WO2019048882A1

WO2019048882A1 - Oligonucleotides and analogues thereof

Info

Publication number: WO2019048882A1
Application number: PCT/GB2018/052556
Authority: WO
Inventors: Tom Brown; Afaf Helmy El-Sagheer
Original assignee: Oxford University Innovation Ltd
Current assignee: Oxford University Innovation Ltd
Priority date: 2017-09-07
Filing date: 2018-09-07
Publication date: 2019-03-14
Anticipated expiration: 2020-03-07
Also published as: GB201714409D0

Abstract

The present invention relates to oligonucleotides or oligonucleotide analogues that are useful for the preparation of genes. The present invention also relates to the use of these oligonucleotides and oligonucleotide analogues in gene synthesis, CRISPR-Cas systems for Gene editing, PCR, rolling circle amplification (RCA) replication, transcription, reverse transcription and translation processes.

Description

OLIGONUCLEOTIDES AND ANALOGUES THEREOF

INTRODUCTION

[0001] The present invention relates to oligonucleotides or oligonucleotide analogues that are useful for the preparation of genes. The present invention also relates to the use of these oligonucleotides and oligonucleotide analogues in gene synthesis, CRISPR-Cas systems for Gene editing, PCR, rolling circle amplification (RCA), replication, transcription, reverse transcription and translation processes.

BACKGROUND OF THE INVENTION

[0002] Gene synthesis is an important and rapidly growing field. The most successful and by far most common methods are based on PCR amplification of synthetic oligonucleotide pools. This methodology is used routinely to produce large DNA constructs up to several kilobases (kb) in length, and has served the biological community well.¹ However, it has limitations, foremost of which is its inability to produce DNA that contains modifications at specific pre-defined loci. Such modified DNA constructs, if available, would be useful in many applications including epigenetics.²

[0003] The limitations of PCR are due to the fact that DNA polymerases cannot discriminate between the canonical deoxyribonucleoside triphosphates (dNTPs) and modified versions. This is because any modified dNTP must possess the same fundamental Watson-Crick base pairing properties as its natural counterpart in order to be incorporated into DNA by polymerase enzymes. Consequently, the natural and unnatural dNTPs compete in an uncontrollable manner. An obvious solution to this problem is to assemble DNA by ligation of pre-synthesized chemically modified oligonucleotides. This would open up new areas of biology, allowing a vast array of modifications to be incorporated into genomic DNA.

[0004] Ligation can be carried out enzymatically,³ but chemical ligation offers an attractive alternative.⁴ It is compatible with large scale applications, radical modifications to the sugars and nucleobases, templated or non-templated reactions, and can be carried out in conditions under which ligase enzymes would not remain functional, including automated nucleic acid assembly. Moreover, chemical ligation is not restricted to the natural phosphodiester backbone of DNA; other backbones can be produced, some of which have been found to be biocompatible.^5,6 The use of chemical ligation to produce modified backbones has other advantages; highly efficient chemical reactions can be chosen, orthogonal ligation chemistries can be used simultaneously for special applications, and successful DNA ligation strategies, once developed, can be applied to the synthesis of long RNA strands⁷ with potential applications in gene editing.⁸

[0005] However, importantly, for any modified DNA ligation chemistry to be useful in biology, the oligonucleotides it produces must be compatible with the synthesis of functional genes, i.e. the oligonucleotides must be stable, capable of association with DNA and RNA and must not give rise to mutations when used as templates for polymerase enzymes. Presently, there exists very few phosphodiester mimic inter- nucleoside linkages that are compatible with polymerase enzymes (e.g. DNA and/or RNA polymerase).

[0006] Thus, to further advance the use of oligonucleotides, in particular modified oligonucleotides, in synthetic biology, and to provide greater access into modified and/or long DNA and RNA molecules for biological, nanotechnological and therapeutic application, there remains a need for new, improved and orthogonal phosphodiester backbones mimics that are compatible with DNA and RNA polymerases. Phosphodiester backbones mimics with such properties would thereby allow for improved methodologies for the synthesis of modified and lengthened DNA and RNA constructs that could be used on an industrial scale.

[0007] The present invention was devised with the foregoing in mind.

SUMMARY OF THE INVENTION

[0008] It has surprisingly been found that the oligonucleotides defined herein are compatible with DNA and RNA polymerase enzymes. As a consequence of this compatibility, the oligonucleotides of the present invention can be used in a wide range of applications that involve DNA and/or RNA synthesis utilising DNA or RNA polymerase enzymes.

[0009] Thus, according to a first aspect of the present invention, there is provided a use of an oligonucleotide or oligonucleotide analogue comprising one or more phosphodiester backbone mimics of Formula III as defined herein, in the synthesis of a gene.

[0010] According to a second aspect of the present invention, there is provided a use of an oligonucleotide or oligonucleotide analogue comprising one or more phosphodiester backbone mimics of Formula III as defined herein, as a template for amplification in a polymerase chain reaction (PCR).

[0011] According to a third aspect of the present invention, there is provided a use of an oligonucleotide or oligonucleotide analogue comprising one or more phosphodiester backbone mimics of Formula III as defined herein, as a template in a DNA replication process.

[0012] According to a fourth aspect of the present invention, there is provided a use of an oligonucleotide or oligonucleotide analogue comprising one or more phosphodiester backbone mimics of Formula III as defined herein, as a template in a transcription process to provide a corresponding RNA transcript, or as a template in a reverse transcription process to provide a corresponding DNA transcript.

[0013] According to a fifth aspect of the present invention, there is provided a use of an oligonucleotide or oligonucleotide analogue comprising one or more phosphodiester backbone mimics of Formula III as defined herein, as template in a translation process to produce a corresponding protein or peptide.

[0014] According to a sixth aspect of the present invention, there is provided a use of an oligonucleotide or oligonucleotide analogue comprising one or more phosphodiester backbone mimics of Formula III as defined herein, as:

(i) antisense DNA or RNA;

(ii) exon skipping DNA or RNA;

(iii) interference RNA (e.g. siRNA); or

(iv) an RNA component of a CRISPR-Cas system (e.g. crRNA, tracrRNA, gRNA).

[0015] According to a seventh aspect of the present invention, there is provided an oligonucleotide or oligonucleotide analogue comprising one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula III as defined herein.

[0016] According to an eighth aspect of the present invention, there is provided a method for amplifying an oligonucleotide or oligonucleotide analogue sequence, the method comprising the steps of:

1) providing an oligonucleotide or oligonucleotide analogue as defined herein; and

2) carrying out a polymerase chain reaction (PCR) using the oligonucleotide of step 1 as a template.

[0017] According to a ninth aspect of the present invention, there is provided a method for replicating an oligonucleotide or oligonucleotide analogue sequence, the method comprising the steps of:

2) carrying out a replication reaction using the oligonucleotide of step 1 as a template. [0018] According to a tenth aspect of the present invention, there is provided a method for producing a ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) sequence comprising the steps of:

2) transcribing the oligonucleotide of step 1 to form a ribonucleic acid (RNA) transcript or deoxyribonucleic acid (DNA) transcript.

[0019] According to an eleventh aspect of the present invention, there is provided a method for preparing a protein or peptide comprising the steps of:

2) translating the oligonucleotide of step 1 to form the protein or peptide.

[0020] According to a twelfth aspect of the present invention, there is provided a process for preparing an oligonucleotide or oligonucleotide, as defined herein.

[0021] According to a thirteenth aspect there is provided a use of an oligonucleotide or oligonucleotide analogue comprising one or more phosphodiester backbone mimics of Formula III as defined herein, to guide one or more proteins of interest to a target DNA or RNA.

[0022] According to a fourteenth aspect there is provided a use of oligonucleotide or oligonucleotide analogue comprising one or more phosphodiester backbone mimics of Formula III as defined herein, as a template in a rolling circle amplification process.

[0023] Features, including optional, suitable, and preferred features in relation to one aspect of the invention may also be features, including optional, suitable and preferred features in relation to any other aspects of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0024] Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of them mean "including but not limited to", and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0025] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0026] The term "alkyl" includes both straight and branched chain alkyl groups. References to individual alkyl groups such as "propyl" are specific for the straight chain version only and references to individual branched chain alkyl groups such as "isopropyl" are specific for the branched chain version only. For example, "(1-6C)alkyl" includes (1-4C)alkyl, (1-3C)alkyl, propyl, isopropyl and f-butyl.

[0027] The term "(m-nC)" or "(m-nC) group" used alone or as a prefix, refers to any group having m to n carbon atoms.

[0028] The term "halo" refers to fluoro, chloro, bromo and iodo.

[0029] Where optional substituents are chosen from "one or more" groups it is to be understood that this definition includes all substituents being chosen from one of the specified groups or the substituents being chosen from two or more of the specified groups.

[0030] The phrase "oligonucleotide or oligonucleotide analogue of the invention" means those oligonucleotides or oligonucleotide analogues which are disclosed herein, both generically and specifically.

[0031] The term "oligonucleotide" refers to a polynucleotide strand. It will be understood that the term oligonucleotide used herein refers to both "short" polynucleotide strands comprising between 2 and 500 nucleotide residues and "long" polynucleotide strands comprising greater than 500 nucleotide residues. It will also be appreciated by those skilled in the art that an oligonucleotide has a 5' and a 3' end and comprises a sequence of nucleosides linked together by inter-nucleoside linkages.

[0032] The terms "oligonucleotide analogue" and "nucleotide analogue" refer to any modified synthetic analogues of oligonucleotides or nucleotides respectively that are known in the art. Examples of oligonucleotide analogues include peptide nucleic acids (PNAs), morpholino oligonucleotides, phosphorothioate oligonucleotides, phosphorodithioate oligonucleotides, alkylphosphonate oligonucleotides, acylphosphonate oligonucleotides and phosphoramidate oligonucleotides.

[0033] The term "nucleobase analogue" refers to any analogues of nucleobases known in the art. The skilled person will appreciate there to be numerous natural and synthetic nucleobase analogues available in the art which could be employed in the present invention. As such, the skilled person will readily be able to identify suitable nucleobase analogues for use in the present invention. Commonly available nucleobase analogues are commercially available from a number of sources (for example, see the Glen Research catalogue (http://www.glenresearch.com/Catalog/contents.php). It will also be appreciated that the term "nucleobase analogue" covers: universal/degenerate bases (e.g. 3-nitropyrrole, 5-nitroindole and hypoxanthine); fluorescent bases (e.g. tricyclic cytosine analogues (tCO, tCS) and 2- aminopurine); base analogues bearing reactive groups selected from alkynes, thiols or amines; and base analogues that can crosslink oligonucleotides to DNA, RNA or proteins (e.g. 5-bromouracil or 3-cyanovinyl carbazole).

[0034] The nucleobase or nucleobase analogue is attached to a sugar moiety (typically ribose or deoxyribose) or a ribose or deoxyribose mimic, for example a chemically modified sugar derivative (e.g. a chemically modified ribose or deoxyribose) or a cyclic group that functions as a synthetic mimic of a ribose or deoxyribose sugar moiety (e.g. the morpholino ring present in morpholino oligonucleotides). The term "nucleoside" is used herein to refer to a moiety composed of a sugar / a ribose or deoxyribose mimic bound to a nucleobase/nucleobase analogue. The term nucleoside as used herein excludes the inter- nucleoside linkage that connects adjacent nucleosides together. An "inter-nucleoside linkage" is a linking group that connects the rings of the sugar / ribose or deoxyribose mimic of adjacent nucleosides.

[0035] The terms "locked nucleic acid", "LIMA" or "locked nucleoside" are used herein to refer to nucleic acids or nucleosides comprising a ribose or deoxyribose moiety in which the conformation of the ribose or deoxyribose ring is fixed or locked in a specific conformation, typically by a bridging group. Typically, the bridging group connects the 2' and 4' carbon atoms of the ribose or deoxyribose rings and locks the ribose or deoxyribose in the 3'-endo conformation (which is often found in A-form duplexes). Examples of locked nucleic acid/nucleoside structures are well known in the art and are commercially available.

Oligonucleotides of the present invention

[0036] According to one aspect of the present invention, there is provided an oligonucleotide or oligonucleotide analogue comprising one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula III, shown below:

Formula III

wherein:

^ e denotes the point of attachment to a 3' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue;

^ f denotes the point of attachment to a 5' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue;

R^3a, R^3b, R^3c and R^3d are independently selected from hydrogen or (1- 4C)alkyl, wherein each (1-4C)alkyl is optionally substituted with one or more NH₂, OH or SH;

R^3e is selected from hydrogen or (1-4C)alkyl;

W2 is selected from O, S or NR^Z, wherein R^z is selected from hydrogen or (1- 4C)alkyl;

m and mi are integers independently selected from 0 to 2; and P2 is an integer selected from 0 to 1 ;

with the proviso that the sum of integers m, mi and P2 is equal to 0, 1 , 2, 3 or 4.

[0037] The inventors have discovered a novel class of phosphodiester mimic inter- nucleoside linkage that is cheap and easy to produce. Furthermore, the inventors have found that oligonucleotides, and analogues thereof, comprising this novel class of phosphodiester mimic inter-nucleoside linkage are both stable and capable of association with complimentary RNA and/or DNA strands. More importantly, the inventors have also discovered that oligonucleotide and oligonucleotide analogues comprising this novel class of phosphodiester mimic inter-nucleoside are compatible with both DNA and RNA polymerases, making them of particular use in numerous biological applications.

[0038] In an embodiment, R^3a, R^3b, R^3c, R^3d and R^3e are independently selected from hydrogen or (1-4C)alkyl. Suitably, R^3a, R^3b, R^3c, R^3d and R^3e are independently selected from hydrogen or methyl. Most suitably, R^3a, R^3b, R^3c, R^3d and R^3e are hydrogen.

[0039] In another embodiment, W2 is selected from O or NR^Z, wherein R^z is selected from hydrogen or (1-4C)alkyl. Suitably, W2 is selected from O or NR^Z, wherein R^z is selected from hydrogen or methyl. More suitably, W2 is selected from O or NH. Most suitably, W2 is oxygen.

[0040] In another embodiment, P2 is 0.

[0041] In another embodiment, the sum of integers m and mi is equal to 0, 1 , 2, 3 or 4. Suitably, the sum of integers m and mi is equal to 0, 1 , 2 or 3. More suitably, the sum of integers m and mi is equal to 0, 1 or 2. Most suitably, the sum of integers m and mi is equal to 0 or 1.

[0042] In another embodiment, the present invention provides an oligonucleotide or oligonucleotide analogue comprising one or more phosphodiester backbone mimic inter- nucleoside linkages of Formula Ilia shown below:

Formula Ilia

wherein:

W2 is selected from O or NH;

m and mi are integers independently selected from 0 to 2; and P2 is an integer selected from 0 or 1 ;

with the proviso that the sum of integers m, mi and P2 is equal to 0, 1 , 2 or 3.

[0043] In another embodiment, the present invention provides an oligonucleotide or oligonucleotide analogue comprising one or more phosphodiester backbone mimic inter- nucleoside linkages of Formula 1Mb shown below:

Formula 1Mb wherein:

R^3a, R^3b, R^3c and R^3d are independently selected from hydrogen or (1-4C)alkyl; and

m and mi are integers independently selected from 0 to 2;

with the proviso that the sum of integers m and mi is equal to 0, 1 , 2 or 3.

[0044] In another embodiment, the present invention provides an oligonucleotide or oligonucleotide analogue comprising one or more phosphodiester backbone mimic inter- nucleoside linkages of Formula lllc shown below:

Formula lllc

wherein:

^ f denotes the point of attachment to a 5' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue; and

m and mi are integers independently selected from 0 to 2;

with the proviso that the sum of integers m and mi is equal to 0, 1 or 2.

[0045] In another embodiment, the present invention provides an oligonucleotide or oligonucleotide analogue comprising one or more phosphodiester backbone mimic inter- nucleoside linkages of the following formula:

wherein: ^ e denotes the point of attachment to a 3' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue; and

^ f denotes the point of attachment to a 5' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue.

[0046] It will be understood that the oligonucleotides or oligonucleotide analogues of the present invention may comprise any number of phosphodiester backbone mimic inter- nucleoside linkages of Formula III. Thus, the oligonucleotide or oligonucleotide analogue of the present invention may comprise one or more, or two or more, or three or more, or four or more, phosphodiester backbone mimic inter-nucleoside linkages of Formula III.

[0047] In an embodiment, the oligonucleotide or oligonucleotide analogue of the present invention comprises between 1 and 5 phosphodiester backbone mimic inter-nucleoside linkages of Formula III. Suitably, the oligonucleotide or oligonucleotide analogue of the present invention comprises between 1 and 4 phosphodiester backbone mimic inter- nucleoside linkages of Formula III. More suitably, the oligonucleotide or oligonucleotide analogue of the present invention comprises between 1 and 3 phosphodiester backbone mimic inter-nucleoside linkages of Formula III. Yet more suitably, the oligonucleotide or oligonucleotide analogue of the present invention comprises one or two phosphodiester backbone mimic inter-nucleoside linkages of Formula III. Most suitably, the oligonucleotide or oligonucleotide analogue of the present invention comprises one phosphodiester backbone mimic inter-nucleoside linkage of Formula III.

[0048] It will also be understood that the oligonucleotides or oligonucleotide analogues of the present invention may also comprises at least one locked nucleoside. Locked nucleosides are well known in the art and include nucleic acids and/or nucleosides comprising a ribose or deoxyribose moiety in which the conformation of the ribose or deoxyribose ring is fixed or "locked" in a specific conformation, typically by a bridging group. A non-limiting list of suitable locked nucleosides which may be used in the present invention are described in K. Singh, S. and J. Wengel (1998). "Universality of LNA-mediated high- affinity nucleic acid recognition." Chemical Communications (12): 1247-1248 and/or Kaur, H., et al. (2007). "Perspectives on Chemistry and Therapeutic Applications of Locked Nucleic Acid (LIMA)." Chemical Reviews 107(1 1): 4672-4697.

[0049] Furthermore, it will also be appreciated that the at least one locked nucleoside may be positioned at either the 3' or 5' end of an inter-nucleoside linkage of Formula III defined herein, or a locked nucleoside may be positioned at both the 3' and 5' end of an inter- nucleoside linkage of Formula I, II or III defined herein. [0050] In an embodiment, the at least one locked nucleoside is positioned at the 3' end of an inter-nucleoside linkage of Formula III defined herein.

[0051] In another embodiment, the at least one locked nucleoside is positioned at the 5' end of an inter-nucleoside linkage of Formula III defined herein.

[0052] In yet another embodiment, the oligonucleotide or oligonucleotide analogue comprises at least two locked nucleosides, with at least one locked nucleoside positioned at the 3' end of an inter-nucleoside linkage of Formula III defined herein and at least one locked nucleoside position at 5' end of an inter-nucleoside linkage of Formula III defined herein.

[0053] The oligonucleotides defined herein may, in some cases, be in the form of linear oligonucleotide stands. Depending on the circumstances, these strands may be single or double oligonucleotide strands. In some embodiments, the oligonucleotides defined herein may be in the form of cyclic strands. Typically, these cyclic oligonucleotide strands are single strands of oligonucleotide in a cyclic form. These cyclic oligonucleotides may be formed from linear strands of oligonucleotide having terminal functional groups capable of reacting with one another to form an inter-nucleoside linkage of formula III as defined herein. These cyclic oligonucleotides can function as templates in rolling circle amplification processes, which are known in the art.

[0054] It will also be appreciated that the oligonucleotides or oligonucleotide analogues of the present invention may also exist in any suitable salt form. A suitable salt of an oligonucleotide or oligonucleotide analogue of the invention is, for example, an acid-addition salt of an oligonucleotide or oligonucleotide analogue of the invention which is sufficiently basic, for example, an acid-addition salt with, for example, an inorganic or organic acid, for example hydrochloric, hydrobromic, sulfuric, phosphoric, trifluoroacetic, formic, citric methane sulfonate or maleic acid. In addition, a suitable salt of an oligonucleotide or oligonucleotide analogue of the invention which is sufficiently acidic is an alkali metal salt, for example a sodium or potassium salt, an alkaline earth metal salt, for example a calcium or magnesium salt, an ammonium salt or a salt with an organic base which affords an acceptable cation, for example a salt with methylamine, dimethylamine or trimethylamine. It is to be understood that the invention encompasses all such salt forms.

[0055] It is also to be understood that certain oligonucleotides or oligonucleotide analogues may exist in solvated as well as unsolvated forms such as, for example, hydrated forms. It is to be understood that the invention encompasses all such solvated forms.

Synthesis [0056] It will be understood that oligonucleotides or oligonucleotide analogues of the present invention may be prepared using any suitable technique known in the art. Particular processes for the preparation of these compounds are described further in the accompanying examples.

[0057] In the description of the synthetic methods described herein and in any referenced synthetic methods that are used to prepare the starting materials, it is to be understood that all proposed reaction conditions, including choice of solvent, reaction atmosphere, reaction temperature, duration of the experiment and workup procedures, can be selected by a person skilled in the art.

[0058] It will be appreciated that during the synthesis of the oligonucleotides or oligonucleotide analogues of the invention in the processes defined herein, or during the synthesis of certain starting materials, it may be desirable to protect certain substituent groups to prevent their undesired reaction. The skilled chemist will appreciate when such protection is required, and how such protecting groups may be put in place, and later removed.

[0059] By way of example, a suitable protecting group for an amino or alkylamino group is, for example, an acyl group, for example an alkanoyi group such as acetyl, an alkoxycarbonyl group, for example a methoxycarbonyl, ethoxycarbonyl or t-butoxycarbonyl group, an arylmethoxycarbonyl group, for example benzyloxycarbonyl, or an aroyl group, for example benzoyl. The deprotection conditions for the above protecting groups necessarily vary with the choice of protecting group. Thus, for example, an acyl group such as an alkanoyi or alkoxycarbonyl group or an aroyl group may be removed by, for example, hydrolysis with a suitable base such as an alkali metal hydroxide, for example lithium or sodium hydroxide. Alternatively, an acyl group such as a te/f-butoxycarbonyl group may be removed, for example, by treatment with a suitable acid as hydrochloric, sulfuric or phosphoric acid or trifluoroacetic acid and an arylmethoxycarbonyl group such as a benzyloxycarbonyl group may be removed, for example, by hydrogenation over a catalyst such as palladium-on-carbon, or by treatment with a Lewis acid for example boron tris(trifluoroacetate). A suitable alternative protecting group for a primary amino group is, for example, a phthaloyi group which may be removed by treatment with an alkylamine, for example dimethylaminopropylamine, or with hydrazine.

[0060] Thus, in one aspect of the present invention, there is provided a process for preparing an oligonucleotide or oligonucleotide analogue as defined herein, the process comprising reacting: B3) one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula E shown below:

wherein:

^ e' denotes the point of attachment to a 3' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue comprising a terminal functional group of Formula E;

R^3a and R^3b are independently selected from hydrogen or (1-4C)alkyl, wherein each (1-4C)alkyl is optionally substituted with one or more NH2, OH or SH; and

m is an integer selected from 0 to 2;

with

one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula F shown below:

wherein:

^ f denotes the point of attachment to a 5' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue comprising a terminal functional group of Formula F;

R^3c and R^3d are independently selected from hydrogen or (1-4C)alkyl, wherein each (1-4C)alkyl is optionally substituted with one or more NH2, OH or SH;

R^3e is selected from and hydrogen or (1-4C)alkyl;

mi is an integer selected from 0 to 2; and P2 is an integer selected from 0 or 1 ;

and wherein the reaction is optionally conducted in the presence of one or more of the following:

i) one or more peptide coupling reagents;

ii) one or more activating agents; and

iii) a catalyst.

[0061] In an embodiment, R^3a, R^3b, R^3c and R^3d are independently selected from hydrogen or (1-4C)alkyl. Suitably, R^3a, R^3b, R^3c and R^3d are independently selected from hydrogen or methyl. Most suitably, R^3a, R^3b, R^3c and R^3d are hydrogen.

[0062] In another embodiment, R^3e is selected from hydrogen or methyl. Suitably, R^3e is hydrogen.

[0063] In another embodiment, W2 is selected from O or NH.

[0064] In an embodiment, P2 is 0.

[0065] In yet another embodiment, m and mi are integer independently selected from 0 or 1. Suitably, m and mi are 0.

[0066] In a further embodiment, the sum of integers m, mi and P2 is equal to 0, 1 , 2, 3 or 4. Suitably, the sum of integers m, mi and P2 is equal to 0, 1 , 2 or 3. More suitably, the sum of integers m, mi and P2 is equal to 0, 1 or 2. Most suitably, the sum of integers m, mi and P2 is equal to 0 or 1.

[0067] In certain embodiments, the reaction between the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula E and the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula F is conducted at a temperature of between 0 °C and 200 °C. Suitably, the reaction between the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula C and the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula D is conducted at a temperature of between 0 °C and 150 °C. More suitably, the reaction between the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula C and the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula D is conducted at a temperature of between 0 °C and 100 °C.

[0068] In another embodiment, the reaction between the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula E and the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula F is carried out in a polar solvent. The polar solvent may be used to solubilise the oligonucleotides comprising functional groups of Formulae E and F and thereby facilitate reaction therebetween. Accordingly, it will be understood that the polar solvent selected will depend on the specific oligonucleotides selected. Suitable polar solvents may include, but are not limited to, water, an aqueous buffered solution (e.g. a solution of sodium phosphate or sodium carbonate), DMF, DMSO, acetonitrile, tetrahydrofuran (THF) and mixtures thereof with aqueous salt solutions.

[0069] In yet a further embodiment, the reaction between the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula E and the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula F is carried out in the presence of a catalyst. It will be understood that a catalyst may be any suitable reagent that helps to promote the rate of the reaction between the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula E and the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula F. Suitably, the catalyst is an acid and/or a base. Most suitably, the catalyst is a base. Non-limiting examples of suitable bases include NaOH, trimethylamine, diisopropylethylamine and N-methylmorpholine.

[0070] In still a further embodiment, the reaction between the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula E and the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula F is carried out in the presence of one or more peptide coupling agents. Any suitable peptide coupling reagent capable of enhancing the reaction between the functional group of Formula E and the functional group of Formula F may be used.

[0071] In another embodiment, the peptide coupling reagent is a carbodiimide-based coupling reagent.

[0072] Suitably, the peptide coupling reagent is selected from 1- [Bis(dimethylamino)methylene]-1 H-1 ,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU), 2-(1 H-benzotriazol-1-yl)-1 ,1 ,3,3-tetramethyluronium hexafluorophosphate (HBTU), (Benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), 4-(4,6-Dimethoxy-1 ,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM), N-Ethoxycarbonyl-2-ethoxy-1 ,2-dihydroquinoline (EEDQ), Ν,Ν'- dicyclohexylcarbodiimide (DCC), Ν,Ν'-diisopropylcarbodiimide (DIC), 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDCI), N-cyclohexyl-N'-isopropylcarbodiimide (CIC) or Ν,Ν'-dicyclopentylcarbodiimide (CPC). More suitably, the peptide coupling reagent is selected from Ν,Ν'-dicyclohexylcarbodiimide (DCC), N.N'-diisopropylcarbodiimide (DIC) or 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI). Most suitably, the peptide coupling reagent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI).

[0073] Additional activating agents such as, for example, hydroxybenzotriazole (HOBt), N- hydroxy 2-phenyl benzimidazole (HOBI), 1-hydroxy-7-azabenzotriazole (HOAt), 1-(2- hydroxyethyl)imidazole, N-hydroxysuccinimide (NHS), N-hydroxysulfosuccinimide (Sulfo- NHS), 4-dimethylaminopyridine (DMAP) and ethyl cyano(hydroxyimino)acetate (Oxyma Pure^®) may also be used together with the peptide coupling reagent defined hereinabove, to further enhance reactivity between the functional group of Formula E and the functional group of Formula F.

[0074] In an embodiment, the activating agent is N-hydroxysuccinimde (NHS), N- hydroxysulfosuccinimide (Sulfo-NHS) or ethyl cyano(hydroxyimino)acetate (Oxyma Pure^®). Suitably, the activating agent is N-hydroxysuccinimde (NHS).

[0075] In another embodiment, the process is carried out in an aqueous medium at a pH within the range of 5 to 9. Suitably, the process of the present invention is carried out at a pH within the range of 6 to 8. Most suitably, the process of the present invention is carried out at a pH within the range of 6.5 to 7.5.

[0076] In an embodiment, a suitable buffer is present to maintain the reaction medium within the pH range 5 to 9. In a further embodiment, the buffer maintains the reaction medium within the pH range 6 to 8. In another embodiment, the buffer maintains the reaction medium within the pH range 6.5 to 7.5 (e.g. pH 7.2).

[0077] It will be understood that any suitable buffer may be used. In an embodiment, the buffer is selected from the group comprising: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), phosphate, acetate, borate, citrate, sulfonic acid, ascorbate, linolenate, carbonate and bicarbonate based buffers. In a further embodiment, the buffer is selected from the group comprising: 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid) (HEPES), phosphate, acetate, carbonate and bicarbonate based buffers. In a particular embodiment, the buffer is 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid) (HEPES), sodium phosphate or sodium carbonate.

[0078] The resultant oligonucleotides formed by the process of the present invention may be isolated and purified using any suitable techniques known in the art. Suitably, the resultant oligonucleotides formed by the process of the present invention may be isolated and purified using column chromatography, for example, using sephadex columns. [0079] In an embodiment, the process is conducted in the presence of a salt (e.g. NaCI). Any suitable concentration of salt may be used. Suitably, the salt is present in a concentration of between 20 mM and 1000 rtiM. More suitably, the salt is present in a concentration between 50 mM and 750 mM. Yet more suitably, the salt is present in a concentration between 100 mM and 500 mM.

[0080] In yet another embodiment, the process is conducted in the presence of a template oligonucleotide. It will be appreciated that the template oligonucleotide will vary in accordance with the terminal functional groups of Formulae E and F that are used. A person skilled in the art will be able to select a suitable template oligonucleotide having a suitable size and sequence to hybridise with the oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula E and the oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula F of the present process. It will be understood that the template oligonucleotide may also comprise synthetic oligonucleotide analogues, such as, for example, peptide nucleic acid (PNA).

[0081] In an embodiment, the template oligonucleotide is a single stranded oligonucleotide or oligonucleotide analogue.

[0082] In other embodiments, the process is conducted in the absence of a template. Processes conducted in the absence of a template will be understood to encompass reactions such as, for example, solution phase reactions and/or solid supported reactions.

[0083] In a further embodiment of the present process, at least one of the oligonucleotides or oligonucleotide analogues to be ligated is attached to a solid support.

[0084] It will be appreciated that any solid support that is suitable for use in oligonucleotide synthesis may be used. In an embodiment, the solid support is selected from controlled pore glass (CPG), silica, hydroxylated methacrylic polymer beads (e.g. Toyopearl® beads), grafted copolymers comprising a crosslinked polystyrene matrix onto which polyethylene glycol is grafted (e.g. Tenagel®) or microporous polystyrene (MPPS). Suitably, the solid support is selected from controlled pore glass (CPG) or microporous polystyrene (MPPS). Most suitably, the solid support is a controlled pore glass (CPG) support.

[0085] In another particular embodiment, the step of reacting together the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula E and the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula F may be repeated sequentially more than once, for example, more than twice, more than three times, more than four times or more than five times, to form an oligonucleotide or oligonucleotide analogue comprising the one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula III. Uses and Applications

[0086] The present invention provides oligonucleotides or oligonucleotide analogues which are capable of being read by DNA and RNA polymerase.

[0087] Accordingly, the process of the present invention enables the synthesis of DNA and RNA constructs containing modified inter-nucleoside linkages for application in, for example, altered gene expression and mutagenic modifications, which may allow for the synthesis of altered proteins and/or suitable fluorescent tags to visualise DNA in cells.

[0088] Thus, according to another aspect of the present invention, there is provided a use of an oligonucleotide or oligonucleotide analogue comprising one or more phosphodiester backbone mimics of Formula III shown below:

Formula III

wherein:

^ e and ^ f independently denote the points of attachment to the

oligonucleotide or oligonucleotide analogue;

R^3a, R^3b, R^3c and R^3d are independently selected from hydrogen or (1- 4C)alkyl, wherein each (1-4C)alkyl is optionally substituted with one or more NH₂, OH or SH

R^3e is selected from hydrogen or (1-4C)alkyl;

m and mi are integers independently selected from 0 to 2; and

P2 is an integer selected from 0 to 1 ;

with the proviso that the sum of integers m, mi and P2 is equal to 0, 1 , 2, 3 or 4;

as:

i) a template for gene synthesis; ii) a template for amplification in a polymerase chain reaction (PCR):

iii) as a template in a DNA replication process;

iv) as a template in a transcription process to provide a corresponding RNA

transcript, or as a template in a reverse transcription process to provide a corresponding DNA transcript;

v) as template in a translation process to produce a corresponding protein or peptide; or

vi) to guide one or more proteins of interest to a target DNA or RNA.

[0089] According to another aspect of the present invention, there is provided a use of an oligonucleotide or oligonucleotide comprising one or more phosphodiester backbone mimics of Formula III shown below:

Formula III

wherein:

^ e and ^ f independently denote the points of attachment to the oligonucleotide or oligonucleotide analogue;

R^3e is selected from hydrogen or (1-4C)alkyl;

m and mi are integers independently selected from 0 to 2; and

P2 is an integer selected from 0 to 1 ;

as:

(i) antisense DNA or RNA;

(ii) exon skipping DNA or RNA; (iii) interference RNA (e.g. siRNA); or

(iv) an RNA component of a CRISPR-Cas system (e.g. crRNA, tracrRNA, gRNA). Illustrative Examples of oligonucleotides in CRISPR-Cas systems

[0090] In general terms, there are two main classes of CRISPR-Cas systems (Makarova et al. Nat Rev Microbiol. 13:722-736 (2015)), which encompass five major types and 16 different subtypes based on cas gene content, cas operon architecture, Cas protein sequences, and process steps (Makarova et al. Biol Direct. 6:38 (201 1); Makarova and Koonin Methods Mol Biol. 1311 :47-75 (2015); Barrangou, R. Genome Biology 16:247 (2015)). This classification in either Class 1 or Class 2 is based upon the Cas genes involved in the interference stage.

[0091] Class 1 systems have a multi-subunit crRNA-effector complex such as Cascade- Cas3, whereas Class 2 systems have a crRNA-effector complex having a single Cas protein, such as Cas9, Cas12 (previously referred to as Cpfl) and Cas 13a (previously referred to as C2c2). For Type II systems there is a second RNA component tracrRNA which hybridises to crRNA to form a crRNA:tracr RNA duplex, these two RNA components may be linked to form single guide RNA.

[0092] RNA components in such CRISPR-Cas systems may be adapted to be an oligonucleotide in accordance with the invention or a dinucleotide of the invention may be comprised within an RNA components of a CRISPR-Cas system. It would be a matter of routine for a person of ordinary skill in the art to synthesise a crRNA, pre-crRNA, tracrRNA or guideRNA comprising a dinucleotide of the invention or having at least one inter- nucleoside linkage which is a triazole linker moiety between two nucleosides with a locked nucleoside positioned at the 3' end of the triazole linker moiety, and which retains the desired function of the RNA component (e.g., to guide the crRNA:effector complex to a target site). Standard methods are known in the art for testing whether oligonucleotides of the invention when used as such CRISPR RNA components retain the desired function (e.g. by comparing the desired function to that of a control CRISPR RNA component which has the same nucleosides without any-triazole linker moieties between nucleosides or locked nucleosides).

[0093] The term "CRISPR RNA components" or "RNA component of a CRISPR-Cas system" is used herein, as in most CRISPR-Cas systems, the nucleic acid sequences which guide the effector protein(s) to a desired target sequence are RNA components. However, CRISPR hybrid DNA/RNA polynucleotides which can also function to guide effector protein(s) to a desired target site in a DNA or RNA sequence are also known in the art - see for example Rueda et al. (Mapping the sugar dependency for rational generation of a DNA- RNA hybrid-guided Cas9 endonuclease, Nature Communications 8, Article Number: 1610 (2017)). Accordingly, reference to CRISPR RNA components herein may also encompass hybrid RNA/DNA components such as crDNA/RNA, tracrDNA/RNA or gDNA/RNA.

[0094] Advantageously the oligonucleotides of the invention may have particular utility in in vivo gene therapy applications. For example, one way of carrying out in vivo therapy using a Type II CRISPR-Cas system involves delivering the Cas9 and tracrRNA via a virus, which can assemble inactive complexes inside of cells. The crRNA can then be administered later to assemble and selectively activate CRISPR/Cas9 complexes, which would then go on to target and edit specific sites in the human genome, such as disease relevant genes (Gagnon and Corey, Proc. Natl. Acad. Sci. USA 112: 15536-15537, 2015; Rahdar, et al, Proc. Natl. Acad. Sci. USA 1 12Έ7110-71 17, 2015). For this gene therapy approach to work the crRNA should be extremely resistant to nucleases and cellular degradation, as well as confer high activity and specificity to the assembled CRISPR/Cas9 complex. Hence, the increased stability of the oligonucleotides of the invention to degradation is highly desirable. Alternatively, crRNA:effector complexes (i.e. CRISPR-Cas complexes, such as CRISPR/Cas9) can be assembled in vitro and directly transfected into cells for genome editing (Liang, et al, J. Biotechnol. 208:44-53, 2015; Zuris, et al, Nat. Biotechnol. 33:73-80, 2015). Special transfection reagents, such as CRISPRMAX (Yu, et al, Biotechnol. Lett. 38:919-929, 2016), have been developed for this purpose. Oligonucleotides of the invention when used as crRNAs may improve this approach by offering stability against degradation.

[0095] Accordingly, the oligonucleotides of the invention when used as CRISPR RNA components can advantageously be used for the various applications of CRISPR-Cas systems known in the art including: gene-editing, gene activation (CRISPRa) or gene interference (CRISPRi), base-editing, multiplex engineering (CRISPRm), DNA amplification, diagnostics (e.g. SKERLOCK or DETECTR), cell recording (e.g. CAMERA), typing bacteria, antimicrobial applications, synthesising new chemicals etc..

[0096] Suitably, in diagnostic applications such as SHERLOCK and DETECTR the oligonucleotides of the invention can be used as RNA components such as the "sacrificial RNA molecules" used to create a signal.

[0097] Furthermore, it is well known that oligonucleotides and analogues thereof may be used therapeutically for the treatment of various diseases and disorders, such as, for example, cancer, genetic disorders and infection. Thus, in another aspect, the present invention provides a use of an oligonucleotide or oligonucleotide analogue, as defined herein, in the treatment of a disease or disorder. In an embodiment, the disease or disorder is cancer. In another embodiment, the disease or disorder is a genetic disorder. In yet another embodiment, the disease or disorder is an infection.

[0098] In a further aspect of the present invention, there is provided a method for the treatment of a disease or disorder, said method involving administering a therapeutically effective amount of an oligonucleotide or oligonucleotide analogue, as defined herein, or a pharmaceutically acceptable salt or solvate thereof. In an embodiment, the disease or disorder is cancer. In a further embodiment, the disease or disorder is a genetic disorder. In another embodiment, the disease or disorder is an infection.

[0099] Additionally, the present invention provides a method for amplifying an oligonucleotide sequence as defined herein.

[00100] The present invention also provides a method of replicating an oligonucleotide sequence, as defined herein.

[00101] Furthermore, the present invention provides a method for producing a ribonucleic acid (RNA) sequence or deoxyribonucleic acid (DNA) sequence as defined here. It will be understood that said method comprises both transcription of a DNA template to produce a complementary RNA strand and reverse transcription (i.e. by reverse transcriptase) of RNA template to produce a complementary DNA strand.

[00102] In certain embodiments, where the oligonucleotide is a template in a transcription process to provide a corresponding RNA transcript, or as a template in a reverse transcription process to provide a corresponding DNA transcript, it will be appreciated that the oligonucleotide may be a cyclic oligonucleotide strand as defined herein which is use in a rolling circle amplification process to produce the corresponding RNA or DNA. Rolling circle amplification processes are known in the art.

[00103] In certain embodiments of the uses and methods described hereinabove, ^ e denotes the point of attachment to a 3' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue and ^ f denotes the point of attachment to a 5' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue.

[00104] It will also be understood that features, including optional, suitable, and preferred features in relation to any one of the aspects of the present invention detailed above (i.e. the oligonucleotides or oligonucleotide analogues of the present invention and/ or process of the present invention) may also be features, including optional, suitable and preferred features in relation to any other aspects of the invention (i.e. the uses of the oligonucleotides or oligonucleotide analogues and/or the methods of the present invention). EXAMPLES

[00105] Embodiments of the invention will be described, by way of example only, with reference to the accompanying drawings, in which:

Figure 1 shows: A) a schematic representation of the concept of single tube gene assembly by phosphoramidate ligation followed by transcription of modified DNA; and B) details of 5^'- phosphoramidate ligation chemistry.

Figure 2 shows the 12% denaturing PAGE analysis of 3'-phosphate/5'-amine oligonucleotides ligation to give the phosphoramidate-containing product. Lane 1 ; phosphoramidate reaction mixture (ODN 1 , 81-mer), lane 2; reference starting material ODN 3. An excess of the amine oligonucleotide (ODN 3) was used, resulting in a residual lower band and complete consumption of the phosphate oligonucleotide.

Figure 3 shows the 12% denaturing PAGE analysis for optimisation of 3'-phosphate/5'-amine oligonucleotides ligation to give the phosphoramidate template (ODN 1 , 81-mer) top bands. Lanes 1-8; reaction mixture after 5, 10, 15, 30, 45, 60 120 and 360 min. An excess of the amine oligonucleotide was used resulting in a residual lower band and consumption of the phosphate oligonucleotide

Figure 4 shows the PCR amplification of the 81-mer phosphoramidate DNA template (ODN 1). Lane 1 ; 50 bp DNA ladder, lane 2; PCR using the phosphoramidate-containing template ODN 1 , lane 3; control PCR without a phosphoramidate linkage.

Figure 5 shows the 6% denaturing PAGE analysis of 3'-phosphate/5'-amine oligonucleotide ligation to give the product containing two phosphoramidate linkages (ODN 5, 303-mer). Lane 1 ; 0.2 M NaCI, 25 mM MgCI₂, lane 2; 50 mM Tris (pH=8.5), 25 mM MgCI₂, 0.2 M NaCI, lane 3; 10 mM phosphate (pH=7.0), 25 mM MgCI₂, 0.2 M NaCI, lane 4; 0.2 M HEPES (pH=7.2), 0.4 M NaCI, 25 mM MgCI₂, lane 5; 0.2 M HEPES (pH=7.2), 0.4 M NaCI, lane 6; 0.4 M NaCI.

Figure 6 shows the PCR amplification of the 303-mer DNA template ODN 5. Lane 1 ; 50 bp DNA ladder, lane 2; control PCR without phosphoramidate linkage, lane 3 and 4; PCR using the phosphoramidate-containing template ODN 5 (303-mer).

Figure 7 shows the sequence alignment of 20 Clones from PCR of ODN 5 (2x phosphoramidate linkages in 303-mer section of EGFP gene, in red with the ligation points in blue). All the sequences are identical indicating the biocompatibility of the phosphoramidate linkage. Only a few mutations were observed and these are far from the ligation points (see Table 1). The mutations could have occurred during sequencing or during oligonucleotide synthesis and purification. Figure 8 shows: A) PCR amplification of the double stranded phosphoramidate EGFP gene (762-mer). Lane 1 ; 100 bp DNA ladder, lane 2; PCR using the double strand phosphoramidate EGFP gene (762-mer), lane 3; control PCR for individual oligos without ligation. B) Representative sequencing data from cloning of the PCR product of the phosphoramidate EGFP gene. The data show that the polymerase faithfully copied the bases around the phosphoramidate ligation sites (shown in red in the sequence text).

Figure 9 shows the data from cloning and sequencing of the PCR product from the phosphoramidate EGFP gene (762-mer) showing the faithful copying at the ligation points (shown in red in the sequence text) (A) and the water mark GTACA (B). All clones show the water mark which was inserted into the sequence of the synthesised EGFP gene as a unique signature to differentiate it from potential contaminant DNA.

Figure 10 shows the data from cloning and sequencing of the PCR product of the phosphoramidate EGFP gene (762-mer). The data show that the polymerase copied the gene faithfully including the bases around the phosphoramidate ligation points (shown in red in the inserted sequence text). Only one deletion mutation was found in this clone.

Figure 11 shows the transcription of 79-mer unmodified and phosphoramidate-containing DNA templates. Lane 1 and 2, reaction using phosphoramidate template (ODN 27) and short coding strand (ODN 33) for 2 and 4 h respectively; lane 3; template ODN 31 lane 4 and 5, reaction using control template (ODN 31) and short coding strand (ODN 33) for 2 and 4 h respectively; Lane 6 and 7, reaction using phosphoramidate template (ODN 27) and long coding strand (ODN 32) for 2 and 4 h respectively; lane 8 and 9, reaction using control template (ODN 31) and long coding strand (ODN 32) for 2 and 4 h respectively. 15% polyacrylamide gel.

Figure 12 shows the ES- Mass spectra of A), the RNA transcripts from the phosphoramidate-containing template (ODN 27) and B), the normal template (ODN 31). The transcripts have the expected 5'-triphosphate and an additional 3'-cytidine. Required mass = 17.236 KD. Found mass, 17.239 (transcript with 5'-triphosphate), 17.261 (transcript with 5'- triphosphate + Na⁺) and 17.566 (transcript with 5'-triphosphate and 3'-cytidine).

Figure 13 shows a schematic representation of how the process of the present invention may be applied using non-templated solid supported chemistry.

Figure 14 shows a schematic representation of how the process of the present invention may be applied using templated solid supported chemistry. Figure 15 shows a schematic representation of how the process of the present invention may be applied using solid supported chemistry to prepare double stranded DNA.

Figure 16 shows: (i) Cyclisation of single-stranded DNA by chemical ligation methods and rolling circle amplification through the non-canonical linkage, (ii) Details of the chemical and enzymatic ligation strategies investigated in this study: A) Formation of an artificial triazole backbone linkage via the Cu(l)-catalysed azide-alkyne cycloaddition (CuAAc) reaction; B) di-imide-mediated formation of an artificial amide backbone linkage; C) di-imide-mediated formation of an artificial phosphoramidate backbone linkage; D) enzyme-catalysed formation of a natural phosphate backbone linkage.

Figure 17 shows: Representative examples of splint-mediated enzymatic and non-splinted chemical cyclisation reactions to give the cyclic oligonucleotide templates 1_P0₄ (A), 1_PA (B), 1_Tz (C) and 1_Am (D). The crude reaction mixtures were analysed by polyacrylamide gel electrophoresis and the gels were imaged by UV shadowing. A) enzymatic cyclisation of linear unmodified 5'-phosphate/3'-hydroxyl oligonucleotide using T4 DNA ligase. B) di-imide- mediated cyclisation of linear 5' -amine/3'-phosphate oligonucleotide, introducing a phosphoramidate linkage. C) CuAAC-mediated cyclisation of linear 5'-azide/3'-alkyne oligonucleotide, introducing a triazole linkage. D) di-imide-mediated cyclisation of linear 5'- amine/3'-carboxyl oligonucleotide, introducing an amide linkage. S = sample loading buffer; 1 = linear oligonucleotide substrate; 2 = crude cyclisation reaction. A and C were analysed by 15% denaturing PAGE whereas B and D were analysed using 15% non-denaturing PAGE. Interestingly, the cyclic products migrate more slowly than the linear oligonucleotides under denaturing PAGE conditions (where urea is present in the gel), whereas the opposite is observed under non-denaturing conditions.

Figure 18: Comparison of the enzymatic and CuAAC-mediated cyclisations used to prepare the cyclic constructs 4_P0₄ and 4_Tz respectively, whose sequences incorporate complex secondary structure. The crude reaction products were analysed using 8% denaturing polyacrylamide gels, which were visualised by post-staining with SYBR Gold (A) or by UV- shadowing (B). A) Splint-mediated enzymatic cyclisation of the linear oligonucleotide in the presence of an increasing concentration of T4 DNA ligase. LT: linear 5'-phosphate/3'- hydroxyl functionalised oligonucleotide substrate. B) Non-templated CuAAC-mediated cyclisation of the linear oligonucleotide in the presence of denaturing organic solvents. LT: linear 5'-azide/3'-alkyne functionalised oligonucleotide precursor. Figure 19: Agarose gel (0.8% agarose) analysis of the products formed during f-29 polymerase catalysed RCA using cyclic templates 1_P0₄, 1_Tz, 1_Am and 1_PA (8 hour amplification time). The 0.8% agarose gel was cast with 0.5X SYBR Gold allowing visualisation of the RCA products. A) Gel image using a short exposure time. B) The same gel imaged using a longer exposure time, enabling visualisation of all products. M: 1 kb DNA ladder; lane 1 : unmodified cyclic template 1_P0₄; lane 2: phosphoramidate-modified cyclic template 1_PA; lane 3: amide-modified cyclic template 1_Am; lane 4: triazole-modified cyclic template 1_Tz.

Figure 20: Proposed mechanism for the formation of double-stranded products during RCA of the triazole- and amide-modified cyclic templates, (i) In the normal RCA mechanism, the amplified single-stranded product is continuously displaced from the cyclic template, resulting in the formation of very long single-stranded products, (ii) An alternative mechanism allows the enzyme to dissociate from the cyclic template oligonucleotide and copy the emerging single-stranded product, resulting in the formation of double-stranded side-products. This process sequesters the cyclic template and inhibits RCA. This mechanism may be favoured in the presence of the unnatural triazole- and amide- backbone linkages, which the enzyme has greater difficulty in reading through.

Figure 21 shows: Probing the nature of the amplified products from Φ-29-mediated RCA of the cyclic templates 1_P0₄, 1_Tz, 1_Am and 1_PA using a fluorescent probe hybridisation assay. Aliquots of the RCA reactions were stopped by heat inactivation at regular two hour intervals for a total period of 20 hours. A Cy3-labelled fluorescent probe with a sequence which is complementary to the RCA products was added to each aliquot and the mixtures were analysed by agarose gel electrophoresis (0.8% agarose). The gels were imaged under the Cy3 fluorescence channel. The gels were subsequently stained with SYBR Gold (Figure 60).

Figure 22 shows: Fluorescence quantification of amplified DNA products from RCA reactions using the cyclic templates 1_P0₄, 1_Am, 1_PA and 1_Tz and the Φ-29 DNA polymerase over a 10 hour time course. EDTA was added to break down the precipitate releasing extended DNA prior to incubation with DNA binding dyes. Measurements are recorded in the presence of a) SYBR Gold, b) SYBR Green I and c) SYBR Green II fluorescent DNA binding dyes. Error bars represent the standard deviation of three measurements taken from a single RCA reaction. The reactions were also repeated in duplicate and the same trends were observed in both cases.

Figure 23 shows: agarose gel (0.8%) analysis of the product distributions from RCA of the triazole-modified cyclic templates 1_Tz and 4_Tz and the unmodified analogues 1_P0₄ and 4_P0₄ using Φ-29 (A) and Bst 2.0 (B) polymerase enzymes after 20 hours. M: 1 kb DNA ladder; lane 1 : unmodified cyclic template 1_P0₄; lane 2: triazole-modified cyclic template 1_Tz; Lane 3: unmodified cyclic template 4_P0₄; Lane 4: triazole-modified cyclic template 4_Tz. Agarose gels were cast with 0.5X SYBR Gold allowing visualisation of the RCA products.

Figure 24: Fluorescence quantification of amplified DNA products from RCA reactions using the cyclic templates (A: 4_P0₄, and 4_Tz; B: 1_P0₄, and 1_Tz) and enzyme over a 10 hour time course. The performances of the Φ-29 and Bst 2.0 DNA polymerases are compared. EDTA was added to break down the precipitate releasing extended DNA prior to incubation with DNA binding dyes. Measurements are recorded in the presence of SYBR Gold, SYBR Green I and SYBR Green II fluorescent DNA binding dyes. Error bars represent the standard deviation of three measurements taken from a single RCA reaction. The reactions were also repeated in duplicate and the same trends were observed in both cases.

Figure 25: SEM images of the DNA-NFs generated from RCA of the cyclic templates 1_P0₄, 1_PA, 1_Am and 1_Tz using the Φ-29 polymerase in the presence of 20mM Mg²⁺ after 8 hours and 20 hours.

Figure 26: SEM images of the DNA-NFs formed from RCA of the cyclic templates 1_P0₄ (top) and 1_PA (bottom) using the Φ-29 polymerase in the presence of different concentrations of Mg²⁺. The RCA reactions were left for 20 hours and the Mg²⁺ concentrations ranged from 10-25 mM

Figure 27. Predicted secondary structures of the circular templates used in this study. (A) template 1 ; (B) template 2; (C) template 3 and (D) template 4. Structures were predicted using the Mfold web server software. (3) The highlighted bases indicate the point of ligation.

Figure 28. Splint-mediated enzymatic cyclisation of template (2_P0₄) using T4 DNA ligase. 8% denaturing PAGE gel analysis of 5'-phosphate/3'-hydroxyl oligonucleotide cyclisation to generate circular template with phosphodiester linkage. Lane 1 : linear 5'-phosphate/3'-hydroxyl functionalised template; Lane 2: cyclisation reaction mixture.

Figure 29. Templated di-imide-mediated cyclisation of linear oligonucleotide (2_PA). 13% denaturing PAGE gel analysis of 5'-amine/3'-phosphate oligonucleotide cyclisation to generate circular template with phosphoramidate linkage. Lane 1 : linear 5'-amine/3'-phosphate functionalised template; Lane 2: cyclisation reaction mixture.

Figure 30. Templated di-imide-mediated cyclisation of template (2_Am). 13% denaturing PAGE gel analysis of 5'-amine/3'-carboxyl oligonucleotide cyclisation to generate circular template with amide linkage. Lane 1 : linear 5'-amine/3'-carboxyl functionalised template; Lane 2: cyclisation reaction mixture.

Figure 31. Non-templated click cyclisation of template (2_Tz). 8% denaturing PAGE gel analysis of 5'-azide/3'-propargyl oligonucleotides cyclisation to generate circular template with triazole linkage. Lane 1 : linear 5'-azide/3'-propargyl functionalised template; Lane 2: cyclisation reaction mixture.

Figure 32. Splint-mediated enzymatic cyclisation of template (3_P0₄) using T4 DNA ligase. 12% denaturing PAGE gel analysis of 5'-phosphate/3'-hydroxyl oligonucleotide cyclisation to generate circular template with phosphodiester linkage. Lane 1 : linear 5'-phosphate/3'-hydroxyl functionalised template; Lane 2: cyclisation reaction mixture.

Figure 33. Templated di-imide-mediated cyclisation of template (3_PA). 15% denaturing PAGE gel analysis of 5'-amine/3'-phosphate oligonucleotide cyclisation to generate circular template with phosphoramidate linkage. Lane 1 : linear 5'-amine/3'- phosphate functionalised template; Lane 2: cyclisation reaction mixture.

Figure 34. Templated di-imide-mediated cyclisation of template (3_Am). 15% denaturing PAGE gel analysis of 5'-amine/3'-carboxyl oligonucleotide cyclisation to generate circular template with amide linkage. Lane 1 : linear 5'-amine/3'-carboxyl functionalised template; Lane 2: cyclisation reaction mixture.

Figure 35. Non-templated click cyclisation of template (3_Tz). 12% denaturing PAGE gel analysis of 5'-azide/3'-propargyl oligonucleotide cyclisation to generate circular template with triazole linkage. Lane 1 : linear 5'-azide/3'-propargyl functionalised template; Lane 2: cyclisation reaction mixture.

Figure 36. Reversed-phase UPLC (UV abs at 260 nm) and mass spectrum (ES^") of cyclic template 1_P0₄, required 15456 Da, found 15458 Da. The peaks show the product and the Na⁺ adduct.

Figure 37. Reversed-phase UPLC (UV abs at 260 nm) and mass spectrum (ES^") of cyclic template 1_PA, required 15457 Da, found 15455 Da. The peaks show the product and the Na⁺ adduct.

Figure 38. Reversed-phase UPLC (UV abs at 260 nm) and mass spectrum (ES^") of cyclic template 1_Am, required 15433 Da, found 15434 Da. The peaks show the product and the Na⁺ adduct.

Figure 39. Reversed-phase UPLC (UV abs at 260 nm) and mass spectrum (ES^") of cyclic template 1_Tz, required 15457 Da, found 15458 Da. The peaks show the product and the Na⁺ adducts. Figure 40. Reversed-phase UPLC (UV abs at 260 nm) and mass spectrum (ES^") of cyclic template 2_P0₄, required 18947 Da, found 18949 Da. The peaks show the product and the acrylonitrile adduct.

Figure 41. Reversed-phase UPLC (UV abs at 260 nm) and mass spectrum (ES^") of cyclic template 2_PA, required 18948 Da, found 18949 Da. The peaks show the product and the Na⁺ adduct.

Figure 42. Reversed-phase UPLC (UV abs at 260 nm) and mass spectrum (ES^") of cyclic template 2_Am, required 18924 Da, found 18924 Da. The peaks show the product and the Na⁺ adduct.

Figure 43. Reversed-phase UPLC (UV abs at 260 nm) and mass spectrum (ES^") of cyclic template 2_Tz, required 18948 Da, found 18950 Da.

Figure 44. Reversed-phase UPLC (UV abs at 260 nm) and mass spectrum (ES^") of cyclic template 3_P0₄, required 14995 Da, found 14996 Da. The peaks show the product and the Na⁺ adduct.

Figure 45. Reversed-phase UPLC (UV abs at 260 nm) and mass spectrum (ES^") of cyclic template 3_PA, required 14997 Da, found 14997 Da. The peaks show the product and the Na⁺ adduct.

Figure 46. Reversed-phase UPLC (UV abs at 260 nm) and mass spectrum (ES^") of cyclic template 3_Am, required 14958 Da, found 14959 Da. The identity of the species with molecular weights of 15030 and 15101 is uncertain. They are tentatively assigned as either partially hydrolysed EDC adducts of the cyclic product, or incompletely deprotected analogues containing residual /V-isobutyryl-functionalised guanine groups.

Figure 47. Reversed-phase UPLC (UV abs at 260 nm) and mass spectrum (ES^") of cyclic template 3_Tz, required 14997 Da, found 14998 Da.

Figure 48. Reversed-phase UPLC (UV abs at 260 nm) and mass spectrum (ES^") of cyclic template 4_P0₄, required 23163 Da, found 23163 Da. The peaks show the product and the Na⁺ adduct.

Figure 49. Reversed-phase UPLC (UV abs at 260 nm) and mass spectrum (ES^") of cyclic template 4_Tz, required 23180 Da, found 23180 Da.

Figure 50. 0.8% agarose gel analysis of original RCA reaction mixture from cyclic 1_P0₄ and cyclic 1_PA. The RCA was performed in the presence of 20 mM Mg²⁺ at different time points using Φ-29 DNA polymerase. Lane 1 : 1 kb DNA ladder; Lane 2: splint (ODN 15x); Lane 3: cyclic unmodified template (1_P0₄); Lane 4: cyclic phosphoramidate template (1_PA).

Figure 51. 0.8% agarose gel analysis of original RCA reaction mixture from cyclic 1_P0₄ and cyclic 1_Am. The RCA was performed in the presence of 20 mM Mg²⁺ at different time points using Φ-29 DNA polymerase. Lane 1 : 1 kb DNA ladder; Lane 2: splint (ODN 15x); Lane 3: splint for 1_Am (ODN 19x).

Figure 52. 0.8% agarose gel analysis of original RCA reaction mixture from cyclic 1_P0₄ and cyclic 1_Tz. The RCA was performed in the presence of 20 mM Mg²⁺ at different time points using Φ-29 DNA polymerase. Lane 1 : 1 kb DNA ladder; Lane 2: splint (ODN 15x).

Figure 53. 0.8% agarose gel analysis of original RCA reaction mixture from cyclic 2_P0₄ and cyclic 2_PA. The RCA was performed in the presence of 20 mM Mg²⁺ at different time points using Φ-29 DNA polymerase. After finishing the RCA reactions, EDTA (20 mM final concentration) was added to the solution in order to break down the precipitate releasing the extended RCA products. Lane 1 : 1 kb DNA ladder; Lane 2: splint (ODN 16x).

Figure 54. 0.8% agarose gel analysis of original RCA reaction mixture from cyclic 2_P0₄ and cyclic 2_Am. The RCA was performed in the presence of 20 mM Mg²⁺ at different time points using Φ-29 DNA polymerase. After finishing the RCA reactions, EDTA (20 mM final concentration) was added to the solution in order to break down the precipitate releasing the extended RCA products. Lane 1 : 1 kb DNA ladder; Lane 2: splint (ODN 16x); Lane 3: splint for 2_Am (ODN 20x).

Figure 55. 0.8% agarose gel analysis of original RCA reaction mixture from cyclic 2_P0₄ and cyclic 2_Tz. The RCA was performed in the presence of 20 mM Mg²⁺ at different time points using Φ-29 DNA polymerase. After finishing the RCA reactions, EDTA (20 mM final concentration) was added to the solution in order to break down the precipitate releasing the extended RCA products. Lane 1 : 1 kb DNA ladder; Lane 2: splint (ODN 16x).

Figure 56. 0.8% agarose gel analysis of RCA supernatant from cyclic 3_P0₄ and cyclic 3_PA. The RCA was performed in the presence of 20 mM Mg²⁺ at different time points using Φ-29 DNA polymerase. After finishing the RCA reactions, EDTA (20 mM final concentration) was added to the solution in order to break down the precipitate releasing the extended RCA products. However, the precipitate was still observed. Lane 1 : 1 kb DNA ladder; Lane 2: splint (ODN 17x). Figure 57. 0.8% agarose gel analysis of RCA supernatant from cyclic 3_P0₄ and cyclic 3_Am. The RCA was performed in the presence of 20 mM Mg²⁺ at different time points using Φ-29 DNA polymerase. After finishing the RCA reactions, EDTA (20 mM final concentration) was added to the solution in order to break down the precipitate releasing the extended RCA products. However, the precipitate was still observed. Lane 1 : 1 kb DNA ladder; Lane 2: splint (ODN 17x); Lane 3: splint for 3_Am (ODN 21x).

Figure 58. 0.8% agarose gel analysis of RCA supernatant from cyclic 3_P0₄ and cyclic 3_Tz. The RCA was performed in the presence of 20 mM Mg²⁺ at different time points using Φ-29 DNA polymerase. After finishing the RCA reactions, EDTA (20 mM final concentration) was added to the solution in order to break down the precipitate releasing the extended RCA products. However, precipitate was still observed. Lane 1 : 1 kb DNA ladder; Lane 2: splint (ODN 17x).

Figure 59. Testing the hybridisation probe assay. 0.8% agarose gel analysis of probing the amplified products from Φ-29-mediated RCA of the cyclic template 1_P0₄ after incubation with fluorescent probes, lane 1 : fluorescent probe ODN 24x; lane 2: fluorescent probe ODN 25x (scrambled version of ODN 24x); lane 3: RCA products without probes; lane 4: after hybridisation of RCA products with fluorescent probe ODN 24x; lane 5: after hybridization of RCA products with scrambled fluorescent probe ODN 25x. The gel was imaged using a G: Box (Syngene) with excitation at 520 nm under the Cy3 fluorescence channel.

Figure 60. Probing the amplified products from Φ-29-mediated RCA of the cyclic template 1_P0₄, 1_PA, 1_Am and 1_Tz using fluorescent probe (ODN 24x). Aliquots of the RCA reactions were stopped at regular two hour intervals for a total period of 20 hours. A Cy3-labelled fluorescent probe with a sequence which is complementary to the RCA products was incubated with each aliquot and the mixtures were analysed by 0.8% agarose gel electrophoresis. The gels were imaged with excitation at 520 nm under the Cy3 fluorescence channel and re-imaged after SYBR Gold staining with excitation at 302 nm using a G:Box (Syngene).

Figure 61. 0.8% agarose gel analysis of precipitate from cyclic 1_P0₄, cyclic 1_PA, cyclic 1_Am and cyclic 1_Tz. The RCA was performed in the presence of 20 mM Mg²⁺ for 8 h using Φ-29 DNA polymerase. M: 1 kb DNA ladder; Lane 2: splint (ODN 15x); Lane 3; splint for cyclic 1_Am (ODN 19x); Lane 3: cyclic 1_P0₄; Lane 4: cyclic 1_PA; Lane 5: cyclic 1_Am; Lane 6: cyclic 1_Tz. Figure 62. 0.8% agarose gel analysis of precipitate from cyclic 1_P0₄, cyclic 1_PA, cyclic 1_Am and cyclic 1_Tz. The RCA was performed in the presence of 20 mM Mg²⁺ for 20 h using Φ-29 DNA polymerase. M: 1 kb DNA ladder; Lane 2: splint (ODN 15x); Lane 3; splint for cyclic 1_Am (ODN 19x); Lane 3: cyclic 1_P0₄; Lane 4: cyclic 1_PA; Lane 5: cyclic 1_Am; Lane 6: cyclic 1_Tz.

Figure 63. 0.8% agarose gel analysis of supernatant from cyclic 1_P0₄ and cyclic 1_PA. The RCA was performed for 20 h at different concentrations of Mg²⁺ using Φ- 29 DNA polymerase. Lane 1 : 1 kb DNA ladder; Lane 2: splint (ODN 15x); Lane 3: cyclic unmodified template (1_P0₄); Lane 4: cyclic phosphoramidate template (1_PA).

Figure 64. 0.8% agarose gel analysis of precipitate from cyclic 1_P0₄ and cyclic 1_PA. The RCA was performed for 20 h at different concentrations of Mg²⁺ using Φ- 29 DNA polymerase. Lane 1 : 1 kb DNA ladder; Lane 2: splint (ODN 15x); Lane 3: cyclic unmodified template (1_P0₄); Lane 4: cyclic phosphoramidate template (1_PA).

Figure 65. 0.8% agarose gel analysis of supernatant from cyclic 2_P0₄ and cyclic 2_PA. The RCA was performed for 20 h at different concentrations of Mg²⁺ using Φ- 29 DNA polymerase. Lane 1 : 1 kb DNA ladder; Lane 2: splint (ODN 16x); Lane 3: cyclic unmodified template (2_P0₄); Lane 4: cyclic phosphoramidate template (2_PA).

Figure 66. 0.8% agarose gel analysis of precipitate from cyclic 2_P0₄ and cyclic 2_PA. The RCA was performed for 20 h at different concentrations of Mg²⁺ using Φ- 29 DNA polymerase. Lane 1 : 1 kb DNA ladder; Lane 2: splint (ODN 16x); Lane 3: cyclic unmodified template (2_P0₄); Lane 4: cyclic phosphoramidate template (2_PA).

Figure 67. 0.8% agarose gel analysis of supernatant from cyclic 3_P0₄ and cyclic 3_PA. The RCA was performed for 20 h at different concentrations of Mg²⁺ using Φ- 29 DNA polymerase. Lane 1 : 1 kb DNA ladder; Lane 2: splint (ODN 17x); Lane 3: cyclic unmodified template (3_P0₄); Lane 4: cyclic phosphoramidate template (3_PA).

Figure 68. 0.8% agarose gel analysis of precipitate from cyclic 3_P0₄ and cyclic 3_PA. The RCA was performed for 20 h at different concentrations of Mg²⁺ using Φ- 29 DNA polymerase. Lane 1 : 1 kb DNA ladder; Lane 2: splint (ODN 17x); Lane 3: cyclic unmodified template (3_P0₄); Lane 4: cyclic phosphoramidate template (3_PA).

Figure 69. SEM image used to measure the size distribution of the particles generated from cyclic 1_P0₄. The RCA was carried out for 20 h in the presence of 10 mM Mg²⁺ using Φ-29 DNA polymerase. ImageJ was used to measure the average diameter of particles: 2.98 ± 0.22 μηι, n = 20 (n refers to the number of measured particles). Figure 70. SEM image used to measure the size distribution of the particles generated from cyclic 1_P0₄. The RCA was carried out for 20 h in the presence of 25 mM Mg²⁺ using Φ-29 DNA polymerase. ImageJ was used to measure the average diameter of particles: 0.89 ± 0.15 μηι, n = 20 (n refers to the number of measured particles).

Figure 71 . SEM image used to measure the size distribution of the particles generated from cyclic 1_PA. The RCA was carried out for 20 h in the presence of 10 mM Mg²⁺ using Φ-29 DNA polymerase. ImageJ was used to measure the average diameter of particles: 3.40 ± 0.35 μηι, n = 23 (n refers to the number of measured particles).

Figure 72. SEM image used to measure the size distribution of the particles generated from cyclic 1_PA. The RCA was carried out for 20 h in the presence of 25 mM Mg²⁺ using Φ-29 DNA polymerase. ImageJ was used to measure the average diameter of particles: 0.77 ± 0.07 μηι, n = 60 (n refers to the number of measured particles).

Figure 73. 0.8% agarose gel analysis of original RCA reaction mixture from cyclic 1_P0₄ and cyclic 1_PA. The RCA was performed in the presence of 10 mM Mg²⁺ at different time points using Φ-29 DNA polymerase. Lane 1 : 1 kb DNA ladder; Lane 2: splint (ODN 15x); Lane 3: cyclic unmodified template (1_P0₄); Lane 4: cyclic phosphoramidate template (1_PA). Arrow indicated the second band which is expected to be the double-stranded DNA formation during RCA process, which had been reported by previous literature. (4) We found that T4 gene 32 protein could not reduce dsDNA production.

Experimental

General method for oligonucleotide synthesis and purification

[00106] Standard DNA phosphoramidites, solid supports, and additional reagents were purchased from Link Technologies Ltd and Applied Biosystems Ltd. 5'- Monornethoxytritylarnino-2'-deoxythyrnidine,3'-[(2-cyanoethyl)-(N,N-diisopropyl)]- phosphoramidite was purchased from Glen Research (Catalog Number: 10-1932-90).

[00107] All oligonucleotides were synthesized on an Applied Biosystems 394 automated DNA/ RNA synthesizer using a standard 0.2 or 1.0 /ymole phosphoramidite cycle of acid- catalyzed detritylation, coupling, capping, and iodine oxidation. Stepwise coupling efficiencies and overall yields were determined by the automated trityl cation conductivity monitoring facility and in all cases were >98.0%.

[00108] All β-cyanoethyl phosphoramidite monomers were dissolved in anhydrous acetonitrile to a concentration of 0.1 M immediately prior to use. The coupling time for normal A, G, C, and T monomers was 60 s, and the coupling time for the 5'-amino dT phosphoramidite monomer was extended to 600 s. Cleavage of the oligonucleotides from the solid support and deprotection was achieved by exposure to concentrated aqueous ammonia solution for 60 min at room temperature followed by heating in a sealed tube for 5 h at 55 °C.

[00109] Purification of oligonucleotides was carried out by reversed-phase HPLC on a Gilson system using a Brownlee Aquapore column (C8, 8 mm x 250 mm, 300A pore) with a gradient of acetonitrile in triethylammonium bicarbonate (TEAB) increasing from 0% to 50% buffer B over 30 min with a flow rate of 4 mL/min (buffer A: 0.1 M triethylammonium bicarbonate, pH 7.0, buffer B: 0.1 M triethylammonium bicarbonate, pH 7.0 with 50% acetonitrile). Elution of oligonucleotides was monitored by ultraviolet absorption at 295 or 300 nm. After HPLC purification, oligonucleotides were freeze dried then dissolved in water without the need for desalting.

[00110] For long oligonucleotides, polyacrylamide gel electrophoresis was used for purification. Oligonucleotide bands were then visualized using a UV lamp and the desired bands excised, crushed and soaked in water overnight at 37 °C. After evaporation, samples were desalted using NAP-25 followed by NAP-10 columns (G.E. Healthcare Life Sciences). All oligonucleotides were characterised by electrospray mass spectrometry using a Bruker micrOTOF II focus ESI-TOF MS instrument in ESI^" mode. Data were processed using MaxEnt.

Demonstration of the compatibility of the phosphoramidate linkage with DNA and RNA polymerase

Synthesis of 81-mer (ODN 1) template with one phosphoramidate ligation point

[00111] Oligonucleotides ODN 2 (1.0 nmol), ODN 3 (1.1 nmol) and splint ODN 4 (1.0 nmol) in 0.2 M HEPES (pH=7.2) with 0.4 M NaCI (80 μΙ_) were annealed by heating at 90 °C for 5 min then cooling slowly to room temperature. A solution of 1-(2-hydroxyethyl) imidazole (1.0 M, 10 μί) (0.1 M final concentration) and EDC.HCI (6.0 M, 10 μί) (0.6 M final concentration) was added to the annealed oligonucleotides and the reaction mixture was kept at room temperature for 2 h. Reagents were removed using NAP-25 gel-filtration column and the ligated DNA was analysed and purified by denaturing 12% polyacrylamide gel electrophoresis (Figure 2). The reaction was scaled up 10-fold and aliquots were taken at different time points (Figure 3).

[00112] The gel showed that the reaction was complete within 45 min and there was no difference in intensity of the product after 2 h.

PCR amplification of the 81-mer phosphoramidate template (ODN 1)

[00113] GoTaq DNA polymerase was used to generate a PCR product from the 81-mer template (ODN 1) which includes one phosphoramidate linkage. Reagents and conditions: 4 μΙ_ of 5x buffer (Promega green PCR buffer) was used in a total reaction volume of 20 μΙ_ with 5 ng of the DNA template, 0.5 mM of each primer, 0.2 mM dNTP and 1.0 unit of GoTaq polymerase. The reaction mixture was loaded onto a 2% agarose gel in 1xTBE buffer. PCR cycling conditions: 95 °C (initial denaturation) for 2 min then 25 cycles of 95 °C (denaturation) for 15 s, 54 °C (annealing) for 20 s and 72 °C (extension) for 30 s. The reaction was then left at 72 °C for 5 min then loaded onto a 2% agarose gel in 1 X Tris/Borate/EDTA buffer (TBE) (Figure 4).

[00114] 5 X Promega green PCR buffer was provided with the enzyme (Promega GoTaq DNA polymerase), pH 8.5 containing 7.5 mM MgC to give a final Mg²⁺ concentration of 1.5 mM. The buffer contains Tris.HCI, KCI and two dyes (blue and yellow) that separate during electrophoresis to monitor the migration process.

Synthesis of 303-mer (ODN 5) template with double phosphoramidate ligation points

[00115] Oligonucleotides ODN 6, ODN 7, ODN 8 with splints ODN 9 and ODN 10 (0.5 nmol of each) in 0.2 M HEPES (pH=7.2) with 0.4 M NaCI (80 μΙ_) were annealed by heating at 90 °C for 5 min then cooling slowly to room temperature. A solution of 1-(2-hydroxyethyl) imidazole (1.0 M, 10 μΙ_) (0.1 M final concentration) and EDC.HCI (6.0 M, 10 μΙ_) (0.6 M final concentration) was added to the annealed oligonucleotides and the reaction mixture was kept at room temperature for 2 h. Reagents were removed using NAP-25 gel-filtration column and the ligated DNA was analysed by denaturing 6% polyacrylamide gel electrophoresis.

[00116] A mixture of 3 nmoles of each oligonucleotide and splints were dissolved in water and then divided into 6 samples and each mixed with 2x buffer. A solution of 1-(2- hydroxyethyl) imidazole (1.0 M, 10 μΙ_) (0.1 M final concentration) and EDC.HCI (6.0 M, 10 μΙ_) (0.6 M final concentration) was added to the annealed oligonucleotides and the reaction mixture was kept at room temperature for 1 h then analysed by denaturing 6% polyacrylamide gel electrophoresis (Figure 5). [00117] The following buffer systems were used:

50 mM Tris (pH=8.5), 25 mM MgCI₂, 0.2 M NaCI; 10 mM phosphate (pH=7.0), 25 mM MgCI₂, 0.2 M NaCI; 0.2 M HEPES (pH=7.2), 0.4 M NaCI, 25 mM MgCI₂ and 0.2 M HEPES (pH=7.2), 0.4 M NaCI.

PCR amplification of the 303-mer double phosphoramidate template ODN 5 using GoTaq DNA polymerase, cloning and sequencing

[00118] Following the above method for PCR amplification of 81-mer ODN 1 , the PCR product was purified by extraction from a 2% agarose gel (Figure 6) using a QIAquick Gel Extraction kit. It was then inserted into vector pCR2.1. TOPO for subcloning. Cloning was carried out using a standard TOPO cloning protocol. Standard automated Sanger DNA sequencing was performed and the data shown in Figure 7.

Table 1 - Mutations found in sequence data in Figure 7

TC: total clones, NMC: non-mutant clones, MC: mutant clones, IM: insertion mutation, DM: deletion mutation, SM: substituted mutation, LPM: ligation point mutation, TB: total number of bases, TM: total number of mutation.

Single tube synthesis of the entire EGFP gene

[00119] The synthesis of the entire EGFP gene in one tube was achieved by mixing 0.1 nmole of each of the 10 oligonucleotides (ODN 15-ODN 24 for both forward and reverse strands), freeze drying them together then re-dissolving them in 100 μΙ_ HEPES Buffer (0.2 M, pH=7.2) with 0.4 M NaCI. The oligonucleotide mixture was annealed by heating at 90 °C for 5 min then cooled slowly to room temperature. EDC.HCI (30 mg) and a solution of 1-(2- hydroxyethyl) imidazole (1.0 M, 30 μΙ_) were added to the annealed oligonucleotides and the reaction mixture was kept at room temperature for 2 h. Reagents were removed using NAP- 25 gel-filtration column and the ligated DNA was analysed by denaturing 4% polyacrylamide gel electrophoresis. The band was cut and DNA was extracted then used in PCR.

PCR amplification of the double stranded phosphoramidate EGFP gene by phosphoramidate ligation using GoTaq DNA polymerase

[00120] A PCR product from the whole EGFP gene duplex was generated using GoTaq DNA polymerase under the same conditions explained above for PCR of 81-mer ODN 1. The PCR product was purified by extraction from a 2% agarose gel (Figure 8A) using a QIAquick Gel Extraction kit. It was then inserted into the vector pCR2.1. Cloning into the TOPO vector was done with a standard TOPO cloning protocol. Automated Sanger DNA sequencing was performed; and the data is shown in Figure 9 and Figure 10. This procedure was carried out by Eurofins GmbH.

[00121] Expected EGFP sequence with the ligation points in bold and a watermark underlined text (SeqID 1):

TCGACGGTACCGCGGGCCCGGGATCCACCGGTCGCCACCATGGTGAGCAAGGGCGA

GGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGG

CCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGAC

CCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACC

ACCCTGACCTACGGTGTACAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACG

ACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAA

GGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGT

GAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCA

CAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAG

AACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGC

TCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCG

ACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCG

ATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGA

GCTGTACAAGTAAAGC.

[00122] Found EGFP sequence watermark in underlined text and only one deletion mutation in bold (SeqID 2) (Figure 10):

CGGTACCGCGGGCCCGGGATCCACCGGTCGCCACCATGGTGAGCAAGGGCGAGGAG

CTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCAC

AAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTG

AAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCC

TGACCTACGGTGTACAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTT

CTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGAC

GACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAAC

CGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAG

CTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACG

GCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGC

CGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAA

CCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCA

CATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTcGGCATGGACGAGCT

GTACAAGTAAAGC. [00123] Two more clones were sequenced and similar results were obtained with a small number of mutations which were far from the ligation points. Two deletion mutations were found in one clone whereas in the other clone, 4 deletion mutation and 2 substitution mutation were found. The mutation could have occurred during sequencing or oligonucleotide synthesis and purification. The mutation rate is consistent with that expected from oligonucleotide syntheses. All clones show the water mark which was inserted in the sequence of the synthesised EGFP as a unique signature to differentiate it from potential contaminant DNA. (Figure 9).

Transcription of phosphoramidate template ODN 27 and control ODN 31

[00124] MegaScript T7 Transcription Kit (ThermoFislher Scientific, cat. no. AM 1333) was used according to the manufacturer's recommended protocol. Reaction mixtures were prepared in the following order at room temperature: phosphoramidate template ODN 27 (5 μΜ, 2.5 μΙ), long coding strand ODN 32 (5 μΜ, 2.75 μΙ), water (14.75 μΙ), reaction buffer (10x, 5 μΙ), ATP (5 μΙ), CTP (5 μΙ), GTP (5 μΙ), UTP (5 μΙ) and enzyme mix (5 μΙ). The reaction mixture was then incubated at 37 °C and 10 μΙ aliquots removed at the specified times and mixed with an equal volume of formamide before storing at -80 °C. Samples were then loaded on 12% denaturing polyacrylamide gel (1x TBE, 7 M urea, W x D x H = 18 x 0.2 x 24.4 cm) at 20 W for 2 h.

[00125] The same reaction was repeated using short coding strand ODN 33 and gave similar results.

[00126] For comparison of efficiency, the experiments were also performed using the control unmodified template ODN 31 using long coding strand ODN 32 and short coding strand ODN 33 (Figure 11).

[00127] Oligonucleotide bands were then visualized using a UV lamp and the desired bands excised, crushed and soaked in buffer (50 mM Tris-HCI, pH 7.5, 25 mM NaCI) overnight at 37 °C. After evaporation of the solvent, samples were desalted using two NAP-25 columns (G.E. Healthcare Life Sciences, cat. no. 17-0852-01). The expected product was confirmed by mass spectrometry of transcripts formed from phosphoramidate-containing and control strands using the long coding strand.

[00128] Transcription reaction (70 μΙ total volume) was performed as above using phosphoramidate (ODN 27) or control (ODN 31) template and long coding strand (ODN 32). The reaction mixture was left for 16 h before mixing with a mixture of phenol:chloroform:isoamyl alcohol (25:24:1 , v/v) (from Invitrogen) to remove excess reagents. The mixture was mixed vigorously and the top layer (transcripts) was removed. The RNA transcripts were precipitated by adding sodium acetate (3 M, 50 μΙ) followed by isopropanol (150 μΙ). The mixture was left at -80 °C for 3 h then centrifuged at 4 °C and 13 RPM for 10 min. The RNA was dried then dissolved in 20 μΙ water where 0.5 μΙ was analysed by mass spectrometry. The crude transcripts gave the same (expected) mass for phosphoramidate and control templates.

Table 2 - Oligonucleotides used in this study

^NT= 5'-amino dT, p=3'-phosphate, X=3'-propargyl-5-Me-dC\

SeqID No. Code Oligonucleotide sequences (5'-3')

SeqID 3 GCATTCGAGCAACGTAAGATCGCTAGCACACAATCTCACACTCTGGA

ODN 1

ATTCACACTGACAATACTGCCGACACACATAACC

SeqID 4 ODN 2 GCATTCGAGCAACGTAAGATCGCfi

SeqID 5 ^AGCACACAATCTCACACTCTGGAATTCACACTGACAATACTGCCGA

ODN 3

CACACATAACC

SeqID 6 ODN 4

TGTGTGCTAGCGATCTTA splint

SeqID 7 AAGCTTTATTAAAATGTCTAAAGGTGAAGAATTATTCACTGGTGTTGT

CCCAATTTTGGTTGAATTAGATGGTGATGTTAATGGTCACAAATTTTC

TGTCTCCGGTGAAGGTGAAGGTGATGCTACTTACGGTAAATTGACCT

ODN 5,

TAAAATTTA I I I GTACTACTGGTAAATTGCCAGTTCCATGGCCAACCT

303-mer

TAGTCACTACTTTCGGTTATGGTGTTCAATG I I I I GCTAGATACCCAG

ATCATATGAAACAACATGACTTTTTCAAGTCTGCCATGCCAGAAGGTT

ATGTTCAAGAAAGAAC

SeqID 8 AAGCTTTATTAAAATGTCTAAAGGTGAAGAATTATTCACTGGTGTTGT

ODN 6 CCCAATTTTGGTTGAATTAGATGGTGATGTTAATGGTCACAAATTTTC

TGTCfi

SeqID 9 ^CCGGTGAAGGTGAAGGTGATGCTACTTACGGTAAATTGACCTTAAA

ODN 7 ATTTATTTGTACTACTGGTAAATTGCCAGTTCCATGGCCAACCTTAGT

CACTACfi

SeqID 10 ^TTCGGTTATGGTGTTCAATGTTTTGCTAGATACCCAGATCATATGAA

ODN 8 ACAACATGACTTTTTCAAGTCTGCCATGCCAGAAGGTTATGTTCAAGA

AAGAAC

SeqID 11 ODN 9 CCATAACCGAAAGTAGTGACTAAG Splint for 303-mer template ligation

SeqID 12 ODN 10 ACCTTCACCGGAGACAGAAAATTT Splint for 303-mer template ligation

SeqID 13 ODN 11 GTTCTTTCTTGAACATAA PCR Primer 1 for 303-mer template

SeqID 14 ODN 12 AAGCTTTATTAAAATGTCTA PCR Primer 2 for 303-mer template

SeqID 15 ODN 13, pTCGACGGTACCGCGGGCCCGGGATCCACCGGTCGCCACCATGGT EGFP GAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGT forward CGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGC strand GAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCA

TCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGAC

CACCCTGACCTACGGTGTACAGTGCTTCAGCCGCTACCCCGACCAC

ATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACG

TCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGAC

CCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCAT

CGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGG

CACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGC

CGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCAC

AACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGA

ACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTA

CCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGC

GATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTC

TCGGCATGGACGAGCTGTACAAGTAAAGC

SeqID 16 pGGCCGCTTTACTTGTACAGCTCGTCCATGCCGAGAGTGATCCCGG

CGGCGGTCACGAACTCCAGCAGGACCATGTGATCGCGCTTCTCGTT

GGGGTCTTTGCTCAGGGCGGACTGGGTGCTCAGGTAGTGGTTGTCG

GGCAGCAGCACGGGGCCGTCGCCGATGGGGGTGTTCTGCTGGTAG

TGGTCGGCGAGCTGCACGCTGCCGTCCTCGATGTTGTGGCGGATCT

TGAAGTTCACCTTGATGCCGTTCTTCTGCTTGTCGGCCATGATATAGA

CGTTGTGGCTGTTGTAGTTGTACTCCAGCTTGTGCCCCAGGATGTTG

ODN 14

CCGTCCTCCTTGAAGTCGATGCCCTTCAGCTCGATGCGGTTCACCAG EGFP

GGTGTCGCCCTCGAACTTCACCTCGGCGCGGGTCTTGTAGTTGCCG

reverse

TCGTCCTTGAAGAAGATGGTGCGCTCCTGGACGTAGCCTTCGGGCA

strand

TGGCGGACTTGAAGAAGTCGTGCTGCTTCATGTGGTCGGGGTAGCG

GCTGAAGCACTGTACACCGTAGGTCAGGGTGGTCACGAGGGTGGGC

CAGGGCACGGGCAGCTTGCCGGTGGTGCAGATGAACTTCAGGGTCA

GCTTGCCGTAGGTGGCATCGCCCTCGCCCTCGCCGGACACGCTGAA

CTTGTGGCCGTTTACGTCGCCGTCCAGCTCGACCAGGATGGGCACC

ACCCCGGTGAACAGCTCCTCGCCCTTGCTCACCATGGTGGCGACCG

GTGGATCCCGGGCCCGCGGTACCG

SeqID 17 JDTCGACGGTACCGCGGGCCCGGGATCCACCGGTCGCCACCATGGT

ODN 15 GAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGT

CGAGCTGGACGGCGACGTAAACGGCCACAAGJD 120-mer

SeqID 18 !^TTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAG

ODN 16 CTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCT

GGCCCACCCTCGTGACCACCCTGACCTACGGTGTACAGTGCTTCAG CCGCTACCCCGACCACAE 154-mer

SeqID 19 ^GAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGT

CCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACC

ODN 17

CGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATC GAGCTGAAGGGCATCGACJD 155-mer

SeqID 20 ^TTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACT

ACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGG

ODN 18

CATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGC GTGCAGCTCGCCGACCACJD 156-mer

SeqID 21 ^ACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCC

ODN 19 GACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCA

ACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGC CGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAAGC 177-mer

SeqID 22 eGGCCGCTTTACTTGTACAGCTCGTCCATGCCGAGAGTGATCCCGG

CGGCGGTCACGAACTCCAGCAGGACCATGTGATCGCGCTTCTCGTT

ODN 20

GGGGTC I I I GCTCAGGGCGGACTGGGTGCTCAGGTAGTGGTTGTCG GGCAGCAGCACGGGGCCGJD 155-mer

SeqID 23 ^CGCCGATGGGGGTGTTCTGCTGGTAGTGGTCGGCGAGCTGCACG

CTGCCGTCCTCGATGTTGTGGCGGATCTTGAAGTTCACCTTGATGCC

ODN 21

GTTCTTCTGCTTGTCGGCCATGATATAGACGTTGTGGCTGTTGTAGTT GTACTCCAGCTTGJD 153-mer

SeqID 24 ^TGCCCCAGGATGTTGCCGTCCTCCTTGAAGTCGATGCCCTTCAGCT

CGATGCGGTTCACCAGGGTGTCGCCCTCGAACTTCACCTCGGCGCG

ODN 22

GGTCTTGTAGTTGCCGTCGTCCTTGAAGAAGATGGTGCGCTCCTGGA CGTAGCCTE 147-mer

SeqID 25 ^CGGGCATGGCGGACTTGAAGAAGTCGTGCTGCTTCATGTGGTCG

GGGTAGCGGCTGAAGCACTGTACACCGTAGGTCAGGGTGGTCACGA

ODN 23

GGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCAGATGAACT TCAGGGTCAGCTTGCCGfi 153-mer

SeqID 26 ^AGGTGGCATCGCCCTCGCCCTCGCCGGACACGCTGAACTTGTGG

CCGTTTACGTCGCCGTCCAGCTCGACCAGGATGGGCACCACCCCGG

ODN 24

TGAACAGCTCCTCGCCCTTGCTCACCATGGTGGCGACCGGTGGATC CCGGGCCCGCGGTACCG 154-mer

SeqID 27 ODN 25 TCGACGGTACCGCGGGCC PCR primer for EGFP forward strand

SeqID 28 ODN 26 GCTTTACTTGTACAGCTCGTCC PCR primer for EGFP reverse strand

SeqID 29 CACCCCGGTGAACAGCTCCTCGCCCTTGCTCACCATGGTGGCGACT

ODN 27

TCTCCCTATAGTGAGTCGTATTAGGACCAGCGT transcription template SeqID 30 I ^NTCGCCCTTGCTCACCATGGTGGCGACTTCTCCCTATAGTGAGTCGT ODN 28 I ^—

\ ATTAGGACCAGCGT

SeqID 31 ODN 29 j CACCCCGGTGAACAGCTCCfi

SeqID 32 ODN 30 GCAAGGGCGAGGAGCTGTTC splint

SeqID 33 \ CACCCCGGTGAACAGCTCCTCGCCCTTGCTCACCATGGTGGCGACT

ODN 31 TCTCCCTATAGTGAGTCGTATTAGGACCAGCGT control for

i transcription

SeqID 34 i ACGCTGGTCCTAATACGACTCACTATAGGGAGAAGTCGCCACCATGG

ODN 32 j

I TGAGCAAGGGCGAGGAGCTGTTCACCGGGGTG long coding strand

SeqID 35 j ACGCTGGTCCTAATACGACTCACTATAGGGAGAAGTCGCC short

ODN 33 I

\ coding strand

SeqID 36 j pppGGGAGAAGUCGCCACCAUGGUGAGCAAGGGCGAGGAGCUGUU

ODN 34 ;

\ CACCGGGGUGc transcript

Table 3 - Oligonucleotide mass spectra

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Enzyme-free synthesis of cyclic single-stranded DNA constructs containing a single triazole, amide or phosphoramidate backbone linkage and their use as templates for rolling circle amplification and nanoflower formation

[00129] Cyclic oligonucleotides are valuable targets with a broad range of potential applications spanning molecular biology and nanotechnology. Of particular importance is their role as templates in the rolling circle amplification (RCA) reaction. We describe three different chemical cyclisation methods for the preparation of single-stranded cyclic DNA constructs. These chemical cyclisation reactions are cheaper to carry out than the enzymatic reaction, and more amenable to preparative scale purification and characterisation of the cyclic product. They can also be performed under denaturing conditions and are therefore particularly valuable for cyclic DNA templates that contain secondary structures. The resulting single-stranded cyclic DNA constructs contain a single non-canonical backbone linkage at the ligation point (triazole, amide or phosphoramidate). They were compared to unmodified cyclic DNA in rolling circle amplification reactions using f-29 and Bst 2.0 DNA polymerase enzymes. The cyclic templates containing a phosphoramidate linkage were particularly well tolerated by f-29 polymerase, consistently performing as well in RCA as the unmodified DNA controls. Moreover, these phosphoramidate-modified cyclic constructs can be readily produced in oligonucleotide synthesis facilities from commercially available precursors. Phosphoramidate ligation therefore holds promise as a practical, scalable method for the synthesis of fully biocompatible cyclic RCA templates. The triazole-modified cyclic templates generally gave lower and more variable yields of RCA products, a significant proportion of which were double-stranded, while the performances of the templates containing an amide linkage lie in between those of the phosphoramidate- and triazole-containing templates.

[00130] Rolling circle amplification (RCA) is an isothermal enzymatic technique that is widely used to make very long single-stranded DNA and RNA.^{1 2} The product contains a specific, tandem-repeating sequence which is encoded by the complementary cyclic template.^3,4 This technique has been harnessed as a simple and powerful method of signal amplification in the development of sensitive detection methods for a variety of nucleic acid, protein, cellular and small molecule targets for diagnostic, biosensing and genotyping purposes.^5-14 RCA has also attracted widespread interest as a tool in the synthesis of functional DNA nanomaterials¹⁵ including origami,¹⁶ nanosprings,¹⁷ nanotubes,¹⁸ templating nanoscaffolds, ^19,20 hydrogels^{21 22} and DNA nanoflowers.²³ Proposed future applications of these materials include biosensors, targeted imaging and drug delivery agents,²⁴ and components of nanoscale computers and electronic circuits. An RCA reaction requires four components: a circular DNA template, a short DNA primer, a polymerase enzyme and deoxynucleotide triphosphates (dNTPs). The cyclic template is most commonly obtained via an enzyme-catalysed cyclisation of a linear DNA strand using DNA ligase enzymes such as T4 ligase or CircLigase. The efficiency of the cyclisation reaction is sensitive to the length of the linear DNA precursor, and is inefficient if the nucleobase sequence leads to secondary structures.^25,26 In addition, the scale on which the cyclisation reaction can be carried out is limited by the cost of the enzyme. In principle these limitations could be circumvented by the use of chemical ligation (Figure 16), which is readily scalable, and would allow the use of a wide variety of buffers, including those in which DNA structures are denatured. However, the creation of a canonical phosphodiester linkage by chemical methods in aqueous media is inherently difficult because the nucleophilicity of the attacking hydroxyl group is similar to that of the surrounding water molecules, which are present in great excess. Although chemical methods for carrying out this transformation have been reported, such methods often rely on the use of acutely toxic reagents such as cyanogen bromide.²⁷ For these reasons artificial DNA backbones which are less challenging to form chemically are an attractive alternative.²⁸ Prevalent amongst these are the triazole,²⁹ amide^30,31 and phosphoramidate^32-34 linkages (Figure 16(ii)). When incorporated into linear oligonucleotides these phosphodiester backbone analogues can be read through by DNA polymerases during linear copying and the polymerase chain reaction (PCR), with accurate transfer of genetic information.²⁸'^32,35 They could potentially be incorporated into cyclic RCA templates provided that they can also be read through by the specific DNA polymerases that are compatible with efficient RCA. However, the use of chemically modified templates in RCA is particularly demanding because it requires the polymerase to accurately read through the modified linkage repeatedly in order to generate very long amplification products. In contrast, during PCR the polymerase needs only to copy the chemically modified template once during the first cycle, after which the reaction can proceed in an exponential manner, with the enzyme almost exclusively copying backbone-unmodified products in all subsequent cycles. Given the widespread importance of the RCA reaction, we undertook to study the cyclisation of DNA using chemical approaches and to examine the suitability of the chemically modified cyclic products as templates during RCA. We then demonstrated that RCA of the cyclic products can be used to prepare DNA nanoflowers.^23,36-41 Finally, we compared the size distribution, morphologies and extent of DNA loading of the DNA nanoflowers assembled from the RCA products of the chemically modified templates.

Experimental

Choice of chemical linkages and template sequences

[00131] In the present study we investigate RCA of cyclic oligonucleotides containing a single triazole, amide or phosphoramidate analogue of the DNA phosphodiester backbone. All three modifications have been previously used in PCR amplification of linear DNA. 28,32,35,42-45 Qf th_ese triazole linkage (Figure 1A) is the most thoroughly studied artificial DNA backbone.²⁹ It can be read through by both DNA and RNA polymerases and has been used in the construction of genes by chemical ligation. There are two reports of cyclisation of linear DNA that contains 5^'-azide and 3^'-alkyne followed by RCA of the cyclic template. The first one, from our laboratory,⁴⁹ shows that cyclisation and subsequent RCA of a 100-mer DNA sequence designed to be free of secondary structure is possible, despite the presence of the 1 ,4-triazole linkage. The second study used a triazole-containing DNA template in an RCA-based fluorogenic assay for microRNA, but the authors did not compare the efficiency of the RCA reaction with that of an unmodified template.⁴⁶ In this work we also investigate amide and phosphoramidate chemical linkages (Figure 16 B and C), both of which can be formed by di-imide coupling chemistry. The phosphoramidate linkage is of particular interest as it closely mimics the steric and electronic properties of the canonical phosphodiester backbone.

[00132] In order to study the above three non-canonical DNA linkages in RCA we designed three cyclic templates of differing base composition, all of which are predicted to incorporate secondary structures including cancer cell surface targeting aptamers (oligonucleotide sequences are given in Table 4, and the predicted structures are shown in Figure 27). Template 1 is based on a randomly generated 50-mer sequence with approximately 50% GC content; template 2 incorporates a dumbbell-shaped double hairpin structure and serves as a template for an aptamer sequence which targets the mucin 1 (MUC1) glycoprotein⁴⁷— an important class of tumour surface marker that is overexpressed on a range of epithelial cancer cells; template 3 is a cytosine-rich sequence encoding a complementary trimeric G quadruplex aptamer which recognises the human epidermal growth factor receptor 2, a major therapeutic target in human cancer treatment.⁴⁸ All four versions of each template were made: an unmodified control containing a natural phosphodiester (P0₄) backbone and analogues containing triazole (Tz), amide (Am) and phosphoramidate (PA) linkages at the point of cyclisation. § In addition we prepared the unmodified and triazole-modified cyclic templates 4_P0₄ and 4_Tz, which incorporate a palindromic sequence. The RCA product of 4_P0₄ has been reported to assemble into a three-dimensional interwoven 'nanoclew' structure that can be used to deliver anticancer drugs.³⁹ Following cyclisation of the linear sequences all of the cyclic templates were purified then subjected to RCA using f-29, a highly processive polymerase with strand displacement activity.⁴⁹

Cyclisation of linear oligonucleotides

[00133] Linear oligonucleotides incorporating the required 5'-phosphate/3'-hydroxyl, 5'-azide/3'-alkyne, 5'-amine/3'-phosphate and 5'-amine/3'-carboxylate modifications were synthesised using automated solid phase phosphoramidite chemistry, as described herein. The linear unmodified control sequences were cyclised to give the cyclic templates 1_P0₄, 2_P0₄, 3_P0₄ and 4_P0₄ using T4 DNA ligase in the presence of a splint oligonucleotide. The splint oligonucleotides incorporate a region which is complementary to each end of the corresponding linear oligonucleotide and hence act as cyclisation templates by bringing the two reacting ends of the linear substrate into close proximity. Cyclisation of the chemically modified linear oligonucleotides was carried out under aqueous CuAAC or di-imide coupling conditions. In almost every case the linear precursor was successfully converted to the desired cyclic product with moderate to excellent efficiency. §§ The linear 5'-azide/3'-alkyne oligonucleotides were cyclised by treatment with CuS0₄ in the presence of sodium ascorbate and tris-hydroxypropyltriazole (THPTA) in pure water or denaturing organic/aqueous solvent mixtures. In all four cases the cyclisation reaction proceeded smoothly in the absence of a templating splint oligonucleotide. The 5'-amine/3'-phosphate oligonucleotides were cyclised by treatment with A/-(3-dimethylaminopropyl)-/\/'- ethylcarbodiimide hydrochloride (EDC HCI) and 2-(hydroxyethyl)imidazole in aqueous 0.2 M HEPES buffer (pH 7.2) using an adaptation of our previously reported phosphoramidate ligation method.³² Similarly the 5'-amine/3'-carboxylate were cyclised using EDC HCI in combination with /V-hydroxysuccinimide in aqueous NaCI or HEPES buffer, also using an adaptation of a previously reported method.²⁸ The phosphoramidate- and amide-modified cyclic constructs 1_PA and 1_Am formed readily in the absence of a templating splint oligonucleotide; for the amide and phosphoramidate sequences 2_PA, 2_Am, 3_PA and 3_Am the cyclisation reactions were found to be less favourable under non-templating conditions and a splint oligonucleotide was therefore added in order to achieve more efficient cyclisation. Crude cyclisation reactions were analysed by poly acrylamide gel- electrophoresis (PAGE). Representative gel images for the template sequences 1_P0₄, 1_PA, 1_Tz and 1_Am are shown in Figure 2 (see Figures 28-35 for PAGE analysis of all other cyclisation reactions).

Table 4 Sequences (5'→ 3') of the cyclic template oligonucleotides used in this study §

SeqID No. Cyclic oligo Sequence of linear oligonucleotide precursor³ Number nucleotide code of bases

SeqID 37 1. _PO₄ ^PTCCCAATTGGGTACGCAGTACCCACAGCAGATGTGACTGTGA 50

ATCGTGAC

SeqID 38 1. _Tz ^ZTCCCAATTGGGTACGCAGTACCCACAGCAGATGTGACTGTGA 50

ATCGTGAC^K

SeqID 39 1. _Am ^MTCCAATTGGGTACGCAGTACCCACAGCAGATGTGACTGTGAA 50

TCGTGACT^X

SeqID 40 1. .PA ^MTCCCAATTGGGTACGCAGTACCCACAGCAGATGTGACTGTGA 50

ATCGTGAC^P

SeqID 41 2. _PO₄ ^PTGTCGTTTTACCCATGTGCTATAGCCACTACTGTCGTTTTACC 62

CATGTGCTATAGCCACTAC

SeqID 42 2. _Tz ^ZTGTCGTTTTACCCATGTGCTATAGCCACTACTGTCGTTTTACCC 62

ATGTGCTATAGCCACTAC^K

SeqID 43 2. _Am ^MTGTCGTTTTACCCATGTGCTATAGCCACTACTGTCGTTTTACC 62

CATGTGCTATAGCCACTAT^X

SeqID 44 2. .PA ^MTGTCGTTTTACCCATGTGCTATAGCCACTACTGTCGTTTTACC 62

CATGTGCTATAGCCACTAC^P

SeqID 45 3. _PO₄ ^PTTACCCCACACCGCTGCCCCCACACCGCTGCCCCCACACCGC 50

TGCCTTAC

SeqID 46 3. _Tz ^ZTTACCCCACACCGCTGCCCCCACACCGCTGCCCCCACACCGC 50

TGCCTTAC^K

SeqID 47 3. _Am ^MTACCCCACACCGCTGCCCCCACACCGCTGCCCCCACACCGC 50

TGCCTTACT^X

SeqID 48 3. .PA ^MTTACCCCACACCGCTGCCCCCACACCGCTGCCCCCACACCG 50

CTGCCTTAC^P

SeqID 49 4. _PO₄ ^PGTTAATATTATTCGACGGGCCTGCTCGAGCTCGAGCTTGCATC 75

GTGCAGCCGAAGCTTGCACGCGTGCTATTAAT

SeqID 50 4. _Tz ^ZTTG C AC G C GTG CTATTAATGTTAATATTATTC GACGGGCCTGC 75

TCGAGCTCGAGCTTGCATCGTGCAGCCGAAG^MeC^K

'P = PO4; = 5'-amine, X = 3'-carboxy, Z = 5'-azide, K = 3'-propargyl, ^MeC = methyl cytosine

[00134] During the EDC-mediated cyclisation reactions to prepare the phosphoramidate-modified templates we observed the formation of adducts of the cyclic products with molecular weights which are 155 mass units higher than those for the expected products by UPLC-mass spectrometry. This is consistent with the chemical addition of EDC to the oligonucleotides. Similar observations have been documented previously: the adducts are proposed to arise from a chemical reaction between the di-imide coupling reagent and the imide group of guanine and thymine DNA bases.^50"54 In support of this we did not observe formation of these adducts upon exposure of poly-adenine and poly- cytosine DNA sequences to 0.1 M EDC in 0.2 M HEPES buffer (pH 7.2) at room temperature, while in contrast a significant degree of EDC adduct formation was observed when the same experiment was performed with sequences containing guanine and thymine bases. Pleasingly it was possible to reverse this unwanted side-reaction by gently heating the crude product mixtures in 0.1 M NaOH after performing the phosphoramidate cyclisation reactions. Interestingly, we did not observe any evidence of the formation of the same EDC adducts during the reactions to prepare the amide-modified cyclic constructs, which were carried out under slightly different conditions and required a lower concentration of the di- imide coupling agent. All of the modified and unmodified cyclic products were readily purified by PAGE. For the splint-mediated cyclisation reactions it was necessary to use denaturing PAGE conditions during purification, while for the non-templated reactions either denaturing or non-denaturing conditions were selected, depending on which gave better separation between the linear oligonucleotide precursor and cyclic oligonucleotide product. Full details of the cyclisation and purification protocols along with analytical data for all of the cyclic constructs are provided as supporting information (Figures 36-49).

[00135] While the efficiency of each cyclisation method described above was observed to vary depending on the individual linear oligonucleotide sequence, it is noteworthy that the chemical ligation methods can offer significant improvements in cyclisation efficiency in cases where the more commonly used enzymatic approach proves problematic. For example, while the T4-DNA ligase-mediated cyclisation of the 'nanoclew' template 4_P0₄ was low-yielding, producing a number of higher molecular weight oligomeric side-products, the analogous CuAAC-mediated cyclisation reaction— when performed in the presence of denaturing organic solvents to disrupt secondary structure— proceeded more cleanly, generating fewer by-products (Figure 18). The efficiency of this chemical cyclisation reaction appears comparable to that of the reported CircLigase ll-mediated cyclisation of the same template;³⁹ however, the chemical cyclisation approach is not limited in scale by the high cost associated with the CircLigase enzyme. The chemical ligation methods discussed here therefore provide a greatly expanded toolkit for the cyclisation of linear oligonucleotides, with the potential to carry out the reactions under varied conditions, in the presence of denaturing agents, at varying salt concentration and temperature, and with significantly reduced cost. In addition, chemical ligation reactions are more robust and more scalable than enzymatic reactions, with attendant enhancements in the ease of purification, isolation and characterisation of the cyclic products.

Rolling circle amplification of the cyclic oligonucleotides [00136] RCA using the using the Φ-29 polymerase. For all fourteen cyclic templates, RCA was carried out using f-29 polymerase, a 16 or 18 nucleobase primer, 2 mM dNTPs and 20 mM Mg²⁺ at 30 °C. §§§ Analysis of the product mixtures using agarose gel- electrophoresis indicated that in all cases the RCA reactions were successful (Figure 19 and Figures 50-58). However, there were significant differences in the quantities and size distributions of the products, which depended on the nature of the modified linkage in the cyclic template. Each reaction produced a detectable quantity of very slow-migrating products which were retarded in the wells of the gel and, in some cases, a second discrete band with higher gel mobility was also observed to form a substantial proportion of the product distribution (Figure 19). By analogy with a recent literature report,⁵⁵ this faster- running band was assigned to double-stranded products, while the material which is retarded in the wells is assigned to the expected very long single-stranded products. A possible mechanism for the formation of double-stranded side-products during RCA is illustrated in Figure 20. It is noteworthy that the faster-running band assigned to double- stranded DNA side- products was not observed after RCA of the cyclic templates 3_P0₄, 3_Am, 3_PA and 3_Tz, which encode a complementary trimeric G quadruplex aptamer sequence (Figures 56-58). One possible explanation is that the mechanism of double- stranded DNA formation is suppressed by the tendency for the emerging RCA product to fold up into a G quadruplex secondary structure. The phosphoramidate modification gave very efficient RCA, consistently yielding product distributions which were indistinguishable from those of the unmodified templates (Figures 19, 50, 53, 56). The amide-modified cyclic templates also appeared to be well-tolerated by the f-29 polymerase, producing similar product distributions to the unmodified control sequences in all cases (Figures 19, 51 , 54, 57). In contrast, the triazole-containing templates gave weaker and more variable results. While the triazole-modified cyclic template 1_Tz performed moderately in comparison to the unmodified and amide- and phosphoramidate-modified analogues, the cyclic templates 2_Tz, 3_Tz and 4_Tz consistently produced much lower yields of very long, slow-migrating RCA products (Figure 19; Figures 52, 55 and 58). The overall performance of the triazole- containing templates therefore appears to be compromised in comparison to those of the unmodified and phosphoramidate-containing analogues. This is a salient observation given that a recent publication uses a triazole-containing cyclic template with f-29 in an RCA-based fluorescent switch-based diagnostic assay for microRNA detection.⁴⁶ It is also interesting that during RCA of the amide- and triazole-modified templates 1_Am, 1_Tz and 2_Am the proportion of double-stranded side-products appears to increase with extended amplification time. In contrast the relative proportions of the bands assigned to single- and double- stranded products respectively appear to stay approximately constant throughout the course of the RCA reactions for all of the unmodified and phosphoramidate-modified templates (Figures 50-54).

[00137] Probing the single-stranded RCA products with complementary fluorescent oligonucleotides. To obtain further information about the nature of the products from the RCA reactions on the modified phosphoramidate-, amide- and triazole- containing cyclic templates we developed a fluorescent probe assay to allow differentiation between single- and double-stranded RCA products by gel-electrophoresis. A single- stranded Cy3-labelled fluorescent hybridisation probe with a sequence which is

complementary to those of the single stranded products obtained from RCA of the cyclic templates 1_P0₄, 1_Tz, 1_PA and 1_Am was synthesised. In order to validate the assay, a scrambled version of the probe was also prepared, and we performed a preliminary control experiment using the complementary and scrambled versions of the hybridisation probe in order to confirm that the complementary probe binds specifically with the RCA product of the cyclic template 1_P0₄ through base paring (Figure 59). RCA of the four cyclic templates 1_P0₄, 1_Tz, 1_PA and 1_Am was then performed using the f-29-polymerase enzyme and individual RCA reactions were stopped by heat inactivation of the polymerase at two hour intervals over a twenty hour time period. The product mixtures were then incubated with the fluorescent hybridisation probe for two hours at room temperature before analysis using agarose gel electrophoresis. The gels were imaged with excitation at 520 nm under the Cy3 fluorescence channel of a Syngene G:BOX imager (Figure 21).

[00138] Finally, the same gels were post-stained with a solution of SYBR Gold, which can sensitively detect both double- and single-stranded DNA, and re-imaged with excitation at 302 nm (Figure 60). The results show that a strong fluorescence signal from the bound Cy3 labelled probe is detected after amplification of the unmodified, amide-modified and phosphoramidate-modified templates 1_P0₄, 1_PA and 1_Am. This demonstrates that the fluorescent probe has successfully hybridised to the RCA products, confirming their single- stranded nature. Conversely, after amplification of the triazole-modified template, no appreciable signal from the Cy3 fluorophore is detected, although some slow-migrating RCA products are weakly detected in the wells of the gel after post-staining with SYBR Gold. This suggests that the products of the RCA reaction using the triazole-modified template contain a significantly lower quantity of both single- and double-stranded DNA.

[00139] Probing the nature and quantity of the RCA products via a fluorometric plate-reading assay. In order to corroborate the results from the gel-imaging we used a fluorometric plate-reading assay to allow a semi-quantitative comparison of the rates and total DNA yields of RCA reactions performed using f-29 polymerase and the cyclic templates 1_P0₄, 1_Tz, 1_PA and 1_Am over a 10 hour timecourse. After the desired time period the reactions were stopped by heat inactivation of the polymerase and then analysed, using three different DNA-binding dyes to provide a fluorescence signal: SYBR gold, SYBR Green I and SYBR Green II. The intensities of the measured fluorescence signals are

approximately proportional to the total concentrations of DNA in each reaction mixture, with some expected deviations as a result of the differing proportions of single- and double- stranded DNA amplification products. In agreement with the gel-imaging results, the reaction rates and product yields for the RCA reactions with the cyclic phosphoramidate-containing template 1_PA are essentially the same as those for the unmodified cyclic template 1_P0₄, and the cyclic amide template 1_Am performs almost as well. The cyclic triazole template 1_Tz is shown to be inferior: at each time-point the measured fluorescence intensities are weaker for the RCA reaction involving the triazole-containing template 1_Tz than for those involving the other three templates, indicating that a lower quantity of DNA is produced when 1_Tz is used as a substrate. By taking into account the differing selectivities of the three DNA binding dyes for double-stranded DNA over single-stranded DNA it also possible to draw inferences about the nature of the products. While all three dyes are known to bind both double- and single-stranded DNA to some extent, SYBR Green I has the most pronounced selectivity for double-stranded DNA over single-stranded DNA.^56-58 The measured fluorescence intensities for the samples containing the presence of the SYBR Gold and SYBR Green II dyes (Figure 22). Hence there is a strong indication that a significant amount of double-stranded DNA is produced in the RCA reaction on the triazole- containing template, which is consistent with the prior gel-imaging results. The same phenomenon is observed for the amide-modified template 1_Am, but to a much smaller extent. This is probably because each time the polymerase encounters the modified linkage in the cyclic template, it can choose between processively reading through the modified linkage or dissociating from the cyclic template and instead copying the RCA product as it emerges, thereby producing double-stranded products (Figure 20).⁵⁵ Since the triazole linkages are seemingly more difficult/slower to read through, it is likely that the alternative mechanism is more strongly favoured in their presence, resulting in the formation of a higher proportion of double-stranded products which would be of shorter length. This process may also sequester the cyclic template, contributing to the lower overall yield of DNA product.

[00140] RCA using the Bst 2.0 DNA polymerase. In light of the disappointing performances of the triazole-containing templates in the f-29-mediated RCA reactions, we also investigated RCA using the Bst 2.0 DNA polymerase for two of the cyclic triazole templates 1_Tz and 4_Tz and their unmodified analogues 1_P0₄ and 4_P0₄, in order to ascertain whether the triazole linkage can be read through more efficiently by this alternative polymerase. The Bst 2.0 enzyme was previously used for RCA of the cyclic template

4_P0₄ ³⁹ and is known to efficiently incorporate modified dNTPs during RCA but its ability to read through modified backbone linkages has not yet been thoroughly tested.⁵⁹'⁶⁰ Agarose electrophoretic analysis of the RCA products indicated that, while the f-29 enzyme again exhibited higher RCA efficiencies with the unmodified templates 1_P0₄ and 4_P0₄ compared to the triazole-containing analogues 1_Tz and 4_Tz, Bst 2.0 was able to process the triazole-modified templates more efficiently (Figure 23). For example, reactions performed using the Bst 2.0 enzyme produced similar yields and product distributions for the triazole-modified and unmodified cyclic templates 1_Tz and 1_P0₄, which are based on a randomly generated sequence (Figure 23B). Moreover, for the nanoclew template sequences 4_Tz and 4_P0₄ the Bst 2.0 DNA enzyme appeared to generate a significantly greater quantity of very long, slow-migrating RCA products from the triazole-containing template 4_Tz than from the unmodified control 4_P0₄. Although unexpected, this result exemplifies the utility of having an available choice of chemical cyclisation methods in cases where the cyclisation and/or amplification of a specific canonical sequence proves to be inefficient. The RCA products from the reactions using the Bst 2.0 polymerase were also quantified via a fluorometric plate-reading assay, which corroborated the gel-imaging results. However it is notable that in all four reactions the Bst 2.0 enzyme generated a higher proportion of double-stranded products than the f-29 enzyme (Figure 24A and 24B)— reflecting the Bst enzyme's inferior processivity and strand displacement activity under the conditions used in our RCA experiments.

Nanoflower formation

[00141] Long RCA products are known to spontaneously self-assemble into densely- packed DNA-inorganic hybrid nanoflower (DNA- NF) structures in the presence of a magnesium pyrophosphate co-precipitant, which is also generated as a by-product of the RCA reaction. The DNA-NF structures are thought to comprise an inorganic magnesium pyrophosphate core onto which the long single-stranded DNA RCA products are adsorbed. These nanoflower structures have several attractive features which are driving their investigation as potential diagnostic and therapeutic agents: they are easy to prepare; they are size-tuneable and biodegradable, yet resistant to digestion by exonuclease enzymes; they can be designed to incorporate a functional aptamer, DNAzyme, restriction enzyme, anti-sense or drug-loading sequence for targeted recognition and delivery; and they have been shown to be capable of cellular transfection.^23,36-41 Scanning electron microscopy (SEM) experiments were used to map the size and morphology of the nanoflowers formed at different time points during the RCA reactions performed using the unmodified and modified cyclic templates. After time periods of eight and twenty hours aliquots of the reactions were removed and heated to inactivate the enzyme. The precipitated nanoflowers were then collected by centrifugation, thoroughly washed with deionised water and subjected to SEM imaging experiments. The extent of DNA loading was also examined by agarose gel electrophoresis (Figures 61 -62). The SEM images (Figures 25-26) indicated that there was no significant difference in the size and morphology of the DNA nanoflowers generated from the four types of cyclic template, even though the triazole containing template gives less efficient RCA. In all cases DNA-NF structures with diameters of > 1 pm and petal-like surface morphologies were formed in the RCA reaction mixture within eight hours, and their sizes were observed to have marginally increase after 20 hours. Variation of the Mg²⁺ concentration was found to have a significant≤10 mM the particles were found to contain negligible quantities of DNA; as the Mg²⁺ concentration was increased from 10-25 mM the extent of DNA loading steadily increased, with a concomitant reduction in the size of the particles from ~3 pm to -0.8 pm (Figures 26 and Figures 63-72). Although the reason for the contraction of the nanoparticles with increasing Mg²⁺ concentration is not immediately obvious, it is possible that this reflects an increased rate of nucleation at higher metal ion concentration, which might be expected to produce a higher number of particles with a smaller average diameter.

Conclusions

[00142] We have shown that single-stranded DNA templates can be efficiently cyclised using three different chemical ligation methods. The resulting cyclic constructs contain a single unnatural triazole, amide or phosphoramidate linkage at the point of ligation. These chemical cyclisation methods offer several advantages over the more limited enzymatic approach: they are cheaper and easier to carry out, allowing for the large-scale synthesis and purification of the cyclic oligonucleotide products; they can also be performed under a wider variety of conditions, including denaturing ones, and are therefore particularly useful for cyclising linear oligonucleotides which contain problematic secondary structure. The chemical cyclisation methods described here therefore provide a versatile expanded toolkit for the preparation of cyclic oligonucleotides, which have an expansive range of potential applications across molecular biology^61-65 and nanotechnology.^66-69 Here we investigated the use of the chemically modified cyclic products as templates for the production of long single-stranded DNA concatemers via the highly demanding rolling circle amplification reaction, an important technique which is currently attracting widespread interest. The cyclic templates containing a non-canonical phosphoramidate backbone linkage performed particularly well during RCA— with overall yields, product distributions and reaction rates which were indistinguishable from those of the analogous unmodified templates. In contrast, the triazole-modified templates were less well tolerated by the f-29 polymerase, showing a tendency to produce double-stranded DNA side-products and varying overall yields. The performance of the templates containing an amide modification falls in between those of the phosphoramidate and triazole linkages. Interestingly, for one of the cyclic template sequences, the Bst 2.0 polymerase enzyme was found to amplify the triazole-modified template with greater efficiency than the unmodified analogue, suggesting that different combinations of chemical ligation method and polymerase enzyme may find application where RCA proves challenging. Of all the cyclic templates investigated, the phosphoramidate-containing versions are the most straightforward to synthesise and could be produced in commercial DNA synthesis facilities from readily available precursors. We propose that phosphoramidate cyclisation is a practical, scalable and convenient method for the production of biocompatible cyclic DNA constructs for use in many applications, including RCA.

Supplemental Experimental Information

General experimental details

[00143] All chemicals were purchased from Sigma-Aldrich unless otherwise specified and used without further purification. T4 DNA ligase was purchased from Promega. NxGen Φ-29 DNA polymerase was purchased from Lucigen. Bst 2.0 DNA polymerase, T4 gene 32 protein, deoxynucleotides (dNTPs) solution mix and Quick-Load® Purple 1 kb DNA ladder were purchased from New England Biolabs (UK). Scanning electron microscopy (SEM) consumables were purchased from Agar scientific. NAP-10 and NAP-25 columns were purchased from G.E. Healthcare Life Sciences. 96-well black, polystyrene, microplates were purchased from Greiner Bio-one. SYBR™ Gold Nucleic Acid Gel Stain, SYBR™ Green I Nucleic Acid Gel Stain and SYBR™ Green II RNA Gel Stain were purchased from ThermoFisher. 5X Nucleic acid sample loading buffer, used for non-denaturing polyacrylamide gel electrophoresis experiments, was purchased from Bio-rad. All RCA reactions were performed using a Bio-rad T100™ thermocycler.

Oligonucleotide synthesis and purification

Table S1 : Sequences of oligonucleotides used in this study

a. Linear oligonucleotides used for cyclisation

Cyclic Linear oligooligonucleotide

Sequence (5'-3') SeqID No. nucleotide precursor

code code

^PTCCCAATTGGGTACGCAGTACCCACAGCAG SeqID 51

1_P0₄ ODN 1x

ATGTGACTGTGAATCGTGAC

^ZTCCCAATTGGGTACGCAGTACCCACAGCAG SeqID 52

1_Tz ODN 2x

ATGTGACTGTGAATCGTGAC^K ^MTC C AATTGG GTAC GC AGTAC CC AC AG C AG A SeqID 53

1_Am ODN 3x

TGTGACTGTGAATCGTGACT^X ^MTCCCAATTGGGTACGCAGTACCCACAGCAG SeqID 54

1_PA ODN 4x

ATGTGACTGTGAATCGTGAC^P ^PTGTCGTTTTACCCATGTGCTATAGCCACTAC SeqID 55

2_P0₄ ODN 5x

TGTCG I I I I ACCCATGTGCTATAGCCACTAC

^ZTGTCGTTTTACCCATGTGCTATAGCCACTAC SeqID 56

2_Tz ODN 6x

TGTCG I I I I ACCCATGTGCTATAGCCACTAC^K ^MTGTCGTTTTACCCATGTGCTATAGCCACTAC SeqID 57

2_Am ODN 7x

TGTCG I I I I ACCCATGTGCTATAGCCACTAT^X ^MTGTCGTTTTACCCATGTGCTATAGCCACTAC SeqID 58

2_PA ODN 8x

TGTCG I I I I ACCCATGTGCTATAGCCACTAC³

^PTTACCCCACACCGCTGCCCCCACACCGCTG SeqID 59

3_P0₄ ODN 9x

CCCCCACACCGCTGCCTTAC

^ZTTACCCCACACCGCTGCCCCCACACCGCTG SeqID 60

3_Tz ODN 10x

CCCCCACACCGCTGCCTTAC^K ^MTACCCCACACCGCTGCCCCCACACCGCTG SeqID 61

3_Am ODN 1 1x

CCCCCACACCGCTGCCTTACT^X ^MTTACCCCACACCGCTGCCCCCACACCGCT SeqID 62

3_PA ODN 12x

GCCCCCACACCGCTGCCTTAC^P ^PGTTAATATTATTCGACGGGCCTGCTCGAGC SeqID 63

4_P0₄ ODN 13x TCGAGCTTGCATCGTGCAGCCGAAGCTTGC

ACGCGTGCTATTAAT

^ZTTGCACGCGTGCTATTAATGTTAATATTATT SeqID 64

4_Tz ODN 14x CGACGGGCCTGCTCGAGCTCGAGCTTGCAT

CGTGCAGCCGAAG^MeC^K

P = P0₄, M = 5'-amine, X = 3'-carboxy, Z = 5'-azide, K

cytosine.

Splints and fluorescence probes used for cyclic templates

Oligonucleotide code Sequence (5'-3') Seq ID No.

ODN 15x (Cyclisation splint for 1_P0₄; SeqID 65

CAATTGGGAGTCACGATT RCA primer for 1_P0₄, 1_Tz and 1_PA)

ODN 16x (Cyclisation splint for 2_P0 ; SeqID 66

AAACGACAGTAGTGGC RCA primer for 2_P0 , 2_Tz and 2_PA)

ODN 17x (Cyclisation splint for 3_P0 ; SeqID 67

TGGGGTAAGTAAGGCA RCA primer for 3_P0 , 3_Tz and 3_PA)

ODN 18x (Cyclisation splint for 4_P0 ; SeqID 68

ATAATATTAAC ATTAATAG C A RCA primer for 4_P0 and 4_Tz)

ODN 19x (Cyclisation splint and RCA SeqID 69

CCAATTGGAAGTCACGAT

primer for 1_Am)

ODN 20x (Cyclisation splint and RCA SeqID 70

AAAACGACAATAGTGGCT

primer for 2_Am) ODN 21x (Cyclisation splint and RCA SeqID 71

TGTGGGGTAAGTAAGGCA

primer for 3_Am)

ODN 22x (Cyclisation splint for 2_PA) AAAACGACAGTAGTGGCT SeqID 72

ODN 23x (Cyclisation splint for 3_PA) GTGGGGTAAGTAAGGCAG SeqID 73

ODN 24x (fluorescence probe) AATTGGGTACGCAGTACC-Cy3 SeqID 74

ODN 25x (scrambled version of SeqID 75

ATGTCAGACCTCGAAGGT-Cy3

fluorescence probe ODN 24x)

General methods for synthesis and purification of linear oligonucleotides

[00144] Oligonucleotides were synthesised on an Applied Biosystems 394 automated DNA/RNA synthesiser using a standard 0.2 μηιοΙ or 1.0 μηιοΙ phosphoramidite cycle of acid- catalysed detritylation, coupling, capping and iodine oxidation. DNA phosphoramidites and reagents were purchased from Link Technologies, Applied Biosystems Ltd or Glen Research. Unless otherwise stated the syntheses were performed using 1000 A controlled pore glass (CPG) solid supports with a particle size of 110 μηι and nucleoside loading of 25- 40 μΓΤΐ/g, purchased from Link technologies. Except where stated, standard β-cyanoethyl phosphoramidite monomers (benzoyl-dA, isobutylryl-dG, benzoyl-dC and dT) were used throughout. During the synthesis of the 3'-carboxy functionalised oligonucleotides, acetyl-dC was used in place of benzoyl-dC. Terminal 5' amino dT modifications were added using the commercially available phosphoramidite monomer 5'-monomethoxytritylamino-2'- deoxythymidine,3'-[(2-cyanoethyl)-(/V,/\/-diisopropyl)]-phosphoramidite, purchased from Glen research. Terminal 5'-azide functionality was introduced by post-synthetic modification of 5'- iodo dT containing oligonucleotides. Terminal 3' phosphate modifications were incorporated using commercially available, pre-packed 3' phosphate SynBase™ CPG 1000/110 columns, purchased from Link technologies. Terminal 3' alkyne functionality was introduced using a 3' propargyl dC or 3' propargyl 5-methyl dC functionalised CPG resin, prepared as described previously. (1) The coupling time for addition of standard A, G, C and T monomers was set to 60 seconds. For addition of modified monomers the coupling time was extended to 600 seconds. Stepwise coupling efficiencies and overall yields were monitored using the instrument's in-built automated trityl cation conductivity measurement facility.

[00145] Except where stated the oligonucleotides were cleaved from the solid support and deprotected by exposure to concentrated aqueous ammonia solution at room temperature (RT) for 60 min followed by heating in a sealed glass vial at 55 °C for 5 hours. The aqueous solution of ammonia was then removed by evaporation prior to oligonucleotide purification. [00146] The oligonucleotides were purified by reverse-phase high performance liquid chromatography (RP-HPLC). All 5' amino functionalised oligonucleotides were purified on a Shimadzu LC-20AP HPLC system with a Waters XBridge OST Ci₈ column (particle size: 2.5 μηι; pore size: 100 A; column dimensions: 1.9 x 50 mm). The following elution buffers were used: buffer A: 0.1 M triethylammonium acetate^; buffer B: 0.1 M triethylammonium acetate(aq) containing 20% CH3CN; gradient 30-70% buffer B over 9 minutes. The flow rate was 15 mL/min. The elution of oligonucleotides was monitored by UV absorption at 260 nm. The pure fractions were combined, evaporated and de-salted using a NAP-25 column followed by a NAP-10 column. All other oligonucleotides were purified on a Gilson system using a Luna 10 μηι C8 100 A pore Phenomenex 10 x 250 mm column with a gradient of acetonitrile in triethylammonium bicarbonate (TEAB) increasing from 0% to 50% buffer B over 20 min with a flow rate of 4 mL/min (buffer A: 0.1 M TEAB, pH 7.5, buffer B: 0.1 M TEAB, pH 7.5 with 50% acetonitrile). The elution of oligonucleotides was monitored by UV absorption at 298 nm. After HPLC purification, the oligonucleotides were freeze dried and then re-dissolved in water without the need for further de-salting.

[00147] All oligonucleotides were characterised by negative-mode UPLC-mass spectrometry using either a Bruker micrOTOF™ II focus ESI-TOF mass spectrometer with an Acquity UPLC system, equipped with a Ethylene Bridged Hybrid (BEH) C18 column (Waters) or a Waters Xevo G2-XS QT of mass spectrometer with an Acquity UPLC system, equipped with an Acquity UPLC oligonucleotide BEH C18 column (particle size: 1.7 μηι; pore size: 130 A; column dimensions: 2.1 x 50 mm). Data were analysed using Waters MassLynx software or Waters UNIFI Scientific Information System software.

[00148] Further details of the syntheses of the chemically modified oligonucleotides are provided below.

[00149] Synthesis of 5 -phosphate oligonucleotides. Terminal 5' phosphate modifications were added using a commercially available 2-[2-(4,4'- dimethoxytrityloxy)ethylsulfonyl]ethyl-(2-cyanoethyl)-(/\/,/\/-diisopropyl)-phosphoramidite, purchased from Link technologies, using an extended coupling time of 600 seconds. The oligonucleotides were cleaved from the solid support, deprotected and purified as described above.

[00150] Synthesis of 3'-alkyne-5'-azide oligonucleotides. 5'-0-(4,4'- Dimethoxytrityl)-3'-0-propargyl-deoxycytidine or 5'-0-(4,4'-dimethoxytrityl)-3'-0-propargyl-5- methyldeoxycytidine on polystyrene solid support, prepared as previously described, (1) was packed into a twist column (Glen Research) and used to synthesise the required sequences in the 3'- to 5'-direction (standard phosphoramidite oligonucleotide synthesis) with 5'-iodo dT as the final nucleotide. The 5'-iodo dT modification was introduced using a commercially available 5'-iodo-2'-deoxythymidine, 3'-[(2-cyanoethyl)-(/V,/\/-diisopropyl)]-phosphoramidite, purchased from Glen research. To convert the 5'-iodo dT to 5'-azido dT, sodium azide (20 mg) was suspended in dry DMF (1 ml) and heated at 70 °C for 20 min followed by cooling down to RT. The supernatant was taken up into a 1 mL syringe, passed back and forth through the column periodically, and then left at 55 °C for 5 h. The column was washed with DMF (3 X 1 mL) followed by acetonitrile (3 X 1 mL) and dried by the passage of a stream of argon gas. The resultant 5'-azide oligonucleotide was cleaved from the solid support, deprotected and purified as described above.

[00151] Synthesis of 5'-amino-3'-phosphate oligonucleotides. Oligonucleotides were synthesised on commercially available, pre-packed 3' phosphate SynBase™ CPG 1000/1 10 columns, purchased from Link technologies. The terminal 5' amino dT modification was added using the commercially available phosphoramidite monomer 5'- monomethoxytritylamino-2'-deoxythymidine,3'-[(2-cyanoethyl)-(/\/,/\/-diisopropyl)]- phosphoramidite, purchased from Glen research, using an extended coupling time of 600 seconds. During addition of the final 5' amino dT nucleotide the acetic anhydride capping step was omitted from the synthesis cycle in order to avoid acetylation of the amino group. The oligonucleotide was cleaved from the solid support, deprotected and purified according to the general procedure described above.

[00152] Synthesis of 5'-amino-3'-carboxy oligonucleotides. In order to introduce the terminal 3' carboxylic acid functionality, oligonucleotides were synthesised on a 3' carboxy dT functionalised polystyrene resin, prepared as previously described. (2) During oligonucleotide synthesis the use of benzoyl-protected dC phosphoramidite was avoided in favour of acetyl-dC, because benzoyl dC is known to undergo a deamination side-reaction under the sodium hydroxide mediated deprotection conditions used in this procedure. The terminal 5' amino dT modification was added using the commercially available phosphoramidite monomer 5'-monomethoxytritylamino-2'-deoxythymidine,3'-[(2-cyanoethyl)- (A/,A/-diisopropyl)]-phosphoramidite, purchased from Glen research, using an extended coupling time of 600 seconds. Prior to the addition of this final nucleotide the resin was treated with a 20% v/v solution of di-isopropylethylamine in acetonitrile for 15 minutes, washed three times with acetonitrile and dried under a stream of argon. During addition of the final 5' amino dT nucleotide the acetic anhydride capping step was omitted from the synthesis cycle in order to avoid acetylation of the amino group. The resin was then treated with 0.5 mL of a solution 0.4 M of NaOH in MeOH:H₂0 4:1 v/v for six hours at RT. At this point 0.5 mL of a solution 0.4 M of NaOH in MeOH:H₂0 1 :4 v/v was added, to give a 0.4 M solution of NaOH in MeOH:H20 1 : 1 v/v. The mixture was allowed to stand at RT for a further 40 hours before being neutralized by addition of 1 mL of triethylammonium acetate buffer (1 M, pH 7.4), concentrated on a rotary evaporator and de-salted using a NAP-25 column. To ensure complete cleavage of all isobutyryl protecting groups, the product was taken up in concentrated aqueous ammonia and heated at 55 °C for 2-5 hours in a sealed glass vial. After concentration of the aqueous ammonia solution in vacuo the oligonucleotide was purified according to the general procedure described above.

Synthesis of cyclic oligonucleotides

[00153] Procedure for denaturing polyacrylamide gel electrophoresis (PAGE).

The DNA samples were mixed with an equal volume of formamide, or with a 20% volume of 5X Nucleic Acid Sample Loading Buffer from Bio-Rad (pH 8, 50 mM Tris-HCI, 25% glycerol, 5 mM ethylenediaminetetraacetic acid (EDTA), 0.2% bromophenol blue, 0.2% xylene cyanole FF) and 7 M urea. Samples denatured by heating at 95 °C for 5 min. The mixture was cooled quickly on ice, and then loaded on denaturing PAGE gel (acrylamide:bisacrylamide 19:1 , 40% acrylamide solution, 7 M urea, prepared in a 1X Tris- borate-EDTA (TBE) buffer (89 mM Tris; 89 mM boric acid; 2 mM EDTA)). The gel was run at either 20 W or 200 V for the desired time period (1-2.5 h) in 1X TBE buffer at RT. Gels were visualised using a G:Box gel documentation system (Syngene). The cyclised oligonucleotide bands were excised, crushed and soaked in MilliQ water (10-15 ml_) overnight at 37 °C with shaking. After filtration and evaporation of the MilliQ water, the cyclised oligonucleotides were desalted using NAP-25, followed by a NAP-10 column. The concentration of cyclised oligonucleotides was measured by using a UV-Vis spectrophotometer (Cary, model 50 Bio, Varian, Australia).

[00154] Procedure for non-denaturing (PAGE). The DNA samples were mixed with 5X Nucleic Acid Sample Loading Buffer from Bio-Rad (4: 1 v/v) (pH 8, 50 mM Tris-HCI, 25% glycerol, 5 mM EDTA, 0.2% bromophenol blue, 0.2% xylene cyanole FF) and loaded onto a non-denaturing PAGE gel (acrylamide:bisacrylamide 19:1 , 40% acrylamide solution, prepared in a 1X TBE buffer). The gel was run at a constant voltage of 150 V in 1X TBE buffer at RT for the desired time length. Gels were visualised using a G:Box gel documentation system (Syngene). The cyclised oligonucleotide bands were excised, crushed and soaked in MilliQ water (10-15 mL) overnight at 37 °C with shaking. After filtration and evaporation of the MilliQ water, the cyclised oligonucleotides were desalted using NAP-25, followed by a NAP-10 column. The concentration of cyclised oligonucleotides was measured by using a UV-Vis spectrophotometer (Cary, model 50 Bio, Varian, Australia).

[00155] T4 DNA ligase-mediated cyclisations to give the cyclic oligonucelotides 1_P0₄, 2_P0₄ and 3_P0₄. A 5'-phosphorylated linear oligonucloetide (ODN 1x, ODN 5x or ODN 9x; 2.4 nmol, 1 eq) and a templating splint oligonucleotide (ODN 15x, ODN 16x or ODN 17x; 7.2 nmol, 3 eq) were annealed in 3.9 mL of 1X T4 DNA ligase buffer (30 mM Tris- HCI, pH 7.8 @ 25 °C, 10 mM MgCI₂, 10 mM DTT, and 1 mM ATP) by heating to 95 °C for 5 min, followed by cooling to RT at a rate of 0.5 °C/min. T4 DNA ligase (100 μΙ_, 31Ι/μΙ_) was then added to the solution which was incubated at RT overnight. The sample was heated to 70 °C for 15 min to denature the enzyme. The volume of reaction mixture was then reduced to 2.5 ml_ using a CentriVap centrifugal concentrator at 55 °C followed by desalting using NAP-25 column. The cyclised oligonucleotides was purified by denaturing PAGE (1_P0₄: 15% PAGE, 2_P0₄: 8% PAGE and 3_P0₄: 12% PAGE) as described above.

[00156] Preparation of the cyclic oligonucleotide 4_P0₄ using T4 DNA ligase. The 5'-phosphorylated linear oligonucleotide ODN 13x (2.4 nmol, 1 eq) and the templating splint oligonucleotide ODN 18x (4.8 nmol, 2 eq) were annealed in 3.9 ml_ of 1X T4 DNA ligase buffer (30 mM Tris-HCI, pH 7.8 @ 25 °C, 10 mM MgCI₂, 10 mM DTT, and 1 mM ATP) by heating to 95 °C for 5 min, followed by cooling to RT at a rate of 0.5 °C/min. T4 DNA ligase (100 μΙ_, 31Ι/μΙ_) was then added and the solution was incubated at RT overnight. The sample was heated to 70 °C for 15 minutes to denature the enzyme. The volume of reaction mixture was reduced to 2.5 ml_ using a CentriVap centrifugal concentrator at 55 °C followed by desalting using NAP-25 column. The cyclised oligonucleotides was purified by 8% denaturing PAGE as described above.

[00157] Preparation of Cu' click catalyst solution. To prepare a solution of Cu' click catalyst, tris-hydroxypropyltriazole ligand (THPTA) (3.6 mg) was dissolved into a solution of sodium ascorbate (12 μηιοΙ in MilliQ water, 24 μΙ_) followed by degassing by bubbling with argon gas (~ 3 min). CuS0₄.5H20 (1.2 μηιοΙ in MilliQ water, 12 μΙ_) was added and the solution was degassed again.

[00158] Preparation of the cyclic oligonucleotides 1_Tz, 2_Tz and 3_Tz containing triazole linkages through CuAAC 'click' ligation. Cu' click catalyst solution (36 μΙ_, prepared as described above) was added to 2.364 ml_ of the linear 5'-azide-3'- propargyl oligonucleotide (ODN 2x, ODN 6x or ODN 10x; 12 nmol in 0.2 M NaCI_(aq),). The reaction mixture was kept at RT for 2.5 h after which reagents were removed by NAP-25 gel filtration. The volume of reaction mixture was concentrated using a CentriVap centrifugal concentrator at 55 °C. The cyclised DNA products were analysed and purified by denaturing PAGE (1_Tz: 15% PAGE, 2_Tz: 8% PAGE and 3_Tz: 12% PAGE) as described above.

[00159] Non-templated cyclisation to give the cyclic oligonucleotide 4_Tz containing a triazole linkage through CuAAC 'click' ligation under denaturing condition. 1.2 nmol of the linear 5'-azide-3'-propargyl oligonucleotide ODN 14x was first dissolved in 236 μΙ_ of the organic solvent/MilliQ water solution (9: 1 , 4:1 or 3:2 v/v of MilliQ water : formamide, DMSO or acetonitrile) and heated to 95 °C for 5 min followed by cooling rapidly on ice. A solution of Cu¹ click catalyst (3.6 μΙ_, prepared as described above) was added to the reaction, and the solution was kept at RT for 2.5 h after which reagents were removed by NAP-10 gel filtration. The volume of reaction mixture was concentrated using a CentriVap centrifugal concentrator at 55 °C. The cyclised DNA products were analysed and purified by 8% denaturing PAGE as described above.

[00160] Non-templated cyclisation to give the modified circular template 1_PA containing a phosphoramidate linkage. The linear 3'-phosphate/5'-amino dT oligonucleotide ODN 4x (10 nmol, 4 μΜ) was dissolved in 2.5 ml_ of 0.2 M HEPES buffer (pH 7.2). 1-(2-Hydroxyethyl)imidazole (0.25 mmol, 0.1 M) was added as a solid, followed by N- (3-dimethylaminopropyl)-/V'-ethylcarbodiimide hydrochloride (EDC HCI) (0.19 mmol, 0.075 M). The solution was allowed to stand at RT for 3 h. An additional portion of EDC HCI (0.19 mmol) was then added and the solution was left at RT for a further 13 h, before removal of the small molecule reagents and buffer by gel-filtration, using a NAP-25 column, followed by a NAP-10 column. The crude product was taken up in 1 mL of 0.1 M NaOH(_aq) and the solution was heated at 55 °C for 6 h, then cooled to RT and de-salted using a NAP- 25 column followed by a NAP-10 column. The crude product mixture was analysed and purified by non-denaturing 15% PAGE as described above.

[00161] Templated cyclisations to give the modified circular templates 2_PA and 3_PA containing a phosphoramidate linkage. An equimolar solution of the linear 3'- phosphate/5'-amino dT oligonucleotide (ODN 8x or ODN 12x; 4 μΜ) and the splint oligonucleotide (ODN 22x or ODN 23x; 4 μΜ) in 0.2 M HEPES buffer (pH 7.2) was heated to 95 °C for 5 min and then allowed to cool slowly to RT over approximately 2 h. 1-(2- Hydroxyethyl)imidazole and EDC HCI were added as solids in one portion to give a 0.2 M concentration of both reagents. The reaction mixture was allowed to stand at RT for 15 h. The reagents and buffer were then removed by gel filtration using a NAP-25 column followed by a NAP-10 column. The crude product mixture was taken up in 1 mL 0.1 M NaOH(_aq) and heated at 55 °C for 5 h. After cooling to RT the NaOH was removed using a NAP-25 column followed by a NAP-10 column and the product mixture was analysed and purified by denaturing PAGE (13% and 15% for 2_PA and 3_PA respectively) as described above.

[00162] Non-templated cyclisation to give the modified circular template 1_Am containing an amide linkage. The linear 3'-carboxy/5'-amino dT oligonucleotide ODN 3x (5 nmol) was dissolved in 1.0 mL of 0.125 M NaCI(_aq). /V-Hydroxysuccinimide (125 of a 25 mM solution in H₂0) and EDC HCI (125 μί of a 100 mM solution in H₂0) were added. The reaction mixture was allowed to stand at RT for 2 h, before removal of reagents and salt using a NAP-25 column. The product mixture was analysed and purified by non-denaturing 15% PAGE as described above.

[00163] Templated cyclisations to give the modified circular template 2_Am containing an amide linkage. An equimolar solution of the linear 3'-carboxy/5'-amino dT oligonucleotide ODN 7x (1 nmol) and the splint oligonucleotide ODN 20x (1 nmol) in 0.2 mL of 0.125 M NaCI(aq) was heated to 90 °C and allowed to cool slowly to RT over approximately 2 h. /V-Hydroxysuccinimide (25 μΙ_ of a 25 mM solution in H₂0) and EDC HCI (25 μΙ_ of a 100 mM solution in H2O) were added. The reaction mixture was allowed to stand at RT for 2 h before removal of reagents and salts using a NAP-25 column. The product mixture was analysed and purified by denaturing 13% PAGE as described above.

[00164] Templated cyclisation to give the modified circular template 3_Am containing an amide linkage. An attempt to prepare the cyclic template 3_Am under the conditions described above for the preparation for 2_Am was unsuccessful. In order to obtain the cyclic template 3_Am the procedure was modified as follows. An equimolar solution of the linear 3'-carboxy/5'-amino dT oligonucleotide ODN 1 1x (5 nmol) and the splint oligonucleotide ODN 21x (5 nmol) in 1.0 ml_ of 0.2 M HEPES buffer (pH 7.2) was heated to 90 °C and allowed to cool slowly to RT over approximately 2 h. /V-Hydroxysuccinimide (125 μΙ_ of a 25 mM solution in H₂0) and EDC HCI (125 μΙ_ of a 100 mM solution in H₂0) were added. The reaction mixture was allowed to stand at RT for 18 h, before removal of the reagent and buffer using a NAP-25 column. The product mixture was analysed and purified by denaturing 15% PAGE as described above. UPLC-MS analysis of the isolated product indicated that it contained inseparable impurities with masses 71 and 142 units higher than that of the correct product. Although the identity of these side-products is uncertain and still under investigation, they are tentatively assigned as either partially hydrolysed EDC adducts of the cyclic product, or incompletely deprotected analogues containing residual N- isobutyryl-functionalised guanine groups.

Table S2: Oligonucleotide mass data

Oligonucleotide Calc. Mass Found Mass

ODN 1x 15473 15474

ODN 2x 15457 15458

ODN 3x 15451 15450

ODN 4x 15474 15474

ODN 5x 18964 18966

ODN 6x 18948 18949

ODN 7x 18942 18942

ODN 8x 18965 18967

ODN 9x 15012 15014

ODN 10x 14997 14998

ODN 1 1x 14976 14975

ODN 12x 15014 15013

ODN 13x 23180 23182

ODN 14x 23180 23182

ODN 15x 5539 5540

ODN 16x 4939 4941 ODN 17x 5010 5012

ODN 18x 6420 6421

ODN 19x 5507 5507

ODN 20x 5540 5540

ODN 21x 5643 5643

ODN 22x 5557 5556

ODN 23x 5670 5670

ODN 24x 6030 6030

ODN 25x 6030 6031

Cyclic ODN 1 (1_P0₄) 15456 15458

Cyclic ODN 2 (1_Tz) 15457 15458

Cyclic ODN 3 (1_Am) 15433 15434

Cyclic ODN 4 (1_PA) 15457 15455

Cyclic ODN 5 (2_P0₄) 18947 18949

Cyclic ODN 6 (2_Tz) 18948 18950

Cyclic ODN 7 (2_Am) 18924 18924

Cyclic ODN 8 (2_PA) 18948 18949

Cyclic ODN 9 (3_P0₄) 14995 14996

Cyclic ODN 10 (3_Tz) 14997 14998

Cyclic ODN 1 1 (3_Am) 14958 14959

Cyclic ODN 12 (3_PA) 14997 14997

Cyclic ODN 13 (4_P0₄) 23163 23163

Cyclic ODN 14 (4_Tz) 23180 23180

Rolling circle amplification

General procedure for agarose gel electrophoresis

The RCA products were analysed using a 0.8% (w/v) agarose gel with the addition of 0.5X SYBR™ Gold (ThermoFisher). 7.5 μΙ_ of sample was mixed with 2.5 μΙ_ of 5X GoTaq® Green buffer (Promega) before loading onto the gel. The gel was run at RT (126 V) in 1X TBE buffer and imaged using a G:Box (Syngene).

General procedures for RCA reactions

Comparing the performance of the different templates and backbones in RCA catalysed using Φ-29 DNA polymerase. The following setup was used for each template described: The cyclic template (3 pmol) and primer (12 pmol) were dissolved in 11.2 μΙ_ of MilliQ water and 2 μΙ_ of MgS0₄ (100 mM) and 2 μΙ_ of 10X Φ-29 DNA polymerase buffer (500 mM Tris-HCI, pH 7.5 @ 25°C, 100 mM (NH₄)₂S0₄, 40 mM DTT, 100 mM MgCI₂) were added. The samples were annealed by heating the mixture to 95 °C for 5 min followed by cooling to RT at a rate of 0.5 °C/min. Φ-29 DNA polymerase (10,000 U/ ml_, 0.8 μΙ_) and dNTPs (10 mM, 4 μΙ_) were added to the above mixture. The samples were incubated at 30 °C for the time described in the manuscript. The enzyme was then inactivated by heating the samples at 65 °C for 10 min before cooling to 4 °C. The RCA reaction mixture was diluted 5 times with MilliQ water and analysed by 0.8% agarose gel electrophoresis before centrifugation.

Quantification of the RCA rate with Φ-29 DNA polymerase using fluorescent probes

RCA reactions were performed as described above for different lengths of time (0 h, 2 h, 4 h, 6 h, 8 h, 10 h, or 20 h) using 1_P0₄, 1_PA, 1_Am and 1_Tz and their corresponding primers. After heat inactivation of the enzyme, the samples were diluted 5 times with 10 mM EDTA in MilliQ water. 10 μΙ_ of each sample was mixed with a 10 μΙ_ solution composed of fluorescent probe (100 μΜ) and NaCI (1 M) and incubated at RT for 2 h. 9 μΙ_ of each sample was mixed with 3 μΙ_ of 5X GoTaq® Green buffer (Promega) and analysed by 0.8% agarose gel electrophoresis (cast without SYBR Gold). The gels were run at RT (100 V) and imaged using a G:Box gel documentation system (Syngene) with excitation at 520 nm under the Cy3 fluorescence channel. Afterwards, the gels were stained with SYBR Gold (1X) at RT for 15 min, and re-imaged with excitation at 302 nm.

RCA reaction catalysed using Φ-29 DNA polymerase with 10 mM Mg²⁺ and single stranded binding protein T4 gene 32 for cyclic templates (1_P04 and 1_PA)

The cyclic template (4.5 pmol) and primer (18 pmol) were dissolved in 19.5 μΙ_ MilliQ water and 3 μΙ_ of 10X Φ-29 DNA polymerase buffer was added. The solution was annealed by heating the mixture to 95 °C for 5 min followed by cooling to RT at a rate of 0.5 °C/min. Φ-29 DNA polymerase (10,000 U/ ml_, 1.2 μΙ_), dNTPs (10 mM, 6 μΙ_) and T4 gene 32 protein (10 mg/ ml_, 0.3 μΙ_) were added to the above mixture (The concentration of Mg²⁺ and T4 gene 32 protein were chosen based on previous literature (4)). The RCA reactions were incubated at 30 °C for 20 h. The enzyme was then inactivated by heating the samples at 65 °C for 10 min before cooling to 4 °C. The original RCA reaction mixture was diluted to 100 μΙ_ with MilliQ water and then analysed by 0.8% agarose gel electrophoresis.

Measurement of fluorescence intensity of the RCA products stained with SYBR Green I, SYBR Green II and SYBR Gold

RCA reactions were performed as described above. After heat inactivation of the polymerase enzyme the samples were diluted 500 times with a solution of 20 mM of EDTA in MilliQ water.100 μΙ_ of buffer (1X TBE, 0.1 % triton X-100, 1X SYBR Green I, 1X SYBR Green II or 1X SYBR Gold) was added into a 96-well plate followed by adding 5 μΙ_ of diluted RCA product. Before each measurement the 96-well plate was shaken three times 10 minutes apart (1 min, 300 RPM). The fluorescence intensity of the solution was then measured using a CLARIOstar microplate reader (BMG LABTECH, Ortenberg, Germany). Three independent readings were taken for each RCA sample. For SYBR Green I measurements the parameters used were Excitation: 492-8; Dichroic filter: 514.2; Emission: 526-8. For SYBR Green II measurements the parameters used were Excitation: 492-8; Dichroic filter: 514.2; Emission: 526-8. For SYBR Gold measurements the parameters used were Excitation: 495-8; Dichroic filter: 514.2; Emission: 537-15.

RCA reaction catalysed using Bst 2.0 DNA polymerase with 20 mM Mg²⁺

The cyclic template (3 pmol) and primer (12 pmol) were dissolved in 10 μΙ_ M ill i water and 3.6 μΙ_ of 100 mM MgS0₄ and 2 μΙ_ of 10X isothermal amplification buffer (200 mM Tris-HCI, pH 8.8 @ 25°C, 100 mM (NH4)₂S0₄, 500 mM KCI, 20 mM MgS04, 0.1 % Tween® 20) were added. The sample was annealed by heating the mixture to 95 °C for 5 min followed by cooling to RT at a rate of 0.5 °C/min. Bst 2.0 DNA polymerase (8000 U/ ml_, 0.4 μΙ_) and dNTPs (10 mM, 4 μΙ_) were then added. The RCA reactions were carried out at 61.5 °C for 20 h. The enzyme was then inactivated by heating the samples at 80 °C for 20 min before cooling to 4 °C. The original RCA reaction mixture was diluted 5 times by MilliQ water and then analysed by 0.8% agarose gel electrophoresis.

DNA nanoflower experiments

Using the cyclic templates to prepare DNA-nanof lowers (DNA-NFs)

A solution of cyclic template (3 pmol), primer (12 pmol), 2 μΙ_ MgS0₄ (100 mM aqueous solution) and 2 μΙ_ of 10X Φ-29 DNA polymerase buffer (500 mM Tris-HCI, pH 7.5 @ 25°C, 100 mM (NH₄)₂S0 , 40 mM DTT, 100 mM MgCI₂) in MilliQ water with a final volume of 15.2 μΙ_ was first annealed by heating the mixture to 95 °C for 5 min followed by cooling to RT at a rate of 0.5 °C/min. Φ-29 DNA polymerase (10,000 U/ ml_, 0.8 μΙ_) and dNTPs (10 mM, 4 μΙ_) were added to the above mixture and briefly vortexed. The samples were incubated at 30 °C for either 8 h or 20 h using a BioRad T100 thermocycler. The enzyme was then inactivated by heating the samples at 65 °C for 10 min before cooling to 4 °C. 80 μΙ_ of MilliQ water was added and the samples were centrifuged. The resultant precipitate was collected by centrifugation and washed 5 times with MilliQ water followed by resuspending in 20 μΙ_ of MilliQ water. 10 μΙ_ of each of the RCA precipitate suspensions were loaded onto silicon wafer chips and dried at 55 °C for 15 min. The samples were then coated with gold before imaging using a Zeiss Sigma 300 Field Emission Gun Scanning Electron Microscope. The resuspended precipitate solution was also analysed by agarose gel electrophoresis.

Comparing the effect of Mg²⁺ concentration on DNA loading of DNA-NFs

A solution of cyclic template (3 pmol), primer (12 pmol), MgS0₄ (either 0 μΙ_, 0.5 μΙ_, 1 μΙ_, 2 μΙ_ or 3 μΙ_ of a 100 mM aqueous solution) and 2 μΙ_ of 10X Φ-29 DNA polymerase buffer (500 mM Tris-HCI, pH 7.5 @ 25°C, 100 mM (NH₄)₂S0₄, 40 mM DTT, 100 mM MgCI₂) in MilliQ water with a final volume of 15.2 μΙ_ was first annealed by heating the mixture to 95 °C for 5 min followed by cooling to RT at a rate of 0.5 °C/min. Φ-29 DNA polymerase (10,000 U/ ml_, 0.8 μΙ_) and dNTPs (10 mM, 4 μΙ_) were added to the above mixture and briefly vortexed. The samples were incubated at 30 °C for 20 h using a BioRad T100 thermocycler. The enzyme was then inactivated by heating the samples at 65 °C for 10 min before cooling to 4 °C. The samples were centrifuged and the supernatant was carefully collected. The resultant precipitate was collected by centrifugation and washed 5 times with MilliQ water followed by resuspending in 20 μΙ_ of MilliQ water. 7.5 μΙ_ of the supernatant or the precipitate suspension were then analysed by 0.8% agarose gel electrophoresis.

Comparing the effect of Mg²⁺ concentration on DNA-NF size

[00165] RCA was performed as described in the 'comparing the effect of Mg²⁺ concentration on DNA loading of DNA-NFs' section above using cyclic templates 1_P0₄ and 1_PA. 10 μΙ_ of each of the RCA precipitate suspensions were loaded onto silicon wafer chips and dried at 55 °C for 15 min. The samples were then coated with gold before imaging using a Zeiss Sigma 300 Field Emission Gun Scanning Electron Microscope.

References to Supplementary Experimental Data

1. El-Sagheer, A.H., Sanzone, A. P., Gao, R., Tavassoli, A. and Brown, T. (2011) Biocompatible artificial DNA linker that is read through by DNA polymerases and is functional in Escherichia coli. Proc. Natl. Acad. Sci., 108, 11338-1 1343.

2. Shivalingam, A., Tyburn, A.E.S., El-Sagheer, A.H. and Brown, T. (2017) Molecular Requirements of High-Fidelity Replication-Competent DNA Backbones for Orthogonal Chemical Ligation. J. Am. Chem. Soc, 139, 1575-1583.

3. Zuker, M. (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res., 31 , 3406-3415.

4. Ducani, C, Bernardinelli, G. and Hogberg, B. (2014) Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA. Nucleic Acids Res., 42, 10596-10604.

[00166] While specific embodiments of the invention have been described for the purpose of reference and illustration, various modifications will be apparent to a person skilled in the art without departing from the scope of the invention as defined by the appended claims.

Claims

1. Use of an oligonucleotide or oligonucleotide analogue comprising one or more

phosphodiester backbone mimics of Formula III shown below:

Formula III

wherein:

R^3e is selected from hydrogen or (1-4C)alkyl;

m and mi are integers independently selected from 0 to 2; and

P2 is an integer selected from 0 to 1 ;

with the proviso that the sum of integers m, mi and P2 is equal to 0, 1 , 2, 3 or 4; in the synthesis of a gene.

2. Use of an oligonucleotide or oligonucleotide analogue comprising one or more

phosphodiester ba :

Formula III wherein:

R^3e is selected from hydrogen or (1-4C)alkyl;

m and mi are integers independently selected from 0 to 2; and

P2 is an integer selected from 0 to 1 ;

with the proviso that the sum of integers m, mi and P2 is equal to 0, 1 , 2, 3 or 4; as a template for amplification in a polymerase chain reaction (PCR).

3. Use of an oligonucleotide or oligonucleotide analogue comprising one or more

phosphodiester ba :

Formula III

wherein:

R^3e is selected from hydrogen or (1-4C)alkyl;

W2 is selected from O, S or NR^Z, wherein R^z is selected from hydrogen or (1- 4C)alkyl; m and mi are integers independently selected from 0 to 2; and

P2 is an integer selected from 0 to 1 ;

as a template in a DNA replication process.

Use of an oligonucleotide or oligonucleotide analogue comprising one or more phosphodiester backbone mimics of Formula III shown below:

Formula III

wherein:

R^3e is selected from hydrogen or (1-4C)alkyl;

m and mi are integers independently selected from 0 to 2; and

P2 is an integer selected from 0 to 1 ;

as a template in a transcription process to provide a corresponding RNA transcript, or as a template in a reverse transcription process to provide a corresponding DNA transcript.

Use of a RNA oligonucleotide or oligonucleotide analogue comprising one or more phosphodiester backbone mimics of Formula III shown below: ^3a j3b O

Formula III

wherein:

R^3e is selected from hydrogen or (1-4C)alkyl;

m and mi are integers independently selected from 0 to 2; and

P2 is an integer selected from 0 to 1 ;

with the proviso that the sum of integers m, mi and P2 is equal to 0, 1 , 2, 3 or 4; as template in a translation process to produce a corresponding protein or peptide.

6. Use of an oligonucleotide or oligonucleotide analogue comprising one or more

phosphodiester bac :

Formula III

wherein:

^ e and ^ f independently denote the points of attachment to the oligonucleotide or oligonucleotide analogue; R^3a, R^3b, R^3c and R^3d are independently selected from hydrogen or (1- 4C)alkyl, wherein each (1-4C)alkyl is optionally substituted with one or more NH₂, OH or SH;

R^3e is selected from hydrogen or (1-4C)alkyl;

m and mi are integers independently selected from 0 to 2; and

P2 is an integer selected from 0 to 1 ;

as:

(i) antisense DNA or RNA;

(ii) exon skipping DNA or RNA; or

(iii) interference RNA (e.g. siRNA).

7. The use according to any one of claims 1 to 6, wherein ^ e denotes the point of attachment to a 3' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue and ^ f denotes the point of attachment to a 5' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue.

8. The use according to any one of claims 1 to 7, wherein W2 is selected from O or NH.

9. The use according to any one of claims 1 to 8, wherein P2 is 0.

10. The use according to any one of claims 1 to 9, wherein R^3a, R^3b, R^3c and R^3d are

independently selected from hydrogen or (1-4C)alkyl.

1 1. The use according to any one of claims 1 to 10, wherein R^3e is hydrogen.

12. The use according to any one of claims 1 to 11 , wherein the sum of integers m, mi and P2 IS equal to 0, 1 or 2.

13. The use according to any one of claims 1 to 12, wherein the oligonucleotide or

oligonucleotide analogue comprises one or more phosphodiester backbone mimics of the formula:

wherein:

^ e denotes the point of attachment to a 3' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue; and

An oligonucleotide or oligonucleotide analogue comprising one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula III, shown below:

Formula III

wherein:

R^3e is selected from hydrogen or (1-4C)alkyl;

15. An oligonucleotide or oligonucleotide analogue according to claim 14, wherein W2 is selected from O or NH.

16. An oligonucleotide or oligonucleotide analogue according to any one of claims 14 or

15, wherein P2 is 0.

17. An oligonucleotide or oligonucleotide analogue according to any one of claims 14 to

16, wherein R^3a, R^3b, R^3c and R^3d are independently selected from hydrogen or (1- 4C)alkyl.

18. An oligonucleotide or oligonucleotide analogue according to any one of claims 14 to

17, wherein R^3e is hydrogen.

19. An oligonucleotide or oligonucleotide analogue according to any one of claims 14 to

18, wherein the sum of integers m, mi and P2 is equal to 0, 1 or 2.

20. An oligonucleotide or oligonucleotide analogue according to any one of claims 14 to

19, wherein the oligonucleotide or oligonucleotide analogue comprises one or more phosphodiester backbone mimics of the formula:

wherein:

21. A method for amplifying an oligonucleotide or oligonucleotide analogue sequence, the method comprising the steps of:

1) providing an oligonucleotide or oligonucleotide analogue as defined in any one of claims 14 to 20; and

2) carrying out a polymerase chain reaction (PCR) using the oligonucleotide or oligonucleotide analogue of step 1 as a template.

22. A method for replicating an oligonucleotide or oligonucleotide analogue sequence, the method comprising the steps of:

2) carrying out a replication reaction using the oligonucleotide or oligonucleotide analogue of step 1 as a template.

23. A method for producing a ribonucleic acid (RNA) sequence comprising the steps of:

2) transcribing the oligonucleotide or oligonucleotide analogue of step 1 to form a ribonucleic acid (RNA) transcript.

24. A method for producing a deoxyribonucleic acid (DNA) sequence comprising the steps of:

2) reverse-transcribing the oligonucleotide or oligonucleotide analogue of step 1 to form a complementary deoxyribonucleic acid (cDNA) sequence.

25. A method for preparing a protein or peptide comprising the steps of:

1) providing an oligonucleotide or oligonucleotide ananlogue as defined in any one of claims 14 to 20; or a RNA oligonucleotide prepared according to claim 23; and 2) translating the oligonucleotide or oligonucleotide analogue of step 1 to form the protein or peptide.

A process for preparing an oligonucleotide or oligonucleotide analogue as defined in claims 14 to 20, the process comprising reacting:

one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula E shown below:

Formula E

wherein:

m is an integer selected from 0 to 2;

with

Formula F

wherein:

^ v denotes the point of attachment to a 5' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue comprising a terminal functional group of Formula F; R^3c and R^3d are independently selected from hydrogen or (1-4C)alkyl, wherein each (1-4C)alkyl is optionally substituted with one or more NH2, OH or SH;

R^3e is selected from and hydrogen or (1-4C)alkyl;

mi is an integer selected from 0 to 2; and

P2 is an integer selected from 0 or 1 ;

i) one or more peptide coupling reagents;

ii) one or more activating agents; and

iii) a catalyst.

27. A process according to claim 26, wherein W2 is selected from O or NH.

28. A process according to any one of claims 26 or 27, wherein P2 is 0.

29. A process according to any one of claims 26 to 28, wherein R^3a, R^3b, R^3c and R^3d are independently selected from hydrogen or (1-4C)alkyl.

30. A process according to any one of claims 26 to 29, wherein R^3e is hydrogen.

31. A process according to any one of claims 26 to 30, wherein the sum of integers m, mi and P2 is equal to 0, 1 or 2.

32. A process according to any one of claims 26 to 31 , wherein at least one of the

oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula E or at least one of the oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula F is attached to a solid support.

33. A process according to claim 32, wherein the solid support is selected from controlled pore glass (CPG), silica, hydroxylated methacrylic polymer beads (e.g. Toyopearl® beads), grafted copolymers comprising a crosslinked polystyrene matrix onto which polyethylene glycol is grafted (e.g. Tenagel®) or microporous polystyrene (MPPS).

34. A process according to any one of claims 26 to 33, wherein the reaction is carried out in the presence of one or more peptide coupling agents.

35. A process according to claim 34, wherein the one or more peptide coupling agents is selected from 1-[Bis(dimethylamino)methylene]-1 H-1 ,2,3-triazolo[4,5-b]pyridinium 3- oxid hexafluorophosphate (HATU), 2-(1 H-benzotriazol-1-yl)-1 , 1 ,3,3- tetramethyluronium hexafluorophosphate (HBTU), (Benzotriazol-1- yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazol-1-yl- oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), 4-(4,6-Dimethoxy-1 ,3,5- triazin-2-yl)-4-methylmorpholinium chloride (DMTMM), N-Ethoxycarbonyl-2-ethoxy-1 ,2- dihydroquinoline (EEDQ), Ν,Ν'-dicyclohexylcarbodiimide (DCC), Ν,Ν'- diisopropylcarbodiimide (DIC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), N-cyclohexyl-N'-isopropylcarbodiimide (CIC) or N.N'-dicyclopentylcarbodiimide (CPC).

36. A process according to any one of claims 26 to 35, wherein the reaction is carried out in the presence of an activating agent selected from hydroxybenzotriazole (HOBt), N- hydroxy 2-phenyl benzimidazole (HOBI), 1-hydroxy-7-azabenzotriazole (HOAt), 1-(2- hydroxyethyl)imidazole, N-hydroxysuccinimide (NHS), N-hydroxysulfosuccinimide (Sulfo-NHS), 4-dimethylaminopyridine (DMAP) or ethyl cyano(hydroxyimino)acetate (Oxyma Pure^®).