded nucleic acid molecules 602, and partially double-stranded nucleic acid molecules 604, in accordance with an embodiment of the present disclosure. At first, one or more linear nucleic acid molecules 602, 604 are provided, optionally a denaturation step is performed, and 5' ends are phosphorylated thereof. At step S1, the 5’ phosphorylated linear nucleic acid molecules 606 are brought into contact with adaptor nucleic acids 608, wherein the adaptor nucleic acids 608 comprise an annealed first and second strand, wherein the first strand comprises a 5’ phosphorylated 5’ portion complementary to the second strand, followed by a hairpin loop followed by a 3’ portion complementary to the second strand, wherein the hairpin loop comprises a recognition site for a nicking < endonuclease, and wherein the second strand comprises a 5’ portion

S comprising random N nucleotides to permit binding to non-universal 3 sequences, followed by a portion that is complementary to the first & 25 strand, followed by a 3’ portion comprising random N nucleotides to z permit binding to non-universal sequences. At step S2, the 5 8 phosphorylated nucleic acid molecules 606 and the adaptor nucleic acids ? 608 are allowed to anneal to generate annealed complexes 610. At step

N S3, the nicks in the annealed complexes 610 are closed using a DNA ligase, thereby joining the 5' phosphorylated nucleic acid molecules 606 to the adaptor nucleic acids 608, to generate ligated complexes 612. At step S4, double-stranded portions of the ligated complexes 612 are nicked with a nicking endonuclease having specificity for said recognition site to generate nicked structures 614. At step S5, a denaturation step is performed such that the nicked structures 614 disintegrate into separate segments 616 having complementary ends. At step S6, intramolecular annealing of the complementary ends of the separate segments 616 are allowed and the separate segments 616 are ligated intramolecularly, thereby obtaining circular nucleic acid molecules 618.

Referring to FIG. 6F, illustrated is an exemplary flow 600 of producing one or more circular nucleic acid molecules 608 from single-stranded synthetic nucleic acid molecules 602 using circular ligation, in accordance with an embodiment of the present disclosure. At step S1, synthetic nucleic acid molecules 602 containing a hairpin loop flanked by single- stranded seguences is brought to contact with a splint oligonucleotide 604 having ends that are complementary to the ends of the synthetic oligonucleotide, and the synthetic nucleic acid molecules 602 are allowed to anneal with the splint oligonucleotides 604, resulting in nicked circular nucleic acid molecules 606 having nick 610 open. At step 2 the nick 610 of the nicked circular nucleic acid molecules 606 is ligated, thereby obtaining circular nucleic acid molecules 608 having nick 610 closed or filled.

N Referring to FIG. 6G, illustrated is an exemplary flow 600 of producing

N one or more circular nucleic acid molecules 604 from single-stranded 3 nucleic acid molecules 602, in accordance with an embodiment of the

N 25 present disclosure. At step S1, linear single-stranded nucleic acid & molecules containing a hairpin loop 602 are synthesised and said nucleic 3 acid molecules 604 are self-ligated to form a circular nucleic acid x molecule 604 using a ligase capable of single-stranded-molecule

N circularization.

Referring to FIG. 7, illustrated is a flow diagram 700 for producing hairpin-looped molecules for targeted circular conversion, in accordance with an embodiment of the present disclosure. At step S1, one or more linear nucleic acid molecules 702 and 704 are provided and optionally a denaturation step is performed. At step S2, the linear single-stranded nucleic acid molecules 706 are brought into the contact and annealed with first adaptor nucleic acid 708 and the second adaptor nucleic acid 710, to generate annealed complexes 712, wherein the first adaptor nucleic acid 708 comprise a 5’ portion and a 3’ portion, and wherein the 3’ portion is complementary to a portion of the linear nucleic acid molecules 706, and wherein the 5” portion is partially complementary to the 3' portion of the second adaptor nucleic acid 710, and wherein the second adaptor nucleic acid 710 comprise a 5’ portion and a 3’ portion, and wherein the 5’ portion is phosphorylated and is complementary to a portion of the linear nucleic acid molecules 706 and wherein the 3’ portion is partially complementary to the 5” portion of the second adaptor nucleic acid 710; wherein the complementary portions of the first adaptor nucleic acid 708 and the second adaptor nucleic acid 710 differ in lengths such that upon annealing of these complementary portions to each other, sticky ends are generated. At step S3, the annealed complexes 712 are rendered double-stranded 714 by filling gaps using a polymerase and nucleotides and a ligation is performed. At step S4, a denaturation is

S performed to provide separated strands 716. At step S5, annealing of ro the complementary portions derived from the first adaptor nucleic acid

Q 25 708 and the second adaptor nucleic acid 710 are allowed to provide a z self-annealed nucleic acid 718. At step S6, the self-annealed nucleic acid > 718 is brought into contact and annealed with a third adaptor nucleic 8 acid 720 comprising a loop and a double-stranded portion. At step S7,

O the double-stranded portion comprises a sticky end that matches with the sticky end of the self-annealed nucleic acid 718. At step S8, the annealed third adaptor nucleic acid is ligated to the self-annealed nucleic acid 718.

Referring to FIG. 8, illustrated is flow diagram 800 for providing single oligonucleotide 822 comprising a hairpin loop, in accordance with an embodiment of the present disclosure. At first, linear nucleic acid molecules 802 comprising: a 5’ portion comprising a linear sequence flanked on each side by one of a pair of complementary single-stranded sequences 804, followed by a single-stranded center portion comprising sequences complementary to a loop adaptor oligonucleotide, followed by a 3’ portion comprising a linear sequence flanked on each side by one of a pair of complementary single-stranded sequences 806, are provided and at step S1, both the pairs of complementary single-stranded sequence are allowed to anneal to form hairpin loops 808, 810 and brought into contact with a loop adaptor oligonucleotide 812 comprising a hairpin loop flanked by single-stranded sequences complementary to the center portion. At step S2, the loop adaptor oligonucleotide 812 is allowed to anneal to the center portion to generate annealed complexes 814. At step S3, the annealed complexes 814 are ligated to obtain closed molecules 816. At step S4, the closed molecules are annealed with a primer 818 complementary to loop derived from the loop adaptor oligonucleotide 812. At step S5, said closed molecules 816 are amplified < starting from said oligonucleotide primer using a rolling circle

S amplification with a strand-displacing polymerase and annealing of 3 complementary sequences are allowed, thereby obtaining concatemeric & 25 molecules 820 comprising single-stranded portions and hairpin loop z structures. At step S6, the double-stranded portions are nicked with a > nicking endonuclease having specificity for said recognition sites 826, 3 828 of the concatemeric molecules 820 comprising single-stranded portions 824 and hairpin loop structures 822 comprising single-stranded portions and hairpin loop structures 822. At step S7, a denaturation step is performed to obtain single oligonucleotides 822 comprising a hairpin loop.

Referring to FIG. 9A, illustrated is a flow diagram 900 for removal of a specific subpopulation of circular nucleic acid molecules from a population of circular nucleic acid molecules 902, in accordance with an embodiment of the present disclosure. A population of at least partially single-stranded circular nucleic acid molecules 902 comprising an endonuclease restriction recognition sequence is provided, wherein said circular nucleic acid molecules 902 optionally comprise two loop sequences separated by two sequences that are capable of annealing to each other such that a double-stranded portion is formed, and in a single-stranded part, a restriction endonuclease recognition site 904. At step S1, said circular nucleic acid molecules 902 are brought into contact with an oligonucleotide primer 906 and annealing is allowed to take place, wherein the oligonucleotide primer 906 is complementary to a single- stranded sequence or a part of the single-stranded sequence that is to be removed from the population of circular nucleic acid molecules 902. At step S2, double-stranded molecules 910 are generated by extending the annealed oligonucleotide primer 906 using a non-strand displacing polymerase 908, rendering the single-stranded restriction endonuclease recognition site double-stranded 912. At step S3, said double-stranded < molecules 910 are cleaved with an endonuclease capable of cleaving the

S endonuclease restriction recognition seguence 914 to generate linearized 3 molecules 916. At step S4, the cleaved double-stranded circular nucleic & 25 acid molecules or the linearized molecule 916 are brought into contact z with exonucleases that further degrade the cleaved double-stranded > circular nucleic acid molecules 916 into nucleotides 918. 3 Referring to FIG. 9B, illustrated is a flow diagram 900 for reduction of a

N specific subpopulation of circular nucleic acid molecules from a population ofcircular nucleic acid molecules 902, in accordance with an embodiment of the present disclosure. A population of at least partially single-stranded circular nucleic acid molecules 902 are provided, wherein said circular nucleic acid molecules 902 optionally comprise two loop sequences separated by two sequences that are capable of annealing to each other such that a double-stranded portion is formed and at step S1, said circular nucleic acid molecules 902 are brought into contact with, a first complementary oligonucleotide primer 906, which is complementary to a target sequence or a part of the target sequence that is to be reduced in frequency in the population of circular nucleic acid molecules 902, wherein the oligonucleotide primer 906 is modified to prevent extension, and wherein the oligonucleotide primer 906 comprises oligonucleotides that cannot be displaced by a strand-displacing polymerase 910, and are allowed to anneal. At step S2, the circular nucleic acid molecules 902 with the annealed first complementary oligonucleotide primers 906, are brought into contact with a second complementary oligonucleotide primer 908, which is complementary to a universal sequence. At step S2, said circular nucleic acid molecules 902 are amplified starting from said second complementary oligonucleotide primer 908 using a rolling circle amplification with a strand-displacing polymerase 910, thus obtaining a modified population of circular nucleic acid molecules wherein molecules containing the target sequence have not been amplified. At step S3, the strand-displacing polymerase 910 is allowed to catalyse a strand synthesis and displacement reaction by rolling circle amplification. The

S polymerase is unable to displace the strong double-strand formed by the ro specific oligonucleotide primer 906, thereby being unable to create the

Q 25 concatemeric amplification product from said the sub-population of the

T circular molecules, leading to reduction of the said sub-population of the > circular nucleic acid molecules in the circle amplification.

S

Claims

1. A method for amplification, and subsequent prolonged storage and/or sequencing, of one or more circular nucleic acid molecules (102), the method comprising the steps of: (i) providing the one or more circular nucleic acid molecules each comprising two single-stranded loop sequences (102A) connected by two complementary sequences that are capable of annealing to each other such that a double-stranded portion (102B) is formed, wherein the double-stranded portion comprises a recognition site for a nicking endonuclease; (ii) contacting said circular nucleic acid molecules with an oligonucleotide primer (104) complementary to one of the two single-stranded loop seguences and allowing annealing to take place; (iii) amplifying said circular nucleic acid molecules starting from said oligonucleotide primer using a rolling circle amplification with a strand-displacing polymerase (106) and allowing annealing of the two complementary seguences that are capable of annealing to each other, thereby obtaining concatemeric molecules (108) comprising single-stranded portions (108A) and hairpin loop x structures (108B) having double-stranded portions (108C) N comprising said recognition site (110) for the nicking 3 endonuclease; & - (iv) nicking the double-stranded portions of the concatemeric a 25 molecules with the nicking endonuclease having specificity for said recognition site; X (v) performing a denaturation step such that the nicked concatemeric molecules disintegrate into separate segments (112) having complementary ends (114 and 116); and

(vi) allowing an intramolecular annealing of the complementary ends of the separate segments and ligating the separate segments intramolecularly, thereby obtaining said circular nucleic acid molecules, wherein the method further comprises performing at least one of: (vii) storing the said circular nucleic acid molecules for a predefined duration; and (viii) subjecting said circular nucleic acid molecules obtained in step (vi) or in step (vii) to a high-throughput sequencing technology.

2. The method according to claim 1, wherein step (i) of providing one or more circular nucleic acid molecules (220) comprises the steps of: (ia) providing linear at least partially double-stranded nucleic acid molecules (202) and optionally performing end-repair of said linear nucleic acid molecules such that blunt-ended or 3’ A-tailed nucleic acid molecules (204) are generated; (ib) contacting said nucleic acid molecules with adaptor nucleic acids (206) comprising double-stranded portions (206B) and two single- stranded portions (206A), wherein the ends of the double-stranded portions of the adaptor nucleic acids are compatible with the ends of said nucleic acid molecules such that they can be joined by ligation; < N < (ic) ligating the adaptor nucleic acids to said linear nucleic acid 3 molecules; 0 N (id) performing a denaturation step such that strands (210) of said I E nucleic acid molecules separate; 00 O 25 (ie) contacting the separated strands with a single oligonucleotide N (212) comprising a hairpin loop, or with a plurality of oligonucleotides Al (214) capable of annealing to form an oligonucleotide complex comprising a hairpin loop, said hairpin loop having said double-

stranded portions comprising the recognition site for a nicking endonuclease, wherein said single oligonucleotide or the oligonucleotide complex comprises 5” and 3’ ends that are complementary to the two single- stranded portions derived from the adaptor nucleic acids; (if) allowing annealing to take place to form annealed complexes (218); (ig) rendering the annealed complexes circular by filling gaps using a polymerase and nucleotides and/or performing a ligation such that circular nucleic acid molecules (220) are generated.

3. The method according to claim 2, wherein step (ie) comprises contacting the separated strands (210) with a single oligonucleotide (212) comprising a hairpin loop comprising the recognition site for a nicking endonuclease, wherein said single oligonucleotide comprises 5" and 3' ends that are complementary to the two single-stranded portions (206A) derived from the adaptor nucleic acids (206).

4. The method according to claim 2, wherein step (ie) comprises contacting the separated strands (210) with plurality of oligonucleotides (214) capable of annealing to form an oligonucleotide complex comprising a hairpin loop comprising a recognition site for a < S nicking endonuclease, wherein the plurality of oligonucleotides ro comprises a first oligonucleotide and a second oligonucleotide, Q and wherein the first oligonucleotide comprises: a 5’ end that consists E of a hairpin loop, a single-stranded portion, and a 3' end that is 2 25 complementary to the two single-stranded portions (206A) derived O 0 from the adaptor nucleic acids (206), and N O N wherein the second oligonucleotide comprises a 5 end that is complementary to the two single-stranded portions derived from the adaptor nucleic acids and comprises a 3' end that is complementary to the single-stranded portion of the first oligonucleotide that is adjacent to the hairpin loop of the first oligonucleotide.

5. The method according to claim 2, wherein step (ie) comprises contacting the separated strands (210) with plurality of oligonucleotides (214) capable of annealing to form an oligonucleotide complex comprising a hairpin loop comprising a recognition site for a nicking endonuclease, wherein the plurality of oligonucleotides comprises: a first oligonucleotide, a second oligonucleotide and a bridge oligonucleotide, wherein the first oligonucleotide comprises: a 5 end that is complementary to a portion of the bridge oligonucleotide, a portion that is complementary to a single-stranded portion of the second oligonucleotide, and a 3’ end that is complementary to the single- stranded portions (206A) derived from the adaptor nucleic acids (206), and wherein the second oligonucleotide comprises: a 5’ end that is complementary to the single-stranded portions derived from the adaptor nucleic acids, a portion that is complementary to a single- stranded portion of the first oligonucleotide and a 3' end that is complementary to a portion of the bridge oligonucleotide, and x wherein the bridge oligonucleotide comprises sequences N complementary to the 5’ end of the first oligonucleotide and the 3’ 3 end of the second oligonucleotide. O Al

I

6. The method according to claim 2, 3 or 4. wherein step (ie) comprises > 25 contacting the separated strands (210) with plurality of O oligonucleotides (214) capable of annealing to form an oligonucleotide X complex comprising a hairpin loop comprising a recognition site for a nicking endonuclease, wherein the plurality of oligonucleotides comprises a first oligonucleotide, a second oligonucleotide, a hairpin loop oligonucleotide and a bridge oligonucleotide or a plurality of bridge oligonucleotides capable of annealing to the hairpin loop oligonucleotide to form a bridge oligo complex, wherein the first oligonucleotide comprises: a 5 end that is complementary to the single-stranded portions (206A) derived from the adaptor nucleic acids (206) and a 3’ end that is complementary to a portion of the bridge oligonucleotide or the bridge oligo complex; wherein the second oligonucleotide comprises: a 5’ end that is complementary to a portion of the bridge oligonucleotide or the bridge oligo complex and a 3' end that is complementary to the single- stranded portions derived from the adaptor nucleic acids at the 3' end, wherein the hairpin loop oligonucleotide comprises, starting from the 5’ end of the molecule, a left bridge oligo-specific sequence, a hairpin loop and a right bridge oligo-specific sequence, and wherein the bridge oligonucleotide or plurality of bridge oligonucleotides contains: a sequence complementary to the 3’ end of the first oligonucleotide, sequences complementary to the left bridge oligo-specific sequence and the right bridge oligo-specific sequence in the hairpin loop oligonucleotide and a sequence complementary to the 5’ end of the first oligonucleotide and the 3’ < end of the second oligonucleotide. S a

7. The method according to claim 1, wherein step (i) of providing one or = more circular nucleic acid molecules (312) comprises the steps of: N I (ia) providing linear at least partially double-stranded nucleic acid a 2 25 molecules (302); O 3 (ib) performing a denaturation step such that strands (304) of said S linear nucleic acid molecules separate; (ic) contacting the separated strands with a single oligonucleotide (306) comprising a hairpin loop, said hairpin loop comprising a recognition site for a nicking endonuclease, wherein said single oligonucleotide comprises 5’ and 3’ ends that are complementary to specific sequences in the separated strands; (id) allowing annealing to take place to form annealed complexes; (ie) rendering the annealed complexes circular by filling gaps using a polymerase and nucleotides; and performing a ligation such that circular nucleic acid molecules are generated.

8. The method according to claim 1, wherein step (i) of providing one or more circular nucleic acid molecules (312) comprises the steps of: (ia) providing linear at least partially double-stranded nucleic acid molecules (302); (ib) performing a denaturation step such that strands (304) of said linear nucleic acid molecules separate; (ic) contacting the separated strands with a plurality of oligonucleotides (308) capable of annealing to form an oligonucleotide complex comprising a hairpin loop, said hairpin loop comprising a recognition site for a nicking endonuclease, wherein the plurality of oligonucleotides comprises a first oligonucleotide and a second oligonucleotide, + 20 wherein the first oligonucleotide comprises: a 5’ end that consists of N < a hairpin loop, a single-stranded portion, and a 3' end that is 3 complementary to a specific sequence in the separated strands, and 0 N wherein the second oligonucleotide comprises a 5 end that is I E- complementary to a specific seguence in the separated strands and 3 25 comprises a 3’ end that is complementary to a single-stranded portion LO I of the first oligonucleotide that is adjacent to the hairpin loop of the O N first oligonucleotide; (id) allowing annealing to take place to form annealed complexes;

(ie) rendering the annealed complexes circular by filling gaps using a polymerase and nucleotides; and performing a ligation such that circular nucleic acid molecules are generated.

9. The method according to claim 1, wherein step (i) of providing one or more circular nucleic acid molecules (310) comprises the steps of: (ia) providing linear at least partially double-stranded nucleic acid molecules (302); (ib) performing a denaturation step such that the strands (304) of said linear nucleic acid molecules separate; (ic) contacting the separated strands with a plurality of oligonucleotides (308) capable of annealing to form an oligonucleotide complex comprising a hairpin loop, said hairpin loop comprising a recognition site for a nicking endonuclease, wherein the plurality of oligonucleotides comprises: a first oligonucleotide, a second oligonucleotide and a bridge oligonucleotide, wherein the first oligonucleotide comprises: a 5 end that is complementary to a portion of the bridge oligonucleotide, a portion that is complementary to a single-stranded portion of the second oligonucleotide, and a 3’ end that is complementary to a specific sequence in the separated strands, and < N wherein the second oligonucleotide comprises: a 5’ end that is N ro complementary to a specific sequence in the separated strands, a e portion that is complementary to a single-stranded portion of the first I oligonucleotide and a 3’ end that is complementary to a portion of the a 2 25 bridge oligonucleotide, and O 3 wherein the bridge oligonucleotide comprises sequences N complementary to the 5’ end of the first oligonucleotide and the 3’ end of the second oligonucleotide; (id) allowing annealing to take place to form annealed complexes;

10. The method according to claim 2, wherein step (ie) comprises contacting the separated strands (210, 410) with a plurality of oligonucleotides (214, 412) capable of annealing to form an oligonucleotide complex comprising a hairpin loop comprising a recognition site for a nicking endonuclease, wherein the plurality of oligonucleotides comprises a hairpin loop oligonucleotide or a plurality of oligonucleotides capable of annealing to form a hairpin loop oligo complex, and a bridge oligonucleotide capable of annealing to the hairpin loop oligonucleotide or a hairpin loop oligo complex to form a bridge oligo complex, wherein the hairpin loop oligonucleotide or the hairpin loop oligo complex comprises, starting from the 5' end of the molecule, a left bridge oligo-specific sequence, a hairpin loop sequence and a right bridge oligo-specific sequence, and wherein the bridge oligonucleotide contains a 5’ end that is complementary to the single-stranded portions (206A) derived from the adaptor nucleic acids (206, 406) at the 3' end, and a 3' end that < is complementary to the single-stranded portions derived from the S adaptor nucleic acids at the 5’ end, and sequences complementary to ro the left bridge oligo-specific sequence and the right bridge oligo- e specific seguence in the hairpin loop oligonucleotide. I a 25

11. The method according to claim 10, wherein the plurality of O oligonucleotides (214, 412) are capable of annealing to form a hairpin 3 loop oligo complex and a bridge oligonucleotide, wherein said plurality N of oligonucleotides comprises:

(a) a first oligonucleotide, a second oligonucleotide and a loop bridging oligonucleotide, wherein the first oligonucleotide comprises: a 5 end that is complementary to a portion of the bridge oligonucleotide, a portion that is complementary to a single-stranded portion of the second oligonucleotide, and a 3’ end that is complementary to the loop bridging oligonucleotide, and wherein the second oligonucleotide comprises: a 5’ end that is complementary to the loop bridging oligonucleotide, a portion that is complementary to a single-stranded portion of the first oligonucleotide and a 3’ end that is complementary to a portion of the bridge oligonucleotide, and wherein the loop bridging oligonucleotide comprises sequences complementary to the first 5’ end of the first oligonucleotide and the 3’ end of the second oligonucleotide, or (b) a first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide comprises: a 5’ end that consists of a hairpin loop, a single-stranded portion and a 3' end that is complementary to the bridge oligonucleotide, and < N wherein the second oligonucleotide comprises a 5 end that is N ro complementary to bridge oligonucleotide and a 3’ end that is e complementary to a single-stranded portion of the first I oligonucleotide that is adjacent to the hairpin loop of the first a 2 25 oligonucleotide.

D N

12.The method according to claim 1, wherein step (i) of providing one or N more circular nucleic acid molecules (520) comprises the steps of:

(ia) providing one or more linear nucleic acid molecules (502), each comprising (a) a sequence of interest, and (b) a first and second pair of complementary single-stranded sequences, wherein each pair of complementary single-stranded sequences is capable of annealing to each other such that a double- stranded stem portion is formed, wherein the sequence of interest is flanked on both ends by a single-stranded sequence of the first pair, and wherein the two single-stranded sequences of the second pair are separated by a loop sequence and wherein a single-stranded sequence of the second pair is linked to a single-stranded sequence of the first pair, optionally via a nucleotide linker; (ib) optionally denaturing the molecules; (ic) allowing the pairs of the complementary sequences to anneal; and (id) joining the 5' and 3’ prime ends of the linear nucleic acid molecule using a ligase or a combination of a polymerase, nucleotides and a ligase to form a circular nucleic acid molecule comprising a double- stranded portion comprising a recognition site for a nicking < N endonuclease.

N LÖ ?

13.The method according to claim 12, wherein the linear nucleic acid 0 N molecules (502) provided in step (ia) are generated synthetically.

I a a 0

14.The method according to claim 1 wherein step (ia) of providing one LO O 25 or more circular nucleic acid molecules (520) comprises the steps of: N N (ia) providing linear at least partially double-stranded nucleic acid molecules (502) and optionally performing end-repair of said linear nucleic acid molecules such that blunt-ended or 3’ A-tailed nucleic acid molecules (504) are generated; (ib) contacting said nucleic acid molecules with adaptor nucleic acids (506) comprising a double-stranded portion and said first and second pair of complementary single-stranded sequences, and wherein ends of the double-stranded portions of the adaptor nucleic acids are compatible with ends of said nucleic acid molecules such that they can be joined by ligation; and (ic) ligating the adaptor nucleic acids to said nucleic acid molecules.

15.The method according to claim 1, wherein step (i) of providing one or more circular nucleic acid molecules (526) comprises the steps of: (ia) providing one or more linear nucleic acid molecules (502, 504), optionally performing a denaturation step, and phosphorylating 5’ ends thereof; (ib) contacting and annealing the 5” phosphorylated linear nucleic acid molecules (506) with first and second adaptor nucleic acids (508, 510), wherein each of the first adaptor nucleic acids (508) comprise a first strand comprising first pair of complementary single-stranded + 20 seguences, which are capable of annealing to each other such that a N < hairpin loop comprising a double-stranded stem portion is formed, 3 followed by a single-stranded half-pair sequence complementary to S the 3’ end of a first strand of the corresponding second adaptor nucleic E acid (510), followed by a double-stranded portion and a single- 2 25 stranded portion attached to a second strand, wherein the single- O 0 stranded portion contains random N nucleotides (512, 514) to permit N N binding to non-universal ends, and wherein the second adaptor nucleic acids comprise a 5' phosphorylated first strand forming a double-stranded portion with a second strand, said first strand further comprising a single-stranded sequence complementary to the half-pair of the first adaptor nucleic acid, and wherein the second strand contains random N nucleotides to permit binding to non-universal ends (ic) allowing the linear nucleic acid molecules and the first and second adaptor nucleic acids (508, 510) to anneal; (id) joining the 5" and 3’ prime ends of the annealed nucleic acid molecule using a ligase.

16. The method according to claim 1, wherein step (i) of providing one or more circular nucleic acid molecules (610) comprises the steps of: (ia) providing linear sticky-ended at least partially double-stranded nucleic acid molecules (602); (ib) contacting said linear sticky-ended nucleic acid molecules with adaptor nucleic acids (604) such that the linear sticky-ended nucleic acid molecules and the adaptor nucleic acids are joined by sticky ends (606), wherein the adaptor nucleic acids comprise a hairpin loop and at least one end compatible with sticky-ends of a corresponding linear sticky- ended nucleic acid molecule; + 20 (ic) ligating the adaptor nucleic acids to the linear sticky-ended nucleic N < acid molecules; LÖ ? (id) performing a denaturation step to generate single-stranded 0 N molecules (608); and I a (ie) ligating the 5' and 3' prime ends of the single-stranded molecules O 25 to form a circular nucleic acid molecule using a ligase capable of N single-stranded-molecule circularization. N

17.The method according to claim 1, wherein step (i) of providing one or more circular nucleic acid molecules (610) comprises the steps of:

(ia) providing linear sticky-ended at least partially double-stranded nucleic acid molecules (602); (ib) contacting said linear sticky-ended nucleic acid molecules with adaptor nucleic acids (604) such that the linear sticky-ended nucleic acid molecules and the adaptor nucleic acids are joined by sticky ends (606); wherein the adaptor nucleic acids comprise a hairpin loop flanked by two double-stranded portions formed via annealing with a linear oligonucleotide, wherein at least one end of the double-stranded portions has sticky ends compatible with sticky-ends of a corresponding linear sticky-ended nucleic acid molecule, (ic) ligating the adaptor nucleic acids to the linear sticky-ended nucleic acid molecules; (id) performing a denaturation step to generate single-stranded molecules (608); and (ie) allowing the single-stranded molecules to self-anneal and ligating the 5' and 3’ prime ends of the single-stranded molecules to form a circular nucleic acid molecule using a ligase capable of single- stranded-molecule circularization.

18.The method according to claim 1, wherein step (i) of providing one or < N more circular nucleic acid molecules (612) comprises the steps of: N 3 (ia) providing linear sticky-ended at least partially double-stranded S nucleic acid molecules (602); I x (ib) contacting such linear sticky-ended nucleic acid molecules with > 25 adaptor nucleic acids (604) such that the linear sticky-ended nucleic 3 acid molecules and the adaptor nucleic acids are joined by sticky ends O N (606);

wherein the adaptor nucleic acids comprise a hairpin loop flanked by a double-stranded portion formed via annealing with a linear oligonucleotide, wherein the double-stranded portion has a sticky end compatible with sticky-ends of a corresponding linear sticky-ended nucleic acid molecule, wherein the hairpin loop is flanked on the other side by a single-stranded sequence complementary to a portion of a splint oligonucleotide (610) and wherein the linear oligonucleotide further comprises a single-stranded sequence complementary to an adjacent portion of said splint oligonucleotide; (ic) ligating the adaptor nucleic acids to the linear sticky-ended nucleic acid molecules; (id) performing a denaturation step to generate single-stranded molecules (608); and (ie) adding the splint oligonucleotide and allowing it to anneal with the single-stranded molecules and ligating the 5” and 3' prime ends of the single-stranded molecules to form a circular nucleic acid molecule (612).

19. The method according to claim 1, wherein step (i) of providing one or more circular nucleic acid molecules (614) comprises the steps of: (ia) providing linear single-stranded nucleic acid molecules (602); + N (ib) contacting the linear single-stranded nucleic acid molecules with N e adaptor nucleic acids (604) and a splint oligonucleotide (606) such e that the linear single-stranded nucleic acid molecules and the adaptor I nucleic acids are joined via the splint oligonucleotide; a 2 25 wherein the adaptor nucleic acids comprise a hairpin loop flanked by O 0 two single-stranded portions, wherein one single-stranded portion is N N complementary to the splint oligonucleotide and wherein the other single-stranded portion is complementary to an end of a corresponding linear single-stranded nucleic acid molecule, and wherein the splint oligonucleotide comprises a portion that is complementary to a single-stranded portion of the adaptor nucleic acid and a portion that is complementary to other end of the corresponding linear single-stranded nucleic acid molecule, (ic) ligating the adaptor nucleic acids to the linear single-stranded nucleic acid molecules (610); (id) optionally performing a denaturation step to dissociate the splint oligonucleotide and generate single-stranded molecules (612); and (ie) allowing the single-stranded molecules to self-anneal and ligating the 5’ and 3’ prime ends of the resulting single-stranded molecules to form circular nucleic acid molecules using a ligase capable of single- stranded-molecule circularization.

20. The method according to claim 1, wherein step (i) of providing one or more circular nucleic acid molecules (618) comprises the steps of: (ia) providing one or more linear nucleic acid molecules (602, 604), optionally performing a denaturation step, and phosphorylating 5’ ends thereof; (ib) contacting the 5’ phosphorylated linear nucleic acid molecules (606) with adaptor nucleic acids (608), wherein the adaptor nucleic acids comprise an annealed first and second strand, + N < wherein the first strand comprises a 5’ phosphorylated portion 3 complementary to the second strand, followed by a hairpin loop S followed by a 3’ portion complementary to the second strand, wherein E the hairpin loop comprises a recognition site for a nicking 2 25 endonuclease, and O LO I wherein the second strand comprises a 5” portion comprising random O N N nucleotides to permit binding to non-universal seguences, followed by a portion that is complementary to the first strand, followed by a

3’ portion comprising random N nucleotides to permit binding to non- universal sequences; (ic) allowing the 5’ phosphorylated nucleic acid molecules and the adaptor nucleic acids to anneal to generate annealed complexes (610); (id) closing the nicks in the annealed complexes using a DNA ligase, thereby joining the 5’ phosphorylated nucleic acid molecules to the adaptor nucleic acids, to generate ligated complexes (612); (ie) nicking double-stranded portions of the ligated complexes with a nicking endonuclease having specificity for said recognition site to generate nicked structures (614); (if) performing a denaturation step such that the nicked structures disintegrate into separate segments (616) having complementary ends; and (ig) allowing intramolecular annealing of the complementary ends of the separate segments and ligating the separate segments intramolecularly, thereby obtaining circular nucleic acid molecules.

21.The method according to claim 1, wherein step (i) of providing one or more circular nucleic acid molecules (608) comprises the steps of: + 20 (ia) synthesizing nucleic acid molecules (602) containing a hairpin N < loop flanked by single-stranded sequences having 5’ and 3’ ends that 3 are complementary to a splint oligonucleotide (604); and 0 N (ib) annealing said nucleic acid molecules with the splint I E- oligonucleotide comprising seguences that are complementary to the 3 25 5" and 3’ ends of the nucleic acid molecules and ligating the ends LO I thereof. O N

22.The method according to claim 1, wherein step (i) of providing one or more circular nucleic acid molecules (604) comprises the steps of: (ia) synthesizing linear single-stranded nucleic acid molecules containing a hairpin loop (602); and (ib) self-ligating said linear nucleic acid molecules to form a circular nucleic acid molecule using a ligase capable of single-stranded- molecule circularization.

23.The method according to claim 1, wherein step (i) of providing one or more circular nucleic acid molecules (724) comprises the steps of: (ia) providing one or more linear nucleic acid molecules (702, 704) and optionally performing a denaturation step, (ib) contacting and annealing the linear nucleic acid molecules (706) with first (708) and second adaptor nucleic acids (710), generating annealed complexes (712) wherein the first adaptor nucleic acids comprise a 5” portion and a 3’ portion, and wherein the 3’ portion is complementary to a portion of the linear nucleic acid molecules, and wherein the 5’ portion is partially complementary to the 3’ portion of the second adaptor nucleic acids, and + 20 wherein the second adaptor nucleic acids comprise a 5” portion and a N < 3" portion, and wherein the 5’ portion is phosphorylated and is 3 complementary to a portion of the linear nucleic acid molecules and S wherein the 3' portion is partially complementary to the 5’ portion of E the second adaptor nucleic acids; 3 25 wherein the complementary portions of the first and second adaptor 3 nucleic acids differ in lengths such that upon annealing of these O N complementary portions to each other sticky ends are generated;

(ic) rendering the annealed complexes double-stranded (714) by filling gaps using a polymerase and nucleotides and performing a ligation; (id) performing a denaturation to provide separated strands (716); (ie) allowing annealing of the complementary portions derived from the first and second adaptor nucleic acids to provide a self-annealed nucleic acid (718); (if) contacting and annealing the self-annealed nucleic acid with a third adaptor nucleic acid (720) comprising a loop and a double- stranded portion, wherein the double-stranded portion comprises a sticky end (722) that matches with the sticky end of the self-annealed nucleic acid; and (ig) ligating the annealed third adaptor nucleic acid to the self- annealed nucleic acid.

24.A method for providing single oligonucleotide (822) comprising a hairpin loop, said method comprising the steps of: (ia) providing linear nucleic acid molecules (802) comprising: a 5’ portion comprising a linear sequence flanked on each side by one of a pair of complementary single-stranded sequences (804), followed + 20 by S N a single-stranded center portion comprising sequences LO ? complementary to a loop adaptor oligonucleotide, followed by 0 N r a 3' portion comprising a linear sequence flanked on each side by one a of a pair of complementary single-stranded seguences (806), LO O 25 wherein both the pairs of complementary single-stranded seguences O (102) (103) contain a recognition site for a nicking endonuclease; (ib) allowing both the pairs of complementary single-stranded seguence to anneal to form hairpin loops (808, 810) and contacting with a loop adaptor oligonucleotide (812) comprising a hairpin loop flanked by single-stranded sequences complementary to the center portion defined in step (ia); (ic) allowing the loop adaptor oligonucleotide to anneal to the center portion to generate annealed complexes (814); (id) ligating the annealed complexes to obtain closed molecules (816); (ie) annealing the closed molecules with an oligonucleotide primer complementary to loop derived from the loop adaptor oligonucleotide; (if) amplifying said closed molecules starting from said oligonucleotide primer using a rolling circle amplification with a strand-displacing polymerase and allowing annealing of complementary sequences, thereby obtaining concatemeric molecules (820) comprising single- stranded portions (824) and hairpin loop structures (822); (ig) nicking the double-stranded portions with a nicking endonuclease having specificity for said recognition sites (826, 828); and (ih) performing a denaturation step to obtain single oligonucleotides comprising a hairpin loop.

25. The method according to claim 3 or 8, wherein the single + 20 oligonucleotide (822) provided has been generated via the method of N < claim 24. 3 n

26.The method according to any one of claims 2 to 12 or 14 to 20 or 24, N r wherein the linear nucleic acid molecules (202, 302, 402, 502, 504, a 602, 702, 704, 802) are provided as a sample, wherein the sample O 25 pertains to a patient sample, clinical sample, food sample, N environmental sample, forensic sample or archeological sample. N

27.The method according to any one of the preceding claims, wherein the oligonucleotide primer (104) provided in step (ii) is a universal primer.

28.The method according to any one of claims 1 to 20, wherein the oligonucleotide primer (104) provided in step (ii) is a target-specific primer.

29. A method for removal of a specific subpopulation of circular nucleic acid molecules from a population of circular nucleic acid molecules (902), the method comprising steps of: (i)providing a population of at least partially single-stranded circular nucleic acid molecules (902) comprising an endonuclease restriction recognition sequence, wherein said circular nucleic acid molecules optionally comprise two loop sequences (904) separated by two sequences that are capable of annealing to each other such that a double-stranded portion is formed; (ii) contacting said circular nucleic acid molecules with an oligonucleotide primer (906) and allowing annealing to take place, wherein the oligonucleotide primer is complementary to a single- stranded seguence or a part of the single-stranded seguence that is to be removed from the population of circular nucleic acid molecules; + S (iii) generating double-stranded molecules (910) by extending the ro annealed oligonucleotide primer (912) using a non-strand displacing e polymerase (908); E (iv) cleaving said double-stranded molecules with an endonuclease 2 25 capable of cleaving the endonuclease restriction recognition seguence O 3 (914) to generate linearized molecules (916); and O N (v) remove linearized molecules from the population of nucleic acid molecules, optionally by exonuclease degradation.

30. A method for reduction of a specific subpopulation of circular nucleic acid molecules from a population of circular nucleic acid molecules (902), the method comprising the steps of: (i)providing a population of at least partially single-stranded circular nucleic acid molecules, wherein said circular nucleic acid molecules optionally comprise two loop sequences separated by two sequences that are capable of annealing to each other such that a double- stranded portion is formed; (ii) contacting said circular nucleic acid molecules and allowing annealing with (a) a first complementary oligonucleotide primer (906), which is complementary to a target seguence or a part of the target seguence that is to be reduced in freguency in the population of circular nucleic acid molecules, wherein the first complementary oligonucleotide primer is modified to prevent extension, and wherein the first complementary oligonucleotide primer comprises oligonucleotides that cannot be displaced by a strand-displacing polymerase (910); and (b) a second complementary oligonucleotide primer (908), which is complementary to a universal seguence; x (iii) amplifying said circular nucleic acid molecules starting from said N second complementary oligonucleotide primer using a rolling circle 3 amplification with a strand-displacing polymerase, thus obtaining a N modified population of circular nucleic acid molecules wherein E: 25 molecules containing the target seguence have not been amplified.

B © 31.The method according to any one of claims 1 to 28, wherein the X method comprises step (viii) and wherein after step (vi), but before step (viii) the method of claim 29 or claim 30 is performed on the population of circular nucleic acid molecules obtained in step (vi).

32.The method according to any one of claims 1 to 28, wherein the method comprises steps (vii) and (viii) and wherein after step (vii),

but before step (viii) the method of claim 29 or claim 30 is performed on the population of circular nucleic acid molecules that was stored in step (vii).

i N O N LÖ ? 0 N I = 00 LO O LO + N O N