HK40003636B - Oligonucleotides comprising modified nucleosides - Google Patents

Description

Oligonucleotides containing modified nucleosides

Cross-references to related applications

This application claims priority to U.S. Provisional Application No. 62/357,623, filed July 1, 2016, and U.S. Provisional Application No. 62/437,592, filed December 21, 2016, each of which is incorporated herein by reference in its entirety for any purpose.

sequence list

This application contains a sequence list, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on June 15, 2017, and is named 01137-0020-00PCT_SL.txt with a size of 21,863 bits.

Technical Field

This disclosure generally relates to the field of oligonucleotides comprising modified nucleosides, such as aptamers capable of binding to target molecules. In some embodiments, the invention relates to oligonucleotides comprising more than one type of base-modified nucleosides, such as aptamers, and methods for preparing and using said aptamers.

Background of the Invention

Modified nucleosides have been used as therapeutic agents, diagnostic agents, and for incorporation into oligonucleotides to improve their properties (e.g., stability).

SELEX (Systematic Evolution of Ligands for Exponential Enrichment) is a method for identifying oligonucleotides (called aptamers) that selectively bind to target molecules. The SELEX method is described, for example, in U.S. Patent No. 5,270,163. The SELEX method involves selecting and identifying oligonucleotides from a random mixture of oligonucleotides to achieve any desired criteria of binding affinity and selectivity. By introducing specific types of modified nucleosides into the oligonucleotides identified during the SELEX method, nuclease stability, electrostatic charge, hydrophilicity, or lipophilicity can be altered to provide differences in the three-dimensional structure and target-binding ability of the oligonucleotides.

Summary of the Invention

In some embodiments, an aptamer is provided comprising at least one first 5-position modified pyrimidine and at least one second 5-position modified pyrimidine, wherein the first 5-position modified pyrimidine and the second 5-position modified pyrimidine are different. In some embodiments, the first 5-position modified pyrimidine is a 5-position modified uridine nucleoside and wherein the second 5-position modified pyrimidine is a 5-position modified cytosine nucleoside. In some embodiments, the first 5-position modified pyrimidine is a 5-position modified cytosine nucleoside and wherein the second 5-position modified pyrimidine is a 5-position modified uridine nucleoside. In some embodiments, the 5-position modified uridine nucleoside comprises a portion selected from a naphthyl moiety, a benzyl moiety, a tyrosine moiety, an indole moiety, and a morpholino moiety at the 5-position. In some embodiments, the 5-position modified cytosine nucleoside comprises a portion selected from a naphthyl moiety, a benzyl moiety, a tyrosine moiety, and a morpholino moiety at the 5-position. In some embodiments, the moiety is covalently linked to the 5-position of a base via a linker comprising a group selected from amide linkers, carbonyl linkers, propynyl linkers, alkyne linkers, ester linkers, urea linkers, carbamate linkers, guanidine linkers, amidine linkers, sulfoxide linkers, and sulfone linkers. In some embodiments, the 5-position modified cytosine nucleoside is selected from NapdC, 2NapdC, TyrdC, and PPdC. In some embodiments, the 5-position modified uracil nucleoside is selected from NapdU, 2NapdU, PPdU, MOEdU, TyrdU, TrpdU, and ThrdU. In some embodiments, the at least one first 5-position modified pyrimidine is NapdC, and the at least one second 5-position modified pyrimidine is selected from NapdU, 2NapdU, PPdU, MOEdU, TyrdU, and ThrdU. In some embodiments, the at least one first 5-position modified pyrimidine is PPdC and the at least one second 5-position modified pyrimidine is selected from NapdU, 2NapdU, PPdU, MOEdU, TyrdU, TrpdU, and ThrdU. In some embodiments, the at least one second 5-position modified pyrimidine is TyrdU. In some embodiments, the first and second 5-position modified pyrimidines can be incorporated by polymerase. In some embodiments, the first and second 5-position modified pyrimidines can be incorporated by KOD DNA polymerase.

In some embodiments, the aptamer binds to a target protein selected from PCSK9, PSMA, ErbB1, ErbB2, FXN, KDM2A, IGF1R, pIGF1R, α1-antitrypsin, CD99, MMP28, and PPIB.

In some embodiments, the aptamer includes a region at its 5' end that is at least 10, at least 15, at least 20, at least 25, or at least 30 nucleotides long, or 5 to 30, 10 to 30, 15 to 30, 5 to 20, or 10 to 20 nucleotides long, wherein the region at the 5' end of the aptamer lacks a 5-position modified pyrimidine. In some embodiments, the aptamer includes a region at its 3' end that is at least 10, at least 15, at least 20, at least 25, or at least 30 nucleotides long, or 5 to 30, 10 to 30, 15 to 30, 5 to 20, or 10 to 20 nucleotides long, wherein the region at the 3' end of the aptamer lacks a 5-position modified pyrimidine. In some embodiments, the aptamers are 20 to 100, or 20 to 90, or 20 to 80, or 20 to 70, or 20 to 60, or 20 to 50, or 30 to 100, or 30 to 90, or 30 to 80, or 30 to 70, or 30 to 60, or 30 to 50, or 40 to 100, or 40 to 90, or 40 to 80, or 40 to 70, or 40 to 60, or 40 to 50 nucleotides in length.

In some embodiments, the aptamer exhibits improved nuclease stability compared to aptamers having the same length and containing unmodified pyrimidines that substitute for each of the first 5-position modified pyrimidines and/or unmodified pyrimidines that substitute for each of the second 5-position modified pyrimidines. In some embodiments, the aptamer has a longer half-life in human serum compared to aptamers having the same length and containing unmodified pyrimidines that substitute for each of the first 5-position modified pyrimidines or unmodified pyrimidines that substitute for each of the second 5-position modified pyrimidines.

In some embodiments, a composition comprising multiple polynucleotides is provided, wherein each polynucleotide comprises at least one first 5-position modified pyrimidine and at least one second 5-position modified pyrimidine, wherein the first 5-position modified pyrimidine and the second 5-position modified pyrimidine are different. In some embodiments, each polynucleotide includes a fixed region at the 5' end of the polynucleotide. In some embodiments, the fixed region at the 5' end of each polynucleotide is at least 10, at least 15, at least 20, at least 25, or at least 30 nucleotides in length, or 5 to 30, 10 to 30, 15 to 30, 5 to 20, or 10 to 20 nucleotides in length. In some embodiments, each polynucleotide includes a fixed region at the 3' end of the polynucleotide. In some embodiments, the fixed region at the 3' end of the polynucleotide is at least 10, at least 15, at least 20, at least 25, or at least 30 nucleotides in length, or 5 to 30, 10 to 30, 15 to 30, 5 to 20, or 10 to 20 nucleotides in length. In some embodiments, the first 5-position modified pyrimidine is a 5-position modified uridine nucleoside, and wherein the second 5-position modified pyrimidine is a 5-position modified cytosine nucleoside. In some embodiments, the first 5-position modified pyrimidine is a 5-position modified cytosine nucleoside, and wherein the second 5-position modified pyrimidine is a 5-position modified uridine nucleoside. In some embodiments, the 5-position modified uridine nucleoside comprises a portion selected from naphthyl, benzyl, tyrosine acyl, tryptophan acyl, indole, and morpholino groups at the 5-position. In some embodiments, the 5-position modified cytosine nucleoside comprises a portion selected from naphthyl, benzyl, tyrosine acyl, and morpholino groups at the 5-position. In some embodiments, the portion is covalently linked to the 5-position of a base via a linker comprising a group selected from amide, carbonyl, propynyl, alkyne, ester, urea, carbamate, guanidine, amidine, sulfoxide, and sulfone links. In some embodiments, the 5-position modified cytosine nucleoside is selected from NapdC, 2NapdC, TyrdC, and PPdC. In some embodiments, the 5-position modified uracil nucleoside is selected from NapdU, PPdU, MOEdU, TyrdU, TrpdU, and ThrdU. In some embodiments, the at least one first 5-position modified pyrimidine is NapdC and the at least one second 5-position modified pyrimidine is selected from NapdU, 2NapdU, PPdU, MOEdU, TyrdU, TrpdU, and ThrdU. In some embodiments, the at least one first 5-position modified pyrimidine is PPdC and the at least one second 5-position modified pyrimidine is selected from NapdU, 2NapdU, PPdU, MOEdU, TrydU, TrpdU, and ThrdU. In some embodiments, the at least one second 5-position modified pyrimidine is TyrdU. In some embodiments, the first and second 5-position modified pyrimidines can be incorporated by polymerase. In some implementations, the first 5-position modified pyrimidine and the second 5-position modified pyrimidine can be incorporated by KOD DNA polymerase.

In some embodiments, each polynucleotide of the composition comprises a random region. In some embodiments, the random region is 20 to 100, or 20 to 90, or 20 to 80, or 20 to 70, or 20 to 60, or 20 to 50, or 20 to 40, or 30 to 100, or 30 to 90, or 30 to 70, or 30 to 60, or 30 to 50, or 30 to 40 nucleotides in length. In some implementations, each polynucleotide is 20 to 100, or 20 to 90, or 20 to 80, or 20 to 70, or 20 to 60, or 20 to 50, or 30 to 100, or 30 to 90, or 30 to 80, or 30 to 70, or 30 to 60, or 30 to 50, or 40 to 100, or 40 to 90, or 40 to 80, or 40 to 70, or 40 to 60, or 40 to 50 nucleotides in length.

In some embodiments, a composition is provided comprising a first aptamer, a second aptamer, and a target, wherein the first aptamer comprises at least one first 5-position modified pyrimidine and at least one second 5-position modified pyrimidine, wherein the second aptamer comprises at least one third 5-position modified pyrimidine, or wherein the second aptamer comprises at least one third 5-position modified pyrimidine and at least one fourth 5-position modified pyrimidine; wherein the first aptamer, the second aptamer, and the target are capable of forming a trimeric complex; and wherein the first 5-position modified pyrimidine and the second 5-position modified pyrimidine are different 5-position modified pyrimidines.

In some embodiments, the first 5-position modified pyrimidine is a 5-position modified uracil nucleoside, and wherein the second 5-position modified pyrimidine is a 5-position modified cytosine nucleoside. In some embodiments, the first 5-position modified pyrimidine is a 5-position modified cytosine nucleoside, and wherein the second 5-position modified pyrimidine is a 5-position modified uracil nucleoside. In some embodiments, the 5-position modified uracil nucleoside comprises a portion selected from naphthyl, benzyl, tyrosine, indole, and morpholino groups at the 5-position. In some embodiments, the 5-position modified cytosine nucleoside comprises a portion selected from naphthyl, benzyl, tyrosine, and morpholino groups at the 5-position. In some embodiments, the portion is covalently linked to the 5-position of a base via a linker comprising a group selected from amide, carbonyl, propynyl, alkyne, ester, urea, carbamate, guanidine, amidine, sulfoxide, and sulfone links. In some embodiments, the 5-position modified cytosine nucleoside is selected from NapdC, 2NapdC, TyrdC, and PPdC. In some embodiments, the 5-position modified cytosine nucleoside is selected from NapdU, 2NapdU, PPdU, MOEdU, TyrdU, TrpdU, and ThrdU. In some embodiments, the at least one first 5-position modified pyrimidine is NapdC and the at least one second 5-position modified pyrimidine is selected from NapdU, 2NapdU, PPdU, MOEdU, TyrdU, TrpdU, and ThrdU. In some embodiments, the at least one first 5-position modified pyrimidine is PPdC and the at least one second 5-position modified pyrimidine is selected from NapdU, 2NapdU, PPdU, MOEdU, TyrdU, TrpdU, and ThrdU. In some embodiments, the at least one second 5-position modified pyrimidine is TyrdU. In some embodiments, the first and second 5-position modified pyrimidines can be incorporated by polymerase. In some implementations, the first 5-position modified pyrimidine and the second 5-position modified pyrimidine can be incorporated by KOD DNA polymerase.

In some embodiments, the third 5-position modified pyrimidine is selected from 5-position modified cytosine nucleosides and 5-position modified pyrimidines. In some embodiments, the third 5-position modified pyrimidine and the fourth 5-position modified pyrimidine are different 5-position modified pyrimidines. In some embodiments, the third 5-position modified pyrimidine is a 5-position modified cytosine nucleoside and the fourth 5-position modified pyrimidine is a 5-position modified uridine nucleoside. In some embodiments, the third 5-position modified cytosine nucleoside is selected from BndC, PEdC, PPdC, NapdC, 2NapdC, NEdC, 2NEdC, and TyrdC. In some embodiments, the 5-position modified uridine nucleoside is selected from BNdU, NapdU, PEdU, IbdU, FBndU, 2NapdU, NEdU, MBndU, BFdU, BTdU, PPdU, MOEdU, TyrdU, TrpdU, and ThrdU.

In some implementations, the target is selected from proteins, peptides, carbohydrates, small molecules, cells, and tissues.

In some implementations, a method is provided that includes:

(a) To bring the aptamer, which can bind to the target molecule, into contact with the sample;

(b) Incubate the aptamer with the sample to allow aptamer-target complex formation;

(c) Enriching the aptamer-target complex in the sample, and

(c) Detecting the presence of the aptamer, the aptamer-target complex, or the target molecule, wherein the detection of the aptamer, the aptamer-target complex, or the target molecule indicates that the target molecule is present in the sample, and wherein the absence of the detection of the aptamer, the aptamer-target complex, or the target molecule indicates that the target molecule is not present in the sample;

The aptamer described herein is a dual-modified aptamer. In some embodiments, the method includes at least one additional step selected from: adding a competing molecule to the sample; capturing the aptamer-target complex on a solid support; and adding the competing molecule and diluting the sample; wherein the at least one additional step occurs after step (a) or step (b). In some embodiments, the competing molecule is selected from polyanionic competitive agents. In some embodiments, the polyanionic competitive agent is selected from oligonucleotides, dextran, DNA, heparin, and dNTPs. In some embodiments, the dextran is dextran sulfated; and the DNA is herring sperm DNA or salmon sperm DNA. In some embodiments, the target molecule is selected from proteins, peptides, carbohydrates, small molecules, cells, and tissues. In some embodiments, the sample is selected from whole blood, white blood cells, peripheral blood mononuclear cells, plasma, serum, saliva, respiration, urine, semen, saliva, meningeal fluid, amniotic fluid, glandular fluid, lymph, papillary aspirate, bronchial aspirate, synovial fluid, joint aspirate, cells, cell extracts, feces, tissue, tissue biopsy, and cerebrospinal fluid.

In some implementations, a method for detecting a target in a sample is provided, the method comprising:

a) Contact the sample with a first aptamer to form a mixture, wherein the first aptamer is capable of binding the target to form a first complex;

b) Incubate the mixture under conditions that allow the formation of the first complex;

c) Contact the mixture with a second aptamer, wherein the second aptamer is capable of binding the first complex to form a second complex;

d) Incubate the mixture under conditions that allow the formation of the second complex;

e) Detect the presence or absence of a first aptamer, a second aptamer, a target, a first complex, or a second complex in the mixture, wherein the presence of the first aptamer, the second aptamer, the target, the first complex, or the second complex indicates that the target is present in the sample;

The first aptamer comprises at least one first 5-position modified pyrimidine and at least one second 5-position modified pyrimidine;

The second aptamer comprises at least one third 5-position modified pyrimidine, or the second aptamer comprises at least one third 5-position modified pyrimidine and at least one fourth 5-position modified pyrimidine;

The first 5-position modified pyrimidine and the second 5-position modified pyrimidine are different 5-position modified pyrimidines.

In some embodiments, the first 5-position modified pyrimidine is a 5-position modified uracil nucleoside, and wherein the second 5-position modified pyrimidine is a 5-position modified cytosine nucleoside. In some embodiments, the first 5-position modified pyrimidine is a 5-position modified cytosine nucleoside, and wherein the second 5-position modified pyrimidine is a 5-position modified uracil nucleoside. In some embodiments, the 5-position modified uracil nucleoside comprises a portion selected from naphthyl, benzyl, tyrosine, indole, and morpholino groups at the 5-position. In some embodiments, the 5-position modified cytosine nucleoside comprises a portion selected from naphthyl, benzyl, tyrosine, and morpholino groups at the 5-position. In some embodiments, the portion is covalently linked to the 5-position of a base via a linker comprising a group selected from amide, carbonyl, propynyl, alkyne, ester, urea, carbamate, guanidine, amidine, sulfoxide, and sulfone links. In some embodiments, the 5-position modified cytosine nucleoside is selected from NapdC, 2NapdC, TyrdC, and PPdC. In some embodiments, the 5-position modified uracil nucleoside is selected from NapdU, 2NapdU, PPdU, MOEdU, TyrdU, TrpdU, and ThrdU. In some embodiments, the at least one first 5-position modified pyrimidine is NapdC and the at least one second 5-position modified pyrimidine is selected from NapdU, 2NapdU, PPdU, MOEdU, TyrdU, and ThrdU. In some embodiments, the at least one first 5-position modified pyrimidine is PPdC and the at least one second 5-position modified pyrimidine is selected from NapdU, 2NapdU, PPdU, MOEdU, TyrdU, TrpdU, and ThrdU. In some embodiments, the at least one second 5-position modified pyrimidine is TyrdU. In some embodiments, the first and second 5-position modified pyrimidines can be incorporated by polymerase. In some implementations, the first 5-position modified pyrimidine and the second 5-position modified pyrimidine can be incorporated by KOD DNA polymerase.

In some embodiments, the third 5-position modified pyrimidine is selected from 5-position modified cytosine nucleosides and 5-position modified pyrimidines. In some embodiments, the third 5-position modified pyrimidine and the fourth 5-position modified pyrimidine are different 5-position modified pyrimidines. In some embodiments, the third 5-position modified pyrimidine is a 5-position modified cytosine nucleoside and the fourth 5-position modified pyrimidine is a 5-position modified uridine nucleoside. In some embodiments, the third 5-position modified cytosine nucleoside is selected from BndC, PEdC, PPdC, NapdC, 2NapdC, NEdC, 2NEdC, and TyrdC. In some embodiments, the 5-position modified uridine nucleoside is selected from BNdU, NapdU, PedU, IbdU, FbndU, 2NapdU, NedU, MbndU, BfdU, BtdU, PpdU, MOEdU, TyrdU, TrpdU, and ThrdU.

In some embodiments, the target molecule is selected from proteins, peptides, carbohydrates, small molecules, cells, and tissues. In some embodiments, the first aptamer, the second aptamer, and the target can form a trimeric complex.

In some embodiments, a method is provided for identifying one or more aptamers capable of binding to a target molecule, the method comprising:

(a) Contacting a library of aptamers with the target molecule to form a mixture and allowing the formation of an aptamer-target complex, wherein the aptamer-target complex is formed when the aptamer has an affinity for the target molecule;

(b) Dispensing (or enriching) the aptamer-target complex from the remainder of the mixture;

(c) Dissociation of the aptamer-target complex; and

(d) Identify one or more aptamers capable of binding to the target molecule;

The aptamer library comprises multiple polynucleotides, each polynucleotide comprising at least one first 5-position modified pyrimidine and at least one second 5-position modified pyrimidine, wherein the first 5-position modified pyrimidine and the second 5-position modified pyrimidine are different 5-position modified pyrimidines. In some embodiments, steps (a), (b), and/or (c) are repeated at least once, twice, three times, four times, five times, six times, seven times, eight times, nine times, or ten times.

In some embodiments, the one or more aptamers capable of binding to the target molecule are amplified. In some embodiments, the mixture comprises a polyanionic competitive molecule. In some embodiments, the polyanionic competitive agent is selected from oligonucleotides, dextran, DNA, heparin, and dNTPs. In some embodiments, the dextran is sulfated dextran; and the DNA is herring sperm DNA or salmon sperm DNA.

In some implementations, the target molecule is selected from proteins, peptides, carbohydrates, small molecules, cells, and tissues.

In some embodiments, each polynucleotide includes a fixed region at the 5' end of the polynucleotide. In some embodiments, the fixed region at the 5' end of each polynucleotide is at least 10, at least 15, at least 20, at least 25, or at least 30 nucleotides in length, or 5 to 30, 10 to 30, 15 to 30, 5 to 20, or 10 to 20 nucleotides in length. In some embodiments, each polynucleotide includes a fixed region at the 3' end of the polynucleotide. In some embodiments, the fixed region at the 3' end of the polynucleotide is at least 10, at least 15, at least 20, at least 25, or at least 30 nucleotides in length, or 5 to 30, 10 to 30, 15 to 30, 5 to 20, or 10 to 20 nucleotides in length.

In some embodiments, the first 5-position modified pyrimidine is a 5-position modified uracil nucleoside, and wherein the second 5-position modified pyrimidine is a 5-position modified cytosine nucleoside. In some embodiments, the first 5-position modified pyrimidine is a 5-position modified cytosine nucleoside, and wherein the second 5-position modified pyrimidine is a 5-position modified uracil nucleoside. In some embodiments, the 5-position modified uracil nucleoside comprises a portion selected from naphthyl, benzyl, tyrosine, indole, and morpholino groups at the 5-position. In some embodiments, the 5-position modified cytosine nucleoside comprises a portion selected from naphthyl, benzyl, tyrosine, and morpholino groups at the 5-position. In some embodiments, the portion is covalently linked to the 5-position of a base via a linker comprising a group selected from amide, carbonyl, propynyl, alkyne, ester, urea, carbamate, guanidine, amidine, sulfoxide, and sulfone links. In some embodiments, the 5-position modified cytosine nucleoside is selected from NapdC, 2NapdC, TyrdC, and PPdC. In some embodiments, the 5-position modified uracil nucleoside is selected from NapdU, PPdU, MOEdU, TyrdU, TrpdU, and ThrdU. In some embodiments, the at least one first 5-position modified pyrimidine is NapdC and the at least one second 5-position modified pyrimidine is selected from NapdU, 2NapdU, PPdU, MOEdU, TrydU, TrpdU, and ThrdU. In some embodiments, the at least one first 5-position modified pyrimidine is PPdC and the at least one second 5-position modified pyrimidine is selected from NapdU, 2NapdU, PPdU, MOEdU, TyrdU, TrpdU, and ThrdU. In some embodiments, the at least one second 5-position modified pyrimidine is TyrdU. In some embodiments, the first and second 5-position modified pyrimidines can be incorporated by polymerase. In some implementations, the first 5-position modified pyrimidine and the second 5-position modified pyrimidine can be incorporated by KOD DNA polymerase.

In some embodiments, each polynucleotide includes a random region. In some embodiments, the random region is 20 to 100, or 20 to 90, or 20 to 80, or 20 to 70, or 20 to 60, or 20 to 50, or 20 to 40, or 30 to 100, or 30 to 90, or 30 to 70, or 30 to 60, or 30 to 50, or 30 to 40 nucleotides in length. In some implementations, each polynucleotide is 20 to 100, or 20 to 90, or 20 to 80, or 20 to 70, or 20 to 60, or 20 to 50, or 30 to 100, or 30 to 90, or 30 to 80, or 30 to 70, or 30 to 60, or 30 to 50, or 40 to 100, or 40 to 90, or 40 to 80, or 40 to 70, or 40 to 60, or 40 to 50 nucleotides in length.

In some implementations, each polynucleotide is an aptamer that binds to a target, and the library contains at least 1,000 aptamers, each containing a different nucleotide sequence.

In some embodiments, an aptamer binding to the PCSK9 protein is provided. In some embodiments, the aptamer comprises the sequence 5'-yGpppG-3', where each y is TyrdU and each p is NapdC. In some embodiments, the aptamer further comprises the sequence 5'-yEAyGA _n pAp-3', where E is selected from y, A, and G; and n is 0 or 1. In some embodiments, n is 0. In some embodiments, the sequence 5'-yEAyGA _n pAp-3' is located at the 5' of the sequence 5'-yGpppG-3'. In some embodiments, E is y.

In some embodiments, an aptamer binding to PCSK9 is provided, wherein the aptamer comprises the sequence 5'-F _n pppAAGRJrpRppW _m -3' (SEQ ID NO: 81), wherein F is selected from r and G; each R is independently selected from G and A; J is selected from r and A; W is selected from r, G, and A; n is 0 or 1; m is 0 or 1; r is PpdC; and p is NapdU. In some embodiments, m is 1. In some embodiments, F is r. In some embodiments, J is r. In some embodiments, W is G.

In some implementations, an aptamer that binds to PCSK9 is provided, wherein the aptamer comprises the sequence 5’-TTppGGpp-3’, wherein each p is NapdC.

In some implementations, the aptamer binding to PCSK9 is 20 to 100, or 20 to 90, or 20 to 80, or 20 to 70, or 20 to 60, or 20 to 50, or 30 to 100, or 30 to 90, or 30 to 80, or 30 to 70, or 30 to 60, or 30 to 50, or 40 to 100, or 40 to 90, or 40 to 80, or 40 to 70, or 40 to 60, or 40 to 50 nucleotides in length.

In some embodiments, the aptamer inhibits PCSK9 binding to LDL-R. In some embodiments, the aptamer inhibits PCSK9 binding to LDL-R, wherein the IC ₅₀ is less than 30 nM, less than 20 nM, or less than 15 nM.

In some embodiments, a method for lowering cholesterol in a subject is provided, the method comprising administering a PCSK9-binding aptamer to the subject in need. In some embodiments, the PCSK9-binding aptamer is the aptamer provided herein. In some embodiments, the cholesterol is low-density lipoprotein (LDL) cholesterol (LDL-C). In some embodiments, the subject has heterozygous familial hypercholesterolemia or clinical atherosclerotic cardiovascular disease (CVD).

The foregoing contents and other objectives, features and advantages of the present invention will become more apparent from the following embodiments, which are carried out with reference to the accompanying drawings.

Brief description of the attached diagram

Figure 1. Selection of nucleic acid aptamers modified with DNA libraries containing C5-modified uracil and cytosine triphosphates. Schematic diagram of selection with two modified bases. An overview of the selection method for multiple modified and unmodified libraries via primer extension reaction using a master-antisense biotinylated template library synthesized using 30N randomized chemical synthesis.

Figure 2. Binding affinity of the 40-mer (30N+5+5) aptamers to PCSK9 generated using multiple modified libraries. Aptamers with an affinity ≥1 nM are highlighted in gray shading, and aptamers showing an affinity of 320 nM indicate no detectable binding at the highest concentration of 32 nM on the binding curve. The black line on each library indicates the median for all aptamers in that library.

Figure 3. Affinity and copy number percentage of dC with single-modification dU or dC compared to dC with two-modification dU. Each point represents one of the aptamers with an affinity value shown on the Y-axis and its copy number percentage on the X-axis. Red dots represent single-modification aptamers, and green dots (hollow and filled) represent two-modification aptamers. Filled green dots represent some Nap-dC/Tyr-dU and PP-dC/Tyr-dU aptamers.

Figures 4A-4B. Truncation of aptamers with single and two base modifications. (A) All high-affinity 40-mer sequences were further truncated to 30-mers of their random region length, with only 5 nucleotides removed from each of the 5' and 3' ends. The percentage of aptamers that maintained or had improved binding affinity for PCSK9 is plotted on the Y-axis. (B) Comparison of affinity and truncation of aptamers with single and two base modifications from each individual library. Aptamers with ≥1 nM affinity are highlighted in gray shading, with the highest average affinity being for aptamers with two base combinations of modifications: PP-dC and PP-dU, Nap-dU, and Tyr-dU.

Figure 5. Target binding specificity of three high-affinity aptamers from various libraries for other protoprotein convertases (PCs). Solution affinity measurements were performed on a total of 33 aptamers (40-mers), of which 11 aptamers had a single modified base (i.e., three aptamers had Nap-dC/dT; three aptamers had dC/Nap-dU; three aptamers had dC/Pp-dU and two aptamers had dC/Ty-dU) and 22 aptamers had a double modified base (i.e., three aptamers had Nap-dC/Nap-dU; three aptamers had Nap-dC/Pp-dU; three aptamers had Nap-dC/Moe-dU; three aptamers had Nap-dC/Tyr-dU; three aptamers had Pp-dC/Pp-dU; three aptamers had Pp-dC/Nap-dU; three aptamers had Pp-Ty-dU and one aptamer had Pp-dC/Moe-dU). The aptamer below the dashed line at the 100 nM affinity indicates no detectable binding at a concentration of 100 nM. Affinity with the remaining PCs (PCSK5, PCSK6, and PCSK8) was not tested.

Figure 6. Species cross-reactivity of aptamers with single and two base modifications. Affinity of truncated 30-mer aptamers (K _{_d} values ≤ 1 nM) with PCSK9 from humans, monkeys, mice, and rats with single modifications (three aptamers) and two modifications (38 aptamers). The single-modified aptamers bind to human and monkey PSSK9, but not to mouse or rat PSKC9. In contrast, the two-modified aptamers bind to human, monkey, mouse, and rat proteins. Percentage of PCSK9 protein identity from each species is provided relative to human PSSK9.

Figures 7A-7C. Sandwich pair screening in bead-based analysis. (A) Schematic diagram of aptamer sandwich pair screening. (B) Sandwich pairs showing a signal greater than or equal to 50-fold at 10 nM PCSK9 concentration compared to those without protein in the buffer. All aptamers tested in the sandwich analysis were 40-mers with Kd ≤ 1 nM. A total of 70 pairs showed a signal ≥ 50-fold. Three sandwich pairs (3 sandwich aptamer pairs/3 single-base modification libraries) were identified when each aptamer in each pair was selected from a single-modification library. In contrast, when one aptamer of each pair is selected from three single-base modified libraries and the other aptamer of the pair is selected from four double-base modified libraries, 22 sandwich pairs are identified (i.e., 22 sandwich aptamer pairs / 3 single-base modified libraries and 4 double-base modified libraries), and when both aptamers of each pair are selected from double-base modified libraries, 45 sandwich pairs are identified (45 sandwich aptamer pairs / 5 double-base modified libraries). (C) Comparison of the number of sandwich pairs for the target protein PCSK9 derived from capture aptamer libraries with single-base modified aptamers and detection aptamer libraries with single-base modified aptamers; capture aptamer libraries with single-base modified aptamers and detection aptamer libraries with two-base modified aptamers; and capture aptamer libraries with two-base modified aptamers and detection aptamer libraries with two-base modified aptamers.

Figures 8A-8D. Sandwich pairs showing PCSK9 concentration-dependent signals in bead-based analysis. (A) Concentration-dependent signals observed with the best-performing capture or primary aptamer paired with the selected secondary or detection aptamer. (B) Concentration-dependent signals observed with the best-performing secondary or detection aptamer paired with the selected primary or capture aptamer. (C) The main sandwich pairs dC/PP-dU aptamer (primary) and Nap-dC/Nap-dU aptamer (secondary), showing signals when the orientation of the aptamers is reversed. (D) Standard curves obtained with recombinant wild-type PCSK9 and gain-of-function mutant PCSK9D374Y. Linear concentration-dependent signals were obtained using sandwich pairs detecting wild-type PCSK9 (circle) and gain-of-function mutant PCSK9D374Y (triangle) proteins.

Figures 9A-9B. Sensitivity of sandwich assays: The efficacy of aptamer sandwich assays (dC/PP-dU aptamer (single) and Nap-dC/Nap-dU aptamer (secondary)) is shown in the detection limit of PCSK9 concentration in the buffer solution. (A) Linear concentration-dependent signal observed with an aptamer sandwich assay having a lower limit of quantitation of approximately 80 pg/mL (LLOQ). (B) Linear concentration-dependent signal observed with an aptamer sandwich assay having an upper limit of quantitation of approximately 10 ng/mL (ULOQ).

Figure 10. Dilution linearity of sandwich assays using dC/PP-dU aptamers (primary) and Nap-dC/Nap-dU aptamers (secondary).

Figure 11. The sandwich assay includes a single-base modified aptamer and two-base modified aptamers (dC/PP-dU aptamer (first-time) and Nap-dC/Nap-dU aptamer (second-time)).

Figure 12. Overexpression of PCSK9 in wild-type HepG2 cells.

Figure 13. Plate-based in vitro PCSK9 inhibition assay: Schematic diagram of PCSK9 inhibition using aptamers.

Figure 14. Suppression screening of aptamers modified with a single base or two bases.

Figures 15A-15B. Two base-modified aptamers with potential therapeutic cross-reactivity in species. (A) Affinity of the 30-base-modified (PP-dC/Nap-dU) rodent cross-reactive aptamer (11733-44, SEQ ID No: 44) with human PCSK9 (filled with blue circles), human gain-of-function mutant PCSK9D374Y (filled with red circles), rhesus monkey PCSK9 (filled with green squares), rat PCSK9 (filled with pink hexagons), mouse PCSK9 (filled with inverted triangles), and a mixed control aptamer (hollow black rhombus). (B) Two base-modified aptamers with potential therapeutic cross-reactivity in species show inhibition of the PCSK9-LDL-R interaction. The aptamer potentially inhibited PCSK9-LDL-R interaction with an _EC50 value of 2.1 nM (blue filled circles) and PCSK9D374Y with an _EC50 value of 3.6 nM (red filled triangles), while the mixed control aptamer showed no inhibition against wild-type PCSK9 (green filled squares) and the gain-of-function mutant PCSK9D374Y (hollow black squares).

Figure 16. Schematic diagram of LDL-uptake reversal assay.

Figure 17. Species-cross-reactive PP-dC/Nap-DU aptamers inhibit LDL-R degradation and increase LDL-R levels on the surface of HepG2 cells by blocking PCSK9-LDL-R interaction. The _EC50 value for LDL-uptake reversal was 13.5 nM with respect to active SOMA polymers (red circles), which was not observed with a mixed control with the same sequence (blue triangles).

Figure 18. Stability of single-modified and double-modified aptamers over time in 90% human serum. Modification patterns of single-C-5 modified or double-C-5 modified aptamers are provided by legend (e.g., X/Y, where X represents dC (unmodified nucleotide), NapdC (Nap), or PPdC (PP); and Y represents dU or dT (unmodified nucleotide, TyrdU (Tyr), NapdU (Nap), PPdU (PP), or MOEdU (MOE)).

Figures 19A-19C. Binding affinities of the 40-mer (30N+5+5) aptamer with ErbB2 (A), ErbB3 (B), and PSMA (C) generated using various modified libraries. Aptamers with an affinity ≥1 nM are highlighted in gray shading, and aptamers showing an affinity of 320 nM indicate no detectable binding at the highest concentration of 32 nM on the binding curve. The black line on each library indicates the median for all aptamers in that library.

Figure 20. Some exemplary 5-position modifications of uracil and cytosine that can be incorporated into the aptamer.

Figure 21. Certain exemplary modifications that may be present at the 5-position of uracil nucleosides. The chemical structure of the C-5 modification includes an exemplary amide bond connecting the modification to the 5-position of the uracil nucleoside. The 5-position moiety shown includes a benzyl moiety (e.g., Bn, PE, and PP), a naphthyl moiety (e.g., Nap, 2Nap, NE), a butyl moiety (e.g., iBu), a fluorobenzyl moiety (e.g., FBn), a tyrosinyl moiety (e.g., Tyr), a 3,4-methylenedioxybenzyl moiety (e.g., MBn), a morpholino moiety (e.g., MOE), a benzofuranyl moiety (e.g., BF), an indole moiety (e.g., Trp), and a hydroxypropyl moiety (e.g., Thr).

Figure 22. Certain exemplary modifications that may be present at the 5-position of the cytosine nucleoside. The chemical structure of the C-5 modification includes an exemplary amide bond connecting the modification to the 5-position of the cytosine nucleoside. The 5-position moiety shown includes a benzyl moiety (e.g., Bn, PE, and PP), a naphthyl moiety (e.g., Nap, 2Nap, NE, and 2NE), and a tyrosine acyl moiety (e.g., Tyr).

Implementation

Unless otherwise noted, technical terms are used as they are in their usual usage. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise clearly indicated herein, the singular terms “a/an” and “the” include multiple references. “Comprising A or B” means comprising A, or B, or A and B. It should be further understood that all base or amino acid dimensions given with respect to nucleic acids or polypeptides, and all molecular weight or molecular mass values, are approximate and provided for descriptive purposes.

Furthermore, the ranges provided herein should be understood as shorthand for all values within the stated range. For example, a range of 1 to 50 should be understood to include any number, combination of numbers, or subrange (and its fractions, unless otherwise clearly specified herein) that comes from groups of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. Unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the stated range and, where appropriate, its fractions (such as tenths and hundredths of an integer). Furthermore, unless otherwise indicated, any figures stated herein with respect to any physical characteristic (such as polymer subunits, dimensions, or thickness) should be understood as any integer included within the stated range. Unless otherwise indicated, as used herein, “about” or “consisting substantially of” means ±20% of the indicated range, value, or structure. As used herein, the terms “comprising” and “including” are open-ended and used synonymously.

Although similar or equivalent methods and materials described herein may be used in the practice or testing of this disclosure, suitable methods and materials are described below. All disclosures, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including the interpretation of terminology) shall prevail. Furthermore, the materials, methods, and examples described are illustrative only and are not intended to be restrictive.

As used herein, the term "nucleotide" refers to a ribonucleotide or deoxyribonucleotide, or a modified form thereof, and analogues thereof. Nucleotides include substances containing purines (e.g., adenine, hypoxanthine, guanine, and their derivatives and analogues) and pyrimidines (e.g., cytosine, uracil, thymine, and their derivatives and analogues). Unless otherwise specifically indicated, as used herein, the term "cytosine nucleoside" is generally used to refer to a ribonucleotide, deoxyribonucleotide, or modified ribonucleotide containing a cytosine base. The term "cytosine nucleoside" includes 2'-modified cytosine nucleosides, such as 2'-fluoro, 2'-methoxy, etc. Similarly, unless otherwise specifically indicated, the term "modified cytosine nucleoside" or a specific modified cytosine nucleoside also refers to a ribonucleotide, deoxyribonucleotide, or modified ribonucleotide (such as 2'-fluoro, 2'-methoxy, etc.) containing a modified cytosine base. Unless otherwise specifically indicated, the term "uracil nucleoside" is generally used to refer to a ribonucleotide, deoxyribonucleotide, or modified ribonucleotide containing a uracil base. The term "uracil nucleoside" includes 2'-modified uracil nucleosides, such as 2'-fluoro, 2'-methoxy, etc. Similarly, unless otherwise specifically indicated, the term "modified uracil nucleoside" or a specific modified uracil nucleoside also refers to a ribonucleotide, deoxyribonucleotide, or modified ribonucleotide (such as 2'-fluoro, 2'-methoxy, etc.) containing a modified uracil base.

As used herein, the terms "5-position modified cytosine nucleoside" or "C-5 modified cytosine nucleoside" refer to a cytosine nucleoside with a modification at the C-5 position, as shown, for example, in Figure 20. Non-limiting exemplary 5-position modified cytosine nucleosides include those shown in Figure 22. Non-limiting exemplary 5-position modified cytosine nucleosides include, but are not limited to, 5-(N-benzylmethylformamide)-2′-deoxycytosine nucleoside (referred to as "BndC" and shown in Figure 21); 5-(N-2-phenylethylformamide)-2′-deoxycytosine nucleoside (referred to as "PedC" and shown in Figure 21); 5-(N-3-phenylpropylformamide)-2′-deoxycytosine nucleoside (referred to as "PPdC" and shown in Figure 21); 5-(N-1-naphthylmethylformamide)-2′-deoxycytosine nucleoside (referred to as "NapdC" and shown in Figure 22). (In Figure 21); 5-(N-2-naphthylmethylformamide)-2′-deoxycytosine nucleoside (referred to as “2NapdC” and shown in Figure 21); 5-(N-1-naphthyl-2-ethylformamide)-2′-deoxycytosine nucleoside (referred to as “NEdC” and shown in Figure 21); 5-(N-2-naphthyl-2-ethylformamide)-2′-deoxycytosine nucleoside (referred to as “2NEdC” and shown in Figure 21); and 5-(N-tyrosinylformamide)-2′-deoxycytosine nucleoside (referred to as TyrdC and shown in Figure 21). In some embodiments, the C5-modified cytosine nucleoside (e.g., in its triphosphate form) can be incorporated into the oligonucleotide by a polymerase (e.g., KOD DNA polymerase).

The chemical modifications of C-5 modified cytosine nucleosides described herein can also be combined with 2′ sugar modifications (e.g., 2′-O-methyl or 2′-fluorine) alone or in any combination, modifications at exocyclic amines, and substitutions of 4-thiocytosine nucleosides and similar modifications.

As used herein, the terms "C-5 modified uracil nucleoside" or "5-position modified uracil nucleoside" refer to a uracil nucleoside with a modification at the C-5 position (typically deoxyuracil nucleoside), as shown, for example, in Figure 20. In some embodiments, the C5 modified uracil nucleoside (e.g., in its triphosphate form) can be incorporated into an oligonucleotide by a polymerase (e.g., KODDNA polymerase). Non-limiting exemplary 5-position modified uracil nucleosides include those shown in Figure 21. Non-limiting exemplary 5-position modified uracil nucleosides include:

5-(N-benzylformamide)-2′-deoxyuridine (BndU),

5-(N-phenylethylformamide)-2′-deoxyuridine (PEdU),

5-(N-Thiophenylmethylformamide)-2′-Deoxyuridine (ThdU),

5-(N-isobutylcarboxamide)-2′-deoxyuridine (iBudU),

5-(N-tyrosinylformamide)-2′-deoxyuridine (TyrdU),

5-(N-3,4-methylenedioxybenzylformamide)-2′-deoxyuridine (MBndU),

5-(N-4-fluorobenzylmethylformamide)-2′-deoxyuridine (FBndU),

5-(N-3-phenylpropylformamide)-2′-deoxyuridine (PPdU),

5-(N-Imidazolylethylformamide)-2′-Deoxyuridine (ImdU)

5-(N-chromocarbamate)-2′-deoxyuridine (TrpdU),

5-(N-R-threonylformamide)-2′-deoxyuridine (ThrdU),

5-(N-[1-(3-trimethylammonium)propyl]formamide)-2′-deoxyuridine chloride,

5-(N-naphthylmethylformamide)-2′-deoxyuridine (NapdU),

5-(N-[1-(2,3-dihydroxypropyl)]formamide)-2′-deoxyuridine),

5-(N-2-phenylmethylformamide)-2′-deoxyuridine (2NapdU),

5-(N-1-Naphthylethylformamide)-2′-Deoxyuridine (NEdU),

5-(N-2-naphthylethylformamide)-2′-deoxyuridine (2NEdU),

5-(N-3-benzofuranylethylformamide)-2′-deoxyuridine (BFdU),

5-(N-3-benzothiopheneylethylformamide)-2′-deoxyuridine (BTdU).

The chemical modifications of C-5 modified uracil nucleosides described herein can also be combined with 2′ sugar modifications (e.g., 2′-O-methyl or 2′-fluorine) alone or in any combination, modifications at exocyclic amines, and substitutions of 4-thiouracil nucleosides and similar modifications.

As used herein, the terms “modify,” “modified,” “modification,” and any variations thereof, when used with reference to oligonucleotides, mean that at least one of the four constitutive nucleotide bases of the oligonucleotide (i.e., A, G, T/U, and C) is an analog or ester of a naturally occurring nucleotide. In some embodiments, the modified nucleotide confers nuclease resistance to the oligonucleotide. Additional modifications may include backbone modifications, methylation, uncommon base pairing combinations (such as isobasisocytosine and isoguanidine), and similar modifications. Modifications may also include 3′ and 5′ modifications, such as capping. Other modifications may include replacing one or more naturally occurring nucleotides with analogs, internucleotide modifications such as those with non-electrolyzed bonds (e.g., methyl phosphonate, triphosphate, phosphoramide, carbamate, etc.) and those with charged bonds (e.g., thiophosphate, dithiophosphate, etc.), those with intercalating agents (e.g., acridine, psoralen, etc.), those containing chelating agents (e.g., metals, radioactive metals, boron, oxidizing metals, etc.), those containing alkylating agents, and those with modifying bonds (e.g., α-anomeric nucleic acids, etc.). Furthermore, any hydroxyl groups normally present on the sugars of nucleotides may be replaced by phosphonate or phosphate groups; protected by standard protecting groups; or activated to prepare additional nucleotides or additional bonds on solid supports. The 5′ and 3′ OH groups may be phosphorylated or replaced with an amine, an organic capping group having about 1 to about 20 carbon atoms, a polyethylene glycol (PEG) polymer in one embodiment ranging from about 10 to about 80 kDa, a PEG polymer in another embodiment ranging from about 20 to about 60 kDa, or other hydrophilic or hydrophobic bio or synthetic polymers.

As used herein, the terms “nucleic acid,” “oligonucleotide,” and “polynucleotide” are used interchangeably to indicate polymers of nucleotides and include DNA, RNA, DNA/RNA hybrids, and modifications of these kinds of nucleic acids, oligonucleotides, and polynucleotides, including the linkage of multiple entities or portions to nucleotide units at any position. The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid” include double-stranded or single-stranded molecules as well as triple-helical molecules. Nucleic acid, oligonucleotide, and polynucleotide are broader terms than the term aptamer, and therefore, the terms nucleic acid, oligonucleotide, and polynucleotide include polymers of nucleotides as aptamers, but are not limited to aptamers.

Polynucleotides may also contain similar forms of ribose or deoxyribose known in the art, including 2′-O-methyl, 2′-O-allyl, 2′-O-ethyl, 2′-O-propyl, 2′-O-CH2CH2OCH3, _2′ -fluorine, 2′- _NH2 or 2′-azido, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars (such as arabinose, xylose or lythose), piperanose, furanose, sedoheptulose, acyclic analogs, and non-basic nucleotide analogs (such as methylriboside). As noted herein, one or more phosphodiester bonds may be replaced by alternative linking groups. These alternative linking groups include embodiments in which _{the phosphate ester is replaced by P(O)S (“thioester”), P(S)S (“dithioester”), (O)NR X2} ⁽ “amide”), P(O) ^RX , P(O)OR ^X′ , CO, or _CH2 (“methylal”), wherein each ^RX or RX ^′ is independently H or optionally contains an ether (-O-) bond, aryl, alkenyl, cycloalkyl, cycloalkenyl, or aralkyl substituted or unsubstituted alkyl group (C1-C20). Not all bonds in the polynucleotide need to be identical. Similar substitutions of sugars, purines, and pyrimidines can facilitate the design of the final product, for example, as can alternative backbone structures (like polyamide backbones).

Polynucleotides can also contain similar forms of carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars (such as arabinose, xylose, or lysoose), piperanose, furanose, sedoheptulose, acyclic analogs, and non-basic nucleotide analogs (such as methylriboside).

If modifications to the nucleotide structure are present, they can be given before or after polymer assembly. The nucleotide sequence can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by binding to labeled components.

As used herein, the term "at least one nucleotide" when referring to the modification of a nucleic acid means one, several, or all of the nucleotides in the nucleic acid, indicating that any or all of any or all of the A, C, T, G, or U in the nucleic acid may be modified or unmodified.

As used herein, “nucleic acid ligand,” “aptamer,” “SOMA polymer,” and “clone” are used interchangeably to indicate a non-naturally present nucleic acid that has the desired effect on a target molecule. The desired effect includes, but is not limited to, binding to the target, catalytically altering the target, reacting with the target in a manner that modifies or alters the target or the target’s functional activity, covalently linking to the target (as in suicide inhibitors), and facilitating a reaction between the target and another molecule. In one embodiment, the effect is a specific binding affinity for a target molecule that is a three-dimensional chemical structure other than a polynucleotide that binds to an aptamer via a mechanism independent of Watson/Crick base pairing or triple helix formation, wherein the aptamer is not a nucleic acid with a known physiological function that the target molecule binds to. Aptamers for a given target include nucleic acids identified by a candidate mixture of nucleic acids, wherein the aptamer is a ligand for the target, the method comprising: (a) contacting the candidate mixture with the target, wherein nucleic acids having increased affinity for the target relative to other nucleic acids in the candidate mixture are dispensed from the remainder of the candidate mixture; (b) dispensing the affinity-increased nucleic acids from the remainder of the candidate mixture; and (c) amplifying the affinity-increased nucleic acids to generate a ligand-enriched mixture of nucleic acids, thereby identifying aptamers for the target molecule. It should be recognized that affinity interactions are a matter of degree; however, in this context, “specific binding affinity” of an aptamer to its target means that the aptamer generally binds to its target with a much higher degree of affinity than it binds to other, non-target components in the mixture or sample. An “aptamer,” “SOMA aggregate,” or “nucleic acid ligand” is a collection of copies of a type or class of nucleic acid molecules having a specific nucleotide sequence. An aptamer may include any suitable number of nucleotides. An “aptamer” refers to more than one such collection of molecules. Different aptamers may have the same or different numbers of nucleotides. Aptamers may be DNA or RNA and may be single-stranded, double-stranded, or contain double-stranded or triple-stranded regions. In some embodiments, aptamers are prepared using the SELEX method as described herein or known in the art.

As used herein, "SOMA polymer" or slow dissociation rate modified aptamer refers to an aptamer with improved dissociation rate characteristics. SOMA polymers can be generated using the improved SELEX method described in U.S. Patent No. 7,947,447 entitled "Method for Generating Aptamers with Improved Off-Rates".

As used herein, an aptamer comprising two different types of 5-position modified pyrimidines or C-5 modified pyrimidines may be referred to as a "dual-modified aptamer," an aptamer having "two modified bases," an aptamer having "two base modifications" or "two modified bases," or an aptamer having "dual-modified bases," and these terms are used interchangeably. The same terminology may also be used for aptamer libraries or aptamer libraries. Therefore, in some embodiments, the aptamer comprises two different 5-position modified pyrimidines, wherein the two different 5-position modified pyrimidines are selected from NapdC and NapdU, NapdC and PPdU, NapdC and MOEdU, NapdC and TyrdU, NapdC and ThrdU, PPdC and PPdU, PPdC and NapdU, PPdC and MOEdU, PPdC and TyrdU, PPdC and ThrdU, NapdC and 2NapdU, NapdC and T rpdU, 2NapdC and NapdU, and 2NapdC and 2NapdU, 2NapdC and PPdU, 2NapdC and TrpdU, 2NapdC and TyrdU, PPdC and 2NapdU, PPdC and TrpdU, PPdC and TyrdU, TyrdC and TyrdU, TrydC and 2NapdU, TyrdC and PPdU, TyrdC and TrpdU, TyrdC and TyrdU, and TyrdC and TyrdU. In some embodiments, the aptamer comprises at least one modified uracil nucleoside and/or thymidine nucleoside and at least one modified cytosine nucleoside, wherein the at least one modified uracil nucleoside and/or thymidine nucleoside is partially modified at the 5-position with a portion selected from naphthyl, benzyl, fluorobenzyl, tyrosinyl, indole, morpholino, isobutyl, 3,4-methylenedioxybenzyl, benzothiophene, and benzofuranyl moieties, and wherein the at least one modified cytosine nucleoside is partially modified at the 5-position with a portion selected from naphthyl, tyrosinyl, and benzyl moieties. In some embodiments, the portion is covalently linked to the 5-position of a base via a linker comprising a group selected from amide linkers, carbonyl linkers, propynyl linkers, alkyne linkers, ester linkers, urea linkers, carbamate linkers, guanidine linkers, amidine linkers, sulfoxide linkers, and sulfone linkers.

As used herein, an aptamer containing a single type of 5-position modified pyrimidine or C-5 modified pyrimidine may be referred to as a "single-modified aptamer," an aptamer with a "single-modified base," an aptamer with a "single-base modification," or an aptamer with a "single-base modification," and these terms are used interchangeably. The same terminology may also be used for aptamer libraries or aptamer collections. As used herein, "protein" is used synonymously with "peptide," "polypeptide," or "peptide fragment." A "purified" polypeptide, protein, peptide, or peptide fragment is substantially free of cellular material or other contaminating proteins derived from cells, tissues, or cell-free sources from which the amino acid sequence was obtained, or, when chemically synthesized, substantially free of chemical precursors or other chemicals.

In some embodiments, the aptamer comprises a first 5-position modified pyrimidine and a second 5-position modified pyrimidine, wherein the first 5-position modified pyrimidine comprises a tyrosine moiety at the 5-position, and the second 5-position modified pyrimidine comprises a naphthyl moiety or a benzyl moiety at the 5-position. In one related embodiment, the first 5-position modified pyrimidine is uracil. In one related embodiment, the second 5-position modified pyrimidine is cytosine. In one related embodiment, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the uracil in the aptamer is modified at the 5-position. In one related embodiment, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the cytosine of the aptamer is modified at position 5.

Modified nucleotides

In some embodiments, the present invention provides oligonucleotides, such as aptamers, comprising two different types of base-modified nucleotides. In some embodiments, the oligonucleotide comprises two different types of 5-position modified pyrimidines. In some embodiments, the oligonucleotide comprises at least one C5-modified cytosine nucleoside and at least one C5-modified uridine nucleoside. In some embodiments, the oligonucleotide comprises two different C5-modified cytosine nucleosides. In some embodiments, the oligonucleotide comprises two different C5-modified uridine nucleosides. Non-limiting exemplary C5-modified uridine nucleosides and cytosine nucleosides are shown, for example, in Formula I and Figure 20. Some non-limiting exemplary C5-modified uridine nucleosides are shown in Figure 21, and some non-limiting exemplary C5-modified cytosine nucleosides are shown in Figure 22.

In some embodiments, the oligonucleotide comprises at least one pyrimidine of formula I:

in

R is independently -(CH ₂ ) _n -, where n is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

R ^X1 independently selects from the following groups:

Wherein, ^* indicates the junction point between the ^RX1 group and the -( _CH2 ) _n- group; and

in,

^RX4 is independently selected from the group consisting of: branched or straight-chain lower alkyl groups (C1-C20); hydroxyl groups; halogens (F, Cl, Br, I); nitriles (CN); boric acids ( _BO₂H₂ ); carboxylic acids ( _COOH ); carboxylic acid esters ( ^COORX₂ ); primary amides ( _CONH₂ ); secondary amides ( ^CONHRX₂ ); ^tertiary amides ( ^{CONRX₂RX₃} ); sulfonamides ( _SO₂NH₂ ); _and N-alkylsulfonamides ( ^SONHRX₂ ).

^RX2 and ^RX3 , each time they appear, are independently selected from the group consisting of: branched or straight-chain lower alkyl groups (C1- _C20 ); phenyl groups ( _C6H5 ); ^RX4 -substituted benzene rings ( ^RX4C6H4 ), wherein _RX4 is defined above; carboxylic acids ( ^COOH ); carboxylic acid esters ( ^COORX5 ), wherein ^RX5 is a branched or straight _- chain lower alkyl group (C1-C20); and cycloalkyl groups, wherein ^RX2 and ^RX3 together form a substituted or unsubstituted 5- or 6-membered ring;

X is independently selected from _the group consisting of: -H, -OH, -OMe, -O _- allyl, -F, -OEt, -OPr, _-OCH2CH2OCH3 , _NH2 , and -azido;

R′ is independently selected from the following groups: -H, -OAc; -OBz; -P(NiPr ₂ )(OCH ₂ CH ₂ CN); and -OSiMe ₂ tBu;

R″ is independently selected from the group consisting of hydrogen, 4,4′-dimethoxytriphenylmethyl (DMT) and triphosphate (-P(O)(OH)-OP(O)(OH)-OP(O)(OH) ₂ ) or their salts;

Z is independently selected from the group consisting of -H, substituted or unsubstituted C(1-4) alkyl groups;

And its salt.

In some embodiments, the oligonucleotide comprises at least one modified pyrimidine as shown in FIG21, wherein each X is independently selected from -H, -OH, -OMe, -O-allyl, -F, -OEt, _-OPr , _-OCH2CH2OCH3 , _NH2 , and _-azido .

In some embodiments, the oligonucleotide comprises at least one modified pyrimidine as shown in FIG22, wherein each X is independently selected from -H, -OH, -OMe, -O-allyl, -F, -OEt, _-OPr , _-OCH2CH2OCH3 , NH2 _{and -azido} .

In some embodiments, the oligonucleotide comprises at least one modified pyrimidine as shown in FIG. 21 and at least one modified pyrimidine as shown in FIG. 22, wherein each X is independently selected from -H, -OH, -OMe, -O-allyl, _-F , -OEt, _-OPr , _{-OCH₂CH₂OCH₃} , _NH₂ , and -azido. Certain non-limiting exemplary pairs of modified pyrimidines are shown in the embodiments described herein.

In some embodiments, the oligonucleotide comprises at least one modified pyrimidine as shown in Figure 20, wherein the 2' _position of the ribose is independently selected from -H, -OH, -OMe, -O-allyl, -F, -OEt, -OPr, _{-OCH₂CH₂OCH₃} , _{NH₂ ,} and -azido. In some embodiments, the oligonucleotide comprises at least two modified pyrimidines as shown in Figure 20, wherein the 2' position of the ribose is independently selected from -H, -OH, -OMe, -O-allyl, -F, _-OEt , _-OPr , _{-OCH₂CH₂OCH₃} , _NH₂ , and -azido.

In some embodiments, the oligonucleotide comprises at least one modified pyrimidine as shown in FIG. 20 and at least one modified pyrimidine as shown in FIG. 21 or FIG. 22, wherein the 2' position of the ribose is independently selected from -H, -OH, -OMe, -O-allyl, -F, -OEt, _-OPr , _{-OCH₂CH₂OCH₃} , _NH₂, and -azido. Certain non-limiting exemplary _pairs of modified pyrimidines are shown in the embodiments described herein.

In any of the embodiments described herein, the oligonucleotide may be an aptamer. In some such embodiments, the oligonucleotide is an aptamer that specifically binds to a target polypeptide.

Preparation of oligonucleotides

The automated synthesis of oligodeoxynucleosides is routine practice in many laboratories (see, for example, Matteucci, MD and Caruthers, MH, (1990) J. Am. Chem. Soc., 103 : 3185-3191, the contents of which are hereby incorporated in whole by reference). The synthesis of oligoribonucleotides is also well known (see, for example, Scaringe, SA et al., (1990) Nucleic Acids Res. 18 : 5433-5441, the contents of which are hereby incorporated in whole by reference). As noted herein, phosphoramides are suitable for the chemical synthesis of modified nucleosides into oligonucleotides, and triphosphates are suitable for the enzymatic synthesis of modified nucleosides into oligonucleotides. (See, for example, Vaught, JD et al. (2004) J. Am. Chem. Soc., 126 : 11231-11237; Vaught, JV et al. (2010) J. Am. Chem. Soc. 132 , 4141-4151; Gait, MJ, “Oligonucleotide Synthesis: a practical approach” (1984) IRL Press (Oxford, UK); Herdewijn, P., “Oligonucleotide Synthesis” (2005) (Humana Press, Totowa, NJ) (each is incorporated herein by reference in its entirety).

SELEX method

The terms SELEX and SELEX method are used interchangeably herein to generally refer to (1) the selection of nucleic acids that interact with target molecules in a desired manner (e.g., by binding to proteins with high affinity) and (2) the combination of amplification of those selected nucleic acids. The SELEX method can be used to identify aptamers with high affinity for specific target molecules or biomarkers.

SELEX generally includes preparing a candidate mixture of nucleic acids, binding the candidate mixture to a desired target molecule to form an affinity complex, separating the affinity complex from unbound candidate nucleic acids, separating and isolating the nucleic acids from the affinity complex, purifying the nucleic acids, and identifying specific aptamer sequences. The method may include multiple rounds to further refine the affinity of selected aptamers. The method may include an amplification step at one or more points in the process. See, for example, U.S. Patent No. 5,475,096 entitled “Nucleic Acid Ligands”. The SELEX method can be used to generate aptamers that covalently bind to their targets and aptamers that non-covalently bind to their targets. See, for example, U.S. Patent No. 5,705,337 entitled “Systematic Evolution of Nucleic Acid Ligands by Exponential Enrichment: Chemi-SELEX”.

The SELEX method can be used to identify high-affinity aptamers containing modified nucleotides that impart improved characteristics to the aptamer, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at ribose and/or phosphate and/or base positions. Aptamers containing modified nucleotides identified by the SELEX method are described in U.S. Patent No. 5,660,985, entitled "High Affinity Nucleic Acid Ligands Containing Modified Nucleotides," which describes oligonucleotides containing nucleotide derivatives chemically modified at the 5′ and 2′ positions of pyrimidine. U.S. Patent No. 5,580,737 (see above) describes highly specific aptamers containing one or more nucleotides modified with 2′-amino (2′- _NH₂ ), 2′-fluoro (2′-F), and/or 2′-O-methyl (2′-OMe). See also U.S. Patent Application Publication No. 20090098549 entitled “SELEX and PHOTOSELEX”, which describes nucleic acid libraries with extended physical and chemical properties and their use in SELEX and photocrosslinked SELEX.

SELEX can also be used to identify aptamers with desired dissociation rate characteristics. See U.S. Patent No. 7,947,447, entitled “Method for Generating Aptamers with Improved Off-Rates,” which is incorporated herein by reference in its entirety, describing an improved SELEX method for generating aptamers capable of binding to target molecules. Methods for generating aptamers and photoaptamers with slower dissociation rates to their respective target molecules are described. These methods involve contacting a candidate mixture with a target molecule, allowing the formation of a nucleic acid-target complex, and performing a slow dissociation rate enrichment method, wherein nucleic acid-target complexes with rapid dissociation rates dissociate and do not re-form, while complexes with slow dissociation rates remain intact. Additionally, these methods include using modified nucleotides in the generation of the candidate nucleic acid mixture to produce aptamers with improved dissociation rate performance (see U.S. Patent No. 8,409,795, entitled “SELEX and PhotoSELEX”). (See also U.S. Patent No. 7,855,054 and U.S. Patent Publication No. 20070166740). These applications are each incorporated herein by reference in their entirety.

"Target" or "target molecule" or "target" in this document refers to any compound in which nucleic acids can function in the desired manner. Target molecules can be, but are not limited to, proteins, peptides, nucleic acids, carbohydrates, lipids, polysaccharides, glycoproteins, hormones, receptors, antigens, antibodies, viruses, pathogens, toxic substances, substrates, metabolites, transition state analogs, cofactors, inhibitors, drugs, dyes, nutrients, growth factors, cells, tissues, any part or fragment of the foregoing, etc. In fact, any chemical or biological effector can be a suitable target. Molecules of any size can serve as targets. Targets can also be modified in some way to enhance the likelihood or strength of the interaction between the target and the nucleic acid. Targets can also include any minor changes to a particular compound or molecule, such as, in the case of proteins, minor changes to the amino acid sequence, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeled component, that do not substantially alter the molecular identity. "Target molecule" or "target" is a collection of copies of a type or class of molecules or multimolecular structures capable of binding aptamers. "Target molecule" or "target" refers to more than one such set of molecules. Embodiments of the SELEX method where the target is a peptide are described in U.S. Patent No. 6,376,190, entitled "Modified SELEX Processes Without Purified Protein". In some embodiments, the target is a protein.

As used herein, the terms "competitive molecule" or "competitive agent" are used interchangeably to refer to any molecule that can form a nonspecific complex with a non-target molecule. In this context, non-target molecules include free aptamers, where, for example, a competitive agent can be used to inhibit the nonspecific binding (rebinding) of said aptamer to another non-target molecule. A "competitive molecule" or "competitive agent" is a collection of copies of a type or class of molecules. A "competitive molecule" or "competitive agent" refers to more than one such collection of molecules. Competitive molecules include, but are not limited to, oligonucleotides, polyanions (e.g., heparin, herring sperm DNA, salmon sperm DNA, tRNA, dextran sulfate, dextran, base-free phosphodiester polymers, dNTPs, and pyrophosphates). In several embodiments, combinations of one or more competitive agents may be used.

As used herein, a “nonspecific complex” refers to a non-covalent association between two or more molecules other than an aptamer and their target molecule. Nonspecific complexes represent interactions between molecular classes. Nonspecific complexes include complexes formed between an aptamer and a non-target molecule, a competitor and a non-target molecule, a competitor and a target molecule, and a target molecule and a non-target molecule.

As used herein, the term "slow dissociation rate enrichment method" refers to a method of altering the relative concentrations of certain components in a candidate mixture such that the relative concentration of an aptamer affinity complex having a slow dissociation rate increases relative to the concentration of an aptamer affinity complex having a faster, less desirable dissociation rate. In one embodiment, the slow dissociation rate enrichment method is a solution-based slow dissociation rate enrichment method. In this embodiment, the solution-based slow dissociation rate enrichment method occurs in solution such that neither the target nor the nucleic acid forming the aptamer affinity complex in the mixture is immobilized on a solid support during the slow dissociation rate enrichment method. In several embodiments, the slow dissociation rate enrichment method may include one or more steps, including adding a competing molecule and incubating with the competing molecule, diluting the mixture, or a combination of these steps (e.g., diluting the mixture in the presence of the competing molecule). Because the effectiveness of slow dissociation rate enrichment methods generally depends on the different dissociation rates of different aptamer affinity complexes (i.e., aptamer affinity complexes formed between the target molecule and different nucleic acids in the candidate mixture), the duration of the slow dissociation rate enrichment method is chosen to maintain a high proportion of aptamer affinity complexes with slow dissociation rates while substantially reducing the number of aptamer affinity complexes with fast dissociation rates. The slow dissociation rate enrichment method can be used in one or more cycles during the SELEX method. When dilution and the addition of a competing agent are used in combination, they can be performed simultaneously or sequentially in any order. The slow dissociation rate enrichment method can be used when the total target (protein) concentration in the mixture is low. In one embodiment, when the slow dissociation rate enrichment method includes dilution, the mixture is diluted as practicably as possible, taking care to recover the nucleic acids retained by the aptamers for subsequent cycles in the SELEX method. In one embodiment, the slow dissociation rate enrichment method includes the use of a competing agent and dilution, thereby allowing the mixture to be diluted to a degree less than might be necessary without the use of a competing agent.

In one embodiment, the slow dissociation rate enrichment method includes the addition of a competing agent, and the competing agent is a polyanion (e.g., heparin or dextran sulfate (dextran)). Heparin or dextran has been used in previous SELEX selections to identify specific aptamers. However, in such methods, heparin or dextran is present during the equilibrium step in which the target and aptamer bind to form a complex. In such methods, the ratio of high-affinity target/aptamer complexes to low-affinity target/aptamer complexes increases due to the increased concentration of heparin or dextran. However, high concentrations of heparin or dextran can reduce the number of high-affinity target/aptamer complexes at equilibrium due to competition for target binding between the nucleic acid and the competing agent. In contrast, the currently described method adds the competing agent after target/aptamer complex formation has been allowed and therefore does not affect the number of complexes formed. Adding the competing agent after equilibrium binding has occurred between the target and aptamer results in a non-equilibrium state that evolves into a new equilibrium with fewer target/aptamer complexes. Before a new equilibrium is achieved, capturing the target/aptamer complex will enrich the sample for aptamers with slow dissociation rates, as the complex with a fast dissociation rate will dissociate first.

In another embodiment, a polyanionic competitive agent (e.g., dextran sulfate or another polyanionic material) is used in the slow dissociation rate enrichment method to facilitate the identification of aptamers resistant to the presence of polyanions. In this context, a "polyanionic resistant aptamer" is an aptamer capable of forming an aptamer/target complex that is less likely to dissociate in a solution also containing the polyanionic resistant material compared to an aptamer/target complex containing a non-polyanionic resistant aptamer. In this way, polyanionic resistant aptamers can be used to perform analytical methods to detect the presence, amount, or concentration of a target in a sample, wherein the detection method includes using a polyanionic material (e.g., dextran sulfate) resistant to the aptamer.

Therefore, in one embodiment, a method for generating polyanion-resistant aptamers is provided. In this embodiment, after a candidate mixture of nucleic acids is contacted with a target, the target and the nucleic acids in the candidate mixture are allowed to reach equilibrium. A polyanion-competitive agent is introduced and incubated in solution for a period of time to ensure that most fast-dissociation-rate aptamers in the candidate mixture dissociate from the target molecule. Moreover, aptamers in the candidate mixture that can dissociate in the presence of the polyanion-competitive agent are released from the target molecule. The mixture is dispensed to isolate high-affinity, slow-dissociation-rate aptamers that have maintained association with the target molecule and to remove any uncomposite material from the solution. The aptamers can then be released from the target molecule and isolated. The isolated aptamers can also be amplified and several additional rounds of selection can be applied to increase the overall performance of the selected aptamers. This method can also be used with minimal incubation time if the selection of slow-dissociation-rate aptamers is not required for a particular application.

Therefore, in one embodiment, a modified SELEX method is provided to identify or generate aptamers with slow (long) dissociation rates, wherein a target molecule and a candidate mixture are contacted and incubated together for a time sufficient to allow equilibrium binding to occur between the target molecule and the nucleic acids contained in the candidate mixture. After equilibrium binding, an excess of a competing molecule (e.g., a polyanionic competitor) is added to the mixture, and the mixture is incubated with the excess competing molecule for a predetermined time. Most aptamers with dissociation rates lower than this predetermined incubation time will dissociate from the target during the predetermined incubation time. Reassociation of these “fast” dissociation rate aptamers with the target is minimized due to the excess of competing molecules that can nonspecifically bind to the target and occupy the target binding site. Most aptamers with longer dissociation rates will remain complexed to the target during the predetermined incubation time. At the end of the incubation time, dispensing the nucleic acid-target complex from the remaining portion of the mixture allows for the separation of the population of aptamers with slow dissociation rates from those with fast dissociation rates. The dissociation step can be used to dissociate slow-dissociation-rate aptamers from their targets and allows for the isolation, identification, sequencing, synthesis, and amplification of slow-dissociation-rate aptamers (individual aptamers or a group of slow-dissociation-rate aptamers) with high affinity and specificity for the target molecule. As with conventional SELEX, aptamer sequences identified from a modified SELEX method can be used to synthesize new candidate mixtures, allowing the steps of contacting, equilibrating binding, adding competing molecules, incubating with competing molecules, and dispensing slow-dissociation-rate aptamers to be repeated many times as needed.

Allowing the candidate mixture to bind to the target in equilibrium before adding the competing agent, followed by adding an excess of the competing agent and incubating it with the competing agent for a predetermined period of time, allows for the selection of aptamer populations with dissociation rates much higher than those previously achieved.

To achieve balanced binding, the candidate mixture may be incubated with the target for at least about 5 minutes, or at least about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, or about 6 hours.

If necessary, a predetermined incubation period may be selected for the mixture of competing molecules with candidate and target molecules, taking into account factors such as target properties and known dissociation rates of the aptamer to the target (if applicable). Predetermined incubation periods may be selected from: at least approximately 5 minutes, at least approximately 10 minutes, at least approximately 20 minutes, at least approximately 30 minutes, at least approximately 45 minutes, at least approximately 1 hour, at least approximately 2 hours, at least approximately 3 hours, at least approximately 4 hours, at least approximately 5 hours, and at least approximately 6 hours.

In other embodiments, dilution is used as a dissociation rate enhancement method and the incubation of the diluted candidate mixture, target molecule/aptamer complex, can be carried out for a predetermined time period, which can be selected from: at least about 5 minutes, at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, and at least about 6 hours.

Embodiments of this disclosure relate to the identification, generation, synthesis, and use of aptamers with slow dissociation rates. These aptamers are those that dissociate from the non-covalent aptamer-target complex at a rate (t _{_1/2} ) higher than the dissociation rate of aptamers typically obtained by conventional SELEX. For mixtures containing non-covalent complexes of aptamers and targets, t _{_1/2} represents the time taken for half of the aptamer to dissociate from the aptamer-target complex. The t _{_1/2} of the slow dissociation rate aptamers according to this disclosure is selected from one of the following: greater than or equal to about 30 minutes; between about 30 minutes and about 240 minutes; between about 30 minutes and about 60 minutes; between about 60 minutes and about 90 minutes; between about 90 minutes and about 120 minutes; between about 120 minutes and about 150 minutes; between about 150 minutes and about 180 minutes; between about 180 minutes and about 210 minutes; between about 210 minutes and about 240 minutes.

The characterization of aptamers identified by the SELEX procedure is their high affinity for their targets. The dissociation constant (k_{_d} ) of the aptamer to its target will be selected from one of the following: less than about 1 μM, less than about 100 nM, less than about 10 nM, less than about 1 nM, less than about 100 pM, less than about 10 pM, or less than about 1 pM.

Oligonucleotide library

In some embodiments, an oligonucleotide library comprising random sequences is provided. Such libraries may be suitable for performing SELEX in some embodiments. In some embodiments, each oligonucleotide in the oligonucleotide library comprises a number of randomized positions, such as at least 20, 25, 30, 35, 40, 45, or 50, or 20-100, 20-80, 20-70, 20-60, 20-50, 20-40, or 30-40 randomized positions. In some embodiments, each oligonucleotide in the oligonucleotide library comprises a fixed sequence sidebanded at the randomized positions. Such fixed sideband sequences may be the same or different from each other (i.e., the 5' sideband sequence and the 3' sideband sequence may be the same or different), and in some embodiments may be the same for all members of the library (i.e., all members of the library may have the same 5' sideband sequence, and/or all members of the library may have the same 3' sideband sequence).

In some embodiments, the randomization position may consist of four or more different nucleotide bases, one or more of which are modified. In some embodiments, all or no nucleotide bases of a certain type are modified (e.g., all cytosine nucleosides in the randomization region are modified, or all are unmodified). In some embodiments, one type of nucleotide base is present in both modified and unmodified forms in the randomization region. In some such embodiments, the randomization position consists of two modified and two unmodified nucleotide bases. In some such embodiments, the randomization position consists of adenine, guanine, C5-modified cytosine nucleosides, and C5-modified uracil nucleosides. Non-limiting exemplary C5-modified cytosine nucleosides and C5-modified uracil nucleosides are shown in Figures 19-21. Oligonucleotide libraries and methods for their preparation are further described, for example, in the embodiments herein.

Exemplary Adapter

In some embodiments, an aptamer is provided that binds to a target molecule. In some embodiments, the target molecule is a target protein. In some embodiments, an aptamer that binds to PCSK9 is provided. In some embodiments, the aptamer that binds to PCSK9 inhibits the binding of PCSK9 to LDL-R. In some such embodiments, the aptamer comprises the sequence 5'-yGpppG-3', where each y is TyrdU and each p is NapdC. In some embodiments, the aptamer further comprises the sequence 5'-yEAyGA _npAp -3', where E is selected from y, A, and G; and n is 0 or 1. In some embodiments, n is 0. In some embodiments, the sequence 5'-yEAyGA _npAp -3' is located at the 5' of the sequence 5'-yGpppG-3'. In some embodiments, E is y.

In some embodiments, an aptamer binding to PCSK9 is provided, wherein the aptamer comprises the sequence 5'-F _n pppAAGRJrpRppW _m -3' (SEQ ID NO: 81), wherein F is selected from r and G; each R is independently selected from G and A; J is selected from r and A; W is selected from r, G, and A; n is 0 or 1; m is 0 or 1; r is PpdC; and p is NapdU. In some embodiments, m is 1. In some embodiments, F is r. In some embodiments, J is r. In some embodiments, W is G.

In some implementations, an aptamer that binds to PCSK9 is provided, wherein the aptamer comprises the sequence 5’-TTppGGpp-3’, wherein each p is NapdC.

In some implementations, the aptamer binding to PCSK9 is 20 to 100, or 20 to 90, or 20 to 80, or 20 to 70, or 20 to 60, or 20 to 50, or 30 to 100, or 30 to 90, or 30 to 80, or 30 to 70, or 30 to 60, or 30 to 50, or 40 to 100, or 40 to 90, or 40 to 80, or 40 to 70, or 40 to 60, or 40 to 50 nucleotides in length.

In some embodiments, the aptamer inhibits the binding of PCSK9 to LDL-R. In some embodiments, the aptamer inhibits the binding of PCSK9 to LDL-R, wherein the IC ₅₀ is less than 30 nM, less than 20 nM, or less than 15 nM.

In some embodiments, a method for lowering cholesterol in a subject is provided, the method comprising administering a PCSK9-binding aptamer to the subject in need. In some embodiments, the PCSK9-binding aptamer is the aptamer provided herein. In some embodiments, the cholesterol is low-density lipoprotein (LDL) cholesterol (LDL-C). In some embodiments, the subject has heterozygous familial hypercholesterolemia or clinical atherosclerotic cardiovascular disease (CVD).

Salt

The preparation, purification, and/or treatment of the corresponding salts of said compounds (e.g., pharmaceutically acceptable salts) may be convenient or necessary. Examples of pharmaceutically acceptable salts are discussed in Berge et al. (1977), “Pharmaceutically Acceptable Salts”, J. Pharm. Sci. 66 : 1-19.

For example, if the compound is anionic, or has a functional group that can be anionic (e.g., -COOH can be -COO-), it can form a salt with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions (such as Na ⁺ and K ⁺ ), alkaline earth metal cations (such as Ca2 ⁺ and Mg2 ^{+ )} , and other cations (such as _Al3 ^{+ ). Examples of suitable organic cations include, but are not limited to, ammonium ions (i.e., NH4+) and substituted ammonium ions (e.g., NH3RX+, NH2RX2+, NHRX3+, NRX4+} ₎ ^{. Some} _{examples of} ^suitable _substituted ^{ammonium ions are} _derived from those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids (such as lysine and arginine). A common example of ^a quaternary ammonium ion is N( _CH3 ) ₄₊ .

If the compound is cationic, or has a functional group that can be cationic (e.g., _-NH₂ can be _-NH₃⁺ ), it can form a salt with a suitable anion. Examples of suitable inorganic anions include, but are not limited to ^, those derived from the following inorganic acids: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfurous acid, nitric acid, nitrous acid, phosphoric acid, and phosphorous acid.

Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetoxybenzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphorsulfonic acid, cinnamic acid, citric acid, ethylenediaminetetraacetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucohepanoic acid, gluconic acid, glutamic acid, glycolic acid, hydroxymaleic acid, hydroxynaphthoic acid, hydroxyethanesulfonic acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, methanesulfonic acid, mucoic acid, oleic acid, oxalic acid, palmitic acid, dihydroxynaphthoic acid, pantothenic acid, phenylacetic acid, phenylsulfonic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, benzenesulfonic acid, and valeric acid. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid and carboxymethyl cellulose.

Unless otherwise specified, references to a particular compound also include its salt form.

Some non-limiting exemplary embodiments

Implementation Scheme 1. An aptamer comprising at least one first 5-position modified pyrimidine and at least one second 5-position modified pyrimidine, wherein the first 5-position modified pyrimidine and the second 5-position modified pyrimidine are different 5-position modified pyrimidines.

Implementation Scheme 2. The aptamer as described in Implementation Scheme 1, wherein the first 5-position modified pyrimidine is a 5-position modified uracil nucleoside and wherein the second 5-position modified pyrimidine is a 5-position modified cytosine nucleoside.

Implementation Scheme 3. The aptamer as described in Implementation Scheme 1, wherein the first 5-position modified pyrimidine is a 5-position modified cytosine nucleoside and wherein the second 5-position modified pyrimidine is a 5-position modified uridine nucleoside.

Implementation Scheme 4. The aptamer as described in Implementation Scheme 2 or Implementation Scheme 3, wherein the 5-position modified uracil nucleoside comprises a portion selected from naphthyl moiety, benzyl moiety, tyrosinyl moiety, indole moiety and morpholino moiety at the 5 position.

Implementation Scheme 5. The aptamer as described in any one of Implementation Schemes 2 to 4, wherein the 5-position modified cytosine nucleoside comprises a portion selected from the naphthyl moiety, benzyl moiety, tyrosinyl moiety, and morpholino moiety at the 5-position.

Implementation Scheme 6. The aptamer as described in any one of Implementation Schemes 2 to 5, wherein the 5-position modified cytosine nucleoside is selected from NapdC, 2NapdC, TyrdC, and PPdC.

Implementation Scheme 7. The aptamer as described in any one of Implementation Schemes 2 to 6, wherein the 5-position modified uracil nucleoside is selected from NapdU, 2NapdU, PPdU, MOEdU, TyrdU, TrpdU, and ThrdU.

Implementation Scheme 8. The aptamer as described in Implementation Scheme 1, wherein the at least one first 5-position modified pyrimidine is NapdC and the at least one second 5-position modified pyrimidine is selected from NapdU, 2NapdU, PPdU, MOEdU, TyrdU, and ThrdU.

Implementation Scheme 9. The aptamer as described in Implementation Scheme 1, wherein the at least one first 5-position modified pyrimidine is PPdC and the at least one second 5-position modified pyrimidine is selected from NapdU, 2NapdU, PPdU, MOEdU, TyrdU, and ThrdU.

Implementation Scheme 10. The aptamer as described in Implementation Scheme 8 or Implementation Scheme 9, wherein the at least one second 5-position modified pyrimidine is TyrdU.

Implementation Scheme 11. An aptamer as described in any one of Implementation Schemes 1 to 10, wherein the aptamer binds to a target protein selected from PCSK9, PSMA, ErbB1, ErbB2, FXN, KDM2A, IGF1R, pIGF1R, α1-antitrypsin, CD99, MMP28, and PPIB.

Implementation Scheme 12. An aptamer as described in any one of Implementation Schemes 1 to 11, wherein the aptamer comprises at least 10, at least 15, at least 20, at least 25, or at least 30 nucleotides in length, or 5 to 30, 10 to 30, 15 to 30, 5 to 20, or 10 to 20 nucleotides in length, at the 5' end of the aptamer, wherein the region at the 5' end of the aptamer lacks a 5-position modified pyrimidine.

Implementation Scheme 13. An aptamer as described in any one of Implementation Schemes 1 to 12, wherein the aptamer comprises at least 10, at least 15, at least 20, at least 25, or at least 30 nucleotides in length, or 5 to 30, 10 to 30, 15 to 30, 5 to 20, or 10 to 20 nucleotides in length, at the 3' end of the aptamer, wherein the region at the 3' end of the aptamer lacks a 5-position modified pyrimidine.

Implementation Scheme 14. An aptamer as described in any one of Implementation Schemes 1 to 13, wherein the aptamer is 20 to 100, or 20 to 90, or 20 to 80, or 20 to 70, or 20 to 60, or 20 to 50, or 30 to 100, or 30 to 90, or 30 to 80, or 30 to 70, or 30 to 60, or 30 to 50 nucleotides in length.

Implementation Scheme 15. A composition comprising a plurality of polynucleotides, wherein each polynucleotide comprises at least one first 5-position modified pyrimidine and at least one second 5-position modified pyrimidine, wherein the first 5-position modified pyrimidine and the second 5-position modified pyrimidine are different 5-position modified pyrimidines.

Implementation Scheme 16. The composition of Implementation Scheme 15, wherein each polynucleotide includes a fixed region at the 5' end of the polynucleotide.

Implementation Scheme 17. The composition of Implementation Scheme 16, wherein the fixed region at the 5' end of each polynucleotide is at least 10, at least 15, at least 20, at least 25 or at least 30 nucleotides in length, or 5 to 30, 10 to 30, 15 to 30, 5 to 20 or 10 to 20 nucleotides in length.

Implementation Scheme 18. The composition of any one of Implementation Schemes 15 to 17, wherein each polynucleotide includes a fixed region at the 3' end of the polynucleotide.

Implementation Scheme 19. The composition of Implementation Scheme 18, wherein the fixed region at the 3' end of the polynucleotide is at least 10, at least 15, at least 20, at least 25 or at least 30 nucleotides in length, or 5 to 30, 10 to 30, 15 to 30, 5 to 20 or 10 to 20 nucleotides in length.

Implementation Scheme 20. The composition of any one of Implementation Schemes 15 to 19, wherein the first 5-position modified pyrimidine is a 5-position modified uracil nucleoside and wherein the second 5-position modified pyrimidine is a 5-position modified cytosine nucleoside.

Implementation Scheme 21. The composition of any one of Implementation Schemes 15 to 19, wherein the first 5-position modified pyrimidine is a 5-position modified cytosine nucleoside and wherein the second 5-position modified pyrimidine is a 5-position modified uridine nucleoside.

Implementation Scheme 22. The composition as described in Implementation Scheme 20 or Implementation Scheme 21, wherein the 5-position modified uracil nucleoside comprises a portion selected from naphthyl moiety, benzyl moiety, tyrosinyl moiety, indole moiety and morpholino moiety at the 5 position.

Implementation Scheme 23. The composition of any one of Implementation Schemes 20 to 22, wherein the 5-position modified cytosine nucleoside comprises a portion selected from naphthyl moiety, benzyl moiety, tyrosinyl moiety and morpholino moiety at the 5 position.

Implementation Scheme 24. The composition of any one of Implementation Schemes 20 to 23, wherein the 5-position modified cytosine nucleoside is selected from NapdC, 2NapdC, TyrdC, and PPdC.

Implementation Scheme 25. A composition of any one of Implementation Schemes 20 to 24, wherein the 5-position modified uracil nucleoside is selected from NapdU, 2NapdU, PPdU, MOEdU, TyrdU, TrpdU, and ThrdU.

Implementation Scheme 26. The composition of Implementation Scheme 15, wherein the at least one first 5-position modified pyrimidine is NapdC and the at least one second 5-position modified pyrimidine is selected from NapdU, 2NapdU, PPdU, MOEdU, TyrdU, TrpdU, and ThrdU.

Implementation Scheme 27. The composition of Implementation Scheme 15, wherein the at least one first 5-position modified pyrimidine is PPdC and the at least one second 5-position modified pyrimidine is selected from NapdU, 2NapdU, PPdU, MOEdU, TrydU, TrpdU, and ThrdU.

Implementation Scheme 28. The composition as described in Implementation Scheme 26 or Implementation Scheme 27, wherein the at least one second 5-position modified pyrimidine is TyrdU.

Implementation Scheme 29. The composition of any one of Implementation Schemes 15 to 28, wherein each polynucleotide comprises a random region.

Implementation Scheme 30. The composition of Implementation Scheme 29, wherein the random regions are 20 to 100, or 20 to 90, or 20 to 80, or 20 to 70, or 20 to 60, or 20 to 50, or 20 to 40, or 30 to 100, or 30 to 90, or 30 to 70, or 30 to 60, or 30 to 50, or 30 to 40 nucleotides in length.

Implementation Scheme 31. The composition of any one of Implementation Schemes 15 to 29, wherein each polynucleotide is 20 to 100, or 20 to 90, or 20 to 80, or 20 to 70, or 20 to 60, or 20 to 50, or 30 to 100, or 30 to 90, or 30 to 80, or 30 to 70, or 30 to 60, or 30 to 50, or 40 to 100, or 40 to 90, or 40 to 80, or 40 to 70, or 40 to 60, or 40 to 50 nucleotides in length.

Implementation Scheme 32. A composition comprising a first aptamer, a second aptamer, and a target,

The first aptamer comprises at least one first 5-position modified pyrimidine and at least one second 5-position modified pyrimidine;

The second aptamer comprises at least one third 5-position modified pyrimidine;

The first aptamer, the second aptamer, and the target can form a trimeric complex; and

The first 5-position modified pyrimidine and the second 5-position modified pyrimidine are different 5-position modified pyrimidines.

Implementation Scheme 33. The composition of Implementation Scheme 32, wherein the first 5-position modified pyrimidine is a 5-position modified uracil nucleoside and wherein the second 5-position modified pyrimidine is a 5-position modified cytosine nucleoside.

Implementation Scheme 34. The composition of Implementation Scheme 32, wherein the first 5-position modified pyrimidine is a 5-position modified cytosine nucleoside and wherein the second 5-position modified pyrimidine is a 5-position modified uridine nucleoside.

Implementation Scheme 35. The composition as described in Implementation Scheme 33 or Implementation Scheme 34, wherein the 5-position modified uracil nucleoside comprises a portion selected from naphthyl moiety, benzyl moiety, tyrosinyl moiety, indole moiety and morpholino moiety at the 5 position.

Implementation Scheme 36. The composition of any one of Implementation Schemes 33 to 35, wherein the 5-position modified cytosine nucleoside comprises a portion selected from naphthyl moiety, benzyl moiety, tyrosinyl moiety and morpholino moiety at the 5 position.

Implementation Scheme 37. The composition of any one of Implementation Schemes 33 to 36, wherein the 5-position modified cytosine nucleoside is selected from NapdC, 2NapdC, TyrdC, and PPdC.

Implementation Scheme 38. The composition of any one of Implementation Schemes 33 to 37, wherein the 5-position modified uracil nucleoside is selected from NapdU, 2NapdU, PPdU, MOEdU, TyrdU, TrpdU, and ThrdU.

Embodiment 39. The composition of Embodiment 32, wherein the at least one first 5-position modified pyrimidine is NapdC and the at least one second 5-position modified pyrimidine is selected from NapdU, 2NapdU, PPdU, MOEdU, TyrdU, TrpdU, and ThrdU.

Implementation Scheme 40. The composition of Implementation Scheme 32, wherein the at least one first 5-position modified pyrimidine is PPdC and the at least one second 5-position modified pyrimidine is selected from NapdU, 2NapdU, PPdU, MOEdU, TyrdU, TrpdU, and ThrdU.

Implementation Scheme 41. The composition as described in Implementation Scheme 39 or Implementation Scheme 40, wherein the at least one second 5-position modified pyrimidine is TyrdU.

Implementation Scheme 42. The composition of any one of Implementation Schemes 32 to 41, wherein the third 5-position modified pyrimidine is selected from 5-position modified cytosine nucleosides and 5-position modified pyrimidines.

Implementation Scheme 43. The composition of Implementation Scheme 42, wherein the third 5-position modified pyrimidine is selected from BndC, PEdC, PPdC, NapdC, 2NapdC, NEdC, 2NEdC, TyrdC, BndU, NapdU, PEdU, IbdU, FBndU, 2NapdU, NEdU, MBndU, BFdU, BTdU, PPdU, MOEdU, TyrdU, TrpdU, and ThrdU.

Implementation Scheme 44. The composition of any one of Implementation Schemes 32 to 43, wherein the target is selected from proteins, peptides, carbohydrates, small molecules, cells, and tissues.

Implementation Scheme 45. A method comprising:

(a) To bring the aptamer, which can bind to the target molecule, into contact with the sample;

(b) Incubate the aptamer with the sample to allow aptamer-target complex formation;

(c) Enriching the aptamer-target complex in the sample, and

(c) Detecting the presence of the aptamer, the aptamer-target complex, or the target molecule, wherein the detection of the aptamer, the aptamer-target complex, or the target molecule indicates that the target molecule is present in the sample, and wherein the absence of the detection of the aptamer, the aptamer-target complex, or the target molecule indicates that the target molecule is not present in the sample;

The aptor described herein is the aptor as described in any one of embodiments 1 to 14.

Implementation Scheme 46. The method of Implementation Scheme 45, wherein the method includes at least one additional step selected from: adding a competing molecule to the sample; capturing the aptamer-target complex on a solid support; and adding the competing molecule and diluting the sample; wherein the at least one additional step occurs after step (a) or step (b).

Implementation Scheme 47. The method as described in Implementation Scheme 46, wherein the competing molecule is selected from polyanionic competing agents.

Implementation Scheme 48. The method of Implementation Scheme 47, wherein the polyanionic competitive agent is selected from oligonucleotides, dextran, DNA, heparin and dNTPs.

Implementation Scheme 49. The method as described in Implementation Scheme 48, wherein the dextran is sulfated dextran; and the DNA is herring sperm DNA or salmon sperm DNA.

Implementation Scheme 50. The method of any one of Implementation Schemes 45 to 49, wherein the target molecule is selected from proteins, peptides, carbohydrates, small molecules, cells, and tissues.

Implementation Scheme 51. The method of any one of Implementation Schemes 45 to 50, wherein the sample is selected from whole blood, white blood cells, peripheral blood mononuclear cells, plasma, serum, saliva, respiration, urine, semen, saliva, meningeal fluid, amniotic fluid, glandular fluid, lymph, papillary aspirate, bronchial aspirate, synovial fluid, joint aspirate, cells, cell extracts, feces, tissue, tissue biopsy, and cerebrospinal fluid.

Implementation Scheme 52. A method for detecting a target in a sample, comprising:

a) Contact the sample with a first aptamer to form a mixture, wherein the first aptamer is capable of binding the target to form a first complex;

b) Incubate the mixture under conditions that allow the formation of the first complex;

c) Contact the mixture with a second aptamer, wherein the second aptamer is capable of binding the first complex to form a second complex;

d) Incubate the mixture under conditions that allow the formation of the second complex;

e) Detect the presence or absence of the first aptamer, the second aptamer, the target, the first complex, or the second complex in the mixture, wherein the presence of the first aptamer, the second aptamer, the target, the first complex, or the second complex indicates that the target is present in the sample;

The first aptamer comprises at least one first 5-position modified pyrimidine and at least one second 5-position modified pyrimidine;

The second aptamer comprises at least one third 5-position modified pyrimidine;

The first 5-position modified pyrimidine and the second 5-position modified pyrimidine are different 5-position modified pyrimidines.

Implementation Scheme 53. The method of Implementation Scheme 52, wherein the first 5-position modified pyrimidine is a 5-position modified uridine and wherein the second 5-position modified pyrimidine is a 5-position modified cytosine.

Implementation Scheme 54. The method of Implementation Scheme 53, wherein the first 5-position modified pyrimidine is a 5-position modified cytosine nucleoside and wherein the second 5-position modified pyrimidine is a 5-position modified uridine nucleoside.

Implementation Scheme 55. The method of Implementation Scheme 53 or Implementation Scheme 54, wherein the 5-position modified uridine nucleoside comprises a portion selected from naphthyl moiety, benzyl moiety, tyrosinyl moiety, indole moiety and morpholino moiety at the 5 position.

Implementation Scheme 56. The method of any one of Implementation Schemes 53 to 55, wherein the 5-position modified cytosine nucleoside comprises a portion selected from naphthyl moiety, benzyl moiety, tyrosinyl moiety and morpholino moiety at the 5 position.

Implementation Scheme 57. The method of any one of Implementation Schemes 53 to 56, wherein the 5-position modified cytosine nucleoside is selected from NapdC, 2NapdC, TyrdC, and PPdC.

Implementation Scheme 58. The method of any one of Implementation Schemes 53 to 57, wherein the 5-position modified uracil nucleoside is selected from NapdU, 2NapdU, PPdU, MOEdU, TyrdU, TrpdU, and ThrdU.

Implementation Scheme 59. The method of Implementation Scheme 52, wherein the at least one first 5-position modified pyrimidine is NapdC and the at least one second 5-position modified pyrimidine is selected from NapdU, 2NapdU, PPdU, MOEdU, TyrdU, TrpdU, and ThrdU.

Implementation Scheme 60. The method of Implementation Scheme 52, wherein the at least one first 5-position modified pyrimidine is PPdC and the at least one second 5-position modified pyrimidine is selected from NapdU, 2NapdU, PPdU, MOEdU, TyrdU, TrpdU, and ThrdU.

Implementation Scheme 61. The method as described in Implementation Scheme 59 or Implementation Scheme 60, wherein the at least one second 5-position modified pyrimidine is TyrdU.

Implementation Scheme 62. The method of any one of Implementation Schemes 52 to 61, wherein the third 5-position modified pyrimidine is selected from 5-position modified cytosine nucleosides and 5-position modified pyrimidines.

Implementation Scheme 63. The method of Implementation Scheme 62, wherein the third 5-position modified pyrimidine is selected from BndC, PEdC, PPdC, NapdC, 2NapdC, NEdC, 2NEdC, TyrdC, BNdU, NapdU, PedU, IbdU, FbndU, 2NapdU, NedU, MbndU, BfdU, BtdU, PpdU, MOEdU, TyrdU, TrpdU, and ThrdU.

Implementation Scheme 64. The method of any one of Implementation Schemes 52 to 63, wherein the target molecule is selected from proteins, peptides, carbohydrates, small molecules, cells, and tissues.

Implementation Scheme 65. The method of any one of Implementation Schemes 52 to 64, wherein the first aptamer, the second aptamer, and the target are capable of forming a trimer complex.

Implementation Scheme 66. A method for identifying one or more aptamers capable of binding to a target molecule, the method comprising:

(a) Contacting a library of aptamers with the target molecule to form a mixture and allowing the formation of an aptamer-target complex, wherein the aptamer-target complex is formed when the aptamer has an affinity for the target molecule;

(b) Dispensing (or enriching) the aptamer-target complex from the remainder of the mixture;

(c) Dissociation of the aptamer-target complex; and

(d) Identify one or more aptamers capable of binding to the target molecule;

The aptamer library contains multiple polynucleotides, each polynucleotide containing at least one first 5-position modified pyrimidine and at least one second 5-position modified pyrimidine, wherein the first 5-position modified pyrimidine and the second 5-position modified pyrimidine are different 5-position modified pyrimidines.

Implementation Scheme 67. The method of Implementation Scheme 66, wherein each polynucleotide includes a fixed region at the 5' end of the polynucleotide.

Implementation Scheme 68. The method of Implementation Scheme 67, wherein the fixed region at the 5' end of each polynucleotide is at least 10, at least 15, at least 20, at least 25 or at least 30 nucleotides in length, or 5 to 30, 10 to 30, 15 to 30, 5 to 20 or 10 to 20 nucleotides in length.

Implementation Scheme 69. The method of any one of Implementation Schemes 66 to 68, wherein each polynucleotide includes a fixed region at the 3' end of the polynucleotide.

Implementation Scheme 70. The method of Implementation Scheme 69, wherein the fixed region at the 3' end of the polynucleotide is at least 10, at least 15, at least 20, at least 25 or at least 30 nucleotides in length, or 5 to 30, 10 to 30, 15 to 30, 5 to 20 or 10 to 20 nucleotides in length.

Implementation Scheme 71. The method of any one of Implementation Schemes 66 to 70, wherein the first 5-position modified pyrimidine is a 5-position modified uracil nucleoside and wherein the second 5-position modified pyrimidine is a 5-position modified cytosine nucleoside.

Implementation Scheme 72. The method of any one of Implementation Schemes 66 to 71, wherein the first 5-position modified pyrimidine is a 5-position modified cytosine nucleoside and wherein the second 5-position modified pyrimidine is a 5-position modified uridine nucleoside.

Implementation Scheme 73. The method as described in Implementation Scheme 71 or Implementation Scheme 72, wherein the 5-position modified uracil nucleoside comprises a portion selected from naphthyl moiety, benzyl moiety, tyrosinyl moiety, indole moiety and morpholino moiety at the 5-position.

Implementation Scheme 74. The method of any one of Implementation Schemes 71 to 73, wherein the 5-position modified cytosine nucleoside comprises a portion selected from naphthyl moiety, benzyl moiety, tyrosinyl moiety and morpholino moiety at the 5 position.

Implementation Scheme 75. The method of any one of Implementation Schemes 71 to 74, wherein the 5-position modified cytosine nucleoside is selected from NapdC, 2NapdC, TyrdC, and PPdC.

Implementation Scheme 76. The method of any one of Implementation Schemes 71 to 75, wherein the 5-position modified uracil nucleoside is selected from NapdU, PPdU, MOEdU, TyrdU, TrpdU, and ThrdU.

Implementation Scheme 77. The method of Implementation Scheme 66, wherein the at least one first 5-position modified pyrimidine is NapdC and the at least one second 5-position modified pyrimidine is selected from NapdU, 2NapdU, PPdU, MOEdU, TrydU, TrpdU, and ThrdU.

Implementation Scheme 78. The method of Implementation Scheme 66, wherein the at least one first 5-position modified pyrimidine is PPdC and the at least one second 5-position modified pyrimidine is selected from NapdU, 2NapdU, PPdU, MOEdU, TyrdU, TrpdU, and ThrdU.

Implementation Scheme 79. The method as described in Implementation Scheme 77 or Implementation Scheme 78, wherein the at least one second 5-position modified pyrimidine is TyrdU.

Implementation scheme 80. The method of any one of implementation schemes 66 to 79, wherein each polynucleotide contains a random region.

Implementation Scheme 81. The method as described in Implementation Scheme 80, wherein the random region is 20 to 100, or 20 to 90, or 20 to 80, or 20 to 70, or 20 to 60, or 20 to 50, or 20 to 40, or 30 to 100, or 30 to 90, or 30 to 70, or 30 to 60, or 30 to 50, or 30 to 40 nucleotides in length.

Implementation Scheme 82. The method of any one of Implementation Schemes 66 to 81, wherein each polynucleotide is 20 to 100, or 20 to 90, or 20 to 80, or 20 to 70, or 20 to 60, or 20 to 50, or 30 to 100, or 30 to 90, or 30 to 80, or 30 to 70, or 30 to 60, or 30 to 50, or 40 to 100, or 40 to 90, or 40 to 80, or 40 to 70, or 40 to 60, or 40 to 50 nucleotides in length.

Implementation Scheme 83. The method of any one of Implementation Schemes 66 to 82, wherein each polynucleotide is an aptamer that binds to a target, and wherein the library contains at least 1,000 aptamers, wherein each aptamer contains a different nucleotide sequence.

Implementation Scheme 84. The method of any one of Implementation Schemes 66 to 83, wherein steps (a), (b) and/or (c) are repeated at least once, twice, three times, four times, five times, six times, seven times, eight times, nine times or ten times.

Implementation Scheme 85. The method of any one of Implementation Schemes 66 to 84, wherein one or more aptamers capable of binding the target molecule are amplified.

Implementation Scheme 86. The method of any one of Implementation Schemes 66 to 85, wherein the mixture comprises a polyanionic competing molecule.

Implementation Scheme 87. The method of Implementation Scheme 86, wherein the polyanionic competitive agent is selected from oligonucleotides, dextran, DNA, heparin and dNTPs.

Implementation Scheme 88. The method as described in Implementation Scheme 87, wherein the dextran is sulfated dextran; and the DNA is herring sperm DNA or salmon sperm DNA.

Implementation Scheme 89. The method of any one of Implementation Schemes 66 to 88, wherein the target molecule is selected from proteins, peptides, carbohydrates, small molecules, cells, and tissues.

Implementation Scheme 90. The aptamer as described in any one of Implementation Schemes 1 to 14, wherein the first 5-position modified pyrimidine and the second 5-position modified pyrimidine are capable of being incorporated by polymerase.

Implementation Scheme 91. The composition of any one of Implementation Schemes 15 to 44, wherein the first 5-position modified pyrimidine and the second 5-position modified pyrimidine are capable of being incorporated by polymerase.

Implementation Scheme 92. The method of any one of Implementation Schemes 45 to 89, wherein the first 5-position modified pyrimidine and the second 5-position modified pyrimidine are capable of being incorporated by polymerase.

Implementation Scheme 93. An aptamer as described in any one of Implementation Schemes 1 to 14 and 90, wherein the aptamer has improved nuclease stability compared to an aptamer having the same length and containing a nucleobase sequence comprising an unmodified pyrimidine replacing each of the first 5-position modified pyrimidines or an unmodified pyrimidine replacing each of the second 5-position modified pyrimidines.

Implementation Scheme 94. An aptamer as described in any one of Implementation Schemes 1 to 14, 90 and 93, wherein the aptamer has a longer half-life in human serum compared to an aptamer having the same length and containing a nucleobase sequence of an unmodified pyrimidine that substitutes for each of the first 5-position modified pyrimidines or an unmodified pyrimidine that substitutes for each of the second 5-position modified pyrimidines.

Example

The following examples are provided to illustrate some embodiments of the invention more fully. However, they should in no way be considered as limiting the broad scope of the invention. Those skilled in the art can readily employ the underlying principles of this discovery to design a variety of compounds without departing from the spirit of the invention.

Example 1: Aptamers containing two modified bases

To compare the relative efficiency of SELEX using two types of modified bases, five single modifications (Nap-dU, PP-dU, MOE-dU, Tyr-dU, and Thr-dU) on dU with unmodified dT as a control, and combinations of modifications on dC (Nap-dC and PP-dC) with unmodified dC as a control were analyzed for a total of 18 starting libraries (Figure 1). The types of modifications tested included hydrophobic aromatic side chains similar to hydrophobic side chains on amino acids. Hydrophilic side chains on dU (MOE-dU and Thr-dU) were also tested. Each of the 18 libraries contained 30 randomized nucleotides, allowing ^≥10¹⁵ different sequences. The libraries were enzymatically synthesized using exonuclease KOD DNA polymerase with native and/or modified nucleotide triphosphates (data not shown).

A 30N randomized library was used instead of the previous 40N randomized library with a single modified dU. Without intending to be bound by any particular theory, it was assumed that increasing the density of modified bases would allow for shorter, high-affinity aptamers. Furthermore, shorter oligonucleotide libraries yielded higher production. The ratio of each nucleotide for dA/dC/dG/dT was 1:1:1:1 (25% each). In all cases, the random regions were flanked by fixed sequences for hybridization PCR amplification primers (Table 2), with additional spacers at the 5' and 3' ends. The master synthetic template was used to generate a modified library in which all dU and/or dC positions were uniformly modified in the alternative primer extension reaction.

A total of 18 enzymatic synthesis libraries were included, containing single-modified dUs (Nap-dU, PP-dU, MOE-dU, Tyr-dU, and/or Thr-dU), with unmodified dT serving as a control; single-modified dCs (Nap-dC and PP-dC), with unmodified dC serving as a control; and combinations of two modified bases: Nap-dC or PP-dC with all possible modified dUs (Nap-dU, PP-dU, MOE-dU, Tyr-dU, and Thr-dU). Qualitative primer extension reactions were performed in solution using an antisense template, radiolabeled 5' primers, and natural or modified nucleotide triphosphates and KOD polymerase (exonuclease) (in triplicate). In a 60 μL primer extension reaction, a 20 pmol biotinylated antisense library was mixed with 40 pmol of 5' cold primer (2X) and trace amounts of ^32P -labeled 5' primers, 0.5 mM native or modified dNTPs, and 0.25 U/mL KOD polymerase (exonuclease) in 1X SQ20 buffer (120 mM Tris-HCl, pH 7.8; 10 mM KCl; ₆ mM ( _NH₄ ) _₂SO₄ ; ₇ mM MgSO₄, 0.1% Triton X-100, and 0.1 mg/mL BSA). The mixture was thermally cooled, followed by the addition of DNA polymerase and the reaction was carried out at 68 °C for 2 h, followed by cooling at 10 °C. Samples from each library reaction, along with free labeled primers, were run on a 10% TBU urea gel. Small aliquots were run on denaturing gels exposed to a fluorescent screen and imaged using a Fuji phosphorograph. Band quantification was performed using ImageGauge 4.0 software, and the results were plotted using Graph Pad Prism 6.05 software. For initial library preparation, large-scale primer extension reactions were performed using master biotinylated antisense random libraries captured on Pierce ^™ high-capacity streptavidin agarose beads (Life Technologies). Lower library yields were obtained for certain combinations of two modified nucleotides compared to a 100% unmodified library DNA (dC/dT) control, for example, 28% ± 1.3% for Nap-dC/Nap-dU, 40% ± 5.2% for Nap-dC/MOE-dU, and 43% ± 2.7% for PP-dC/Nap-dU. The frequencies of each nucleotide were calculated from sequencing results obtained from the initial library and master-antisense random plates used to generate single and two-base-modified libraries. The main random antisense template (30N) was chemically synthesized at a scale of 1 μM using a 1:1:1:1 ratio of dA:dG:dC:dT (TriLink Biotechnologies). Initial random single-base and two-base modified libraries were enzymatically synthesized in a large-scale reaction and used in selection experiments. These libraries, along with enrichment pools, were sequenced, and nucleotide frequencies were plotted for all four bases in the 30N random region at a total of 100%. No significant deviation in nucleotide frequencies was observed when the base composition of the libraries was determined using deep sequencing, compared to the initial synthetic native DNA template library and the enzymatically synthesized unmodified DNA control library (data not shown).

The library was used to select aptamers that bind to PCSK9. The selection was performed substantially as previously reported using dextran sulfate as a polyanionic competitive agent, for a total of six rounds, with incremental target dilutions applied during each successive round of selection. See Table 1 (R1 = Round 1, R2 = Round 2, etc.). Selection was initiated by mixing a modified random library (or an unmodified control) (≥1000 pmol) with the human recombinant His-tagged target protein PCSK9, present at a concentration of 0.5 μM in a 100 μL volume. The selected complexes were partitioned onto magnetic His-tagged traps, unbound sequences were washed, the selected aptamers were eluted, and PCR amplification was performed using all native nucleotides and 3' biotin-primers. Native double-stranded DNA with biotinylated at the 3' end was trapped on MyOne ^™ streptavidin C1 beads, the sense strand was eliminated by alkaline denaturation, and the enrichment pool was regenerated by replacement with modified dC and/or dU in the primer extension reaction, and the selection cycle was repeated with diluted protein. The protein concentration used for the next round of SELEX is determined based on the signal-to-background ratio calculated from the critical cycle time (Ct) value for each sample.

Table 1: In vitro selection criteria

The 5' primer used for amplification contains an (AT4)-tail and the 3' primer contains an (A-biotin)2-T8-tail (SEQ ID NO: 82), which avoids the need to add modified dC or dU during the synthesis of the modified library.

Table 2: Sequences of natural DNA templates and primers used in SELEX

Following six rounds of selection, aptamers containing native nucleotides were deep sequenced using an Ion Torrent PGM instrument. Sequence analysis was performed using custom software with partial batch alignment. Data from sequence analysis of all enrichment pools demonstrated that the combination of two modified libraries resulted in greater diversity of enriched sequences compared to a single modified library (data not shown). To test the binding affinity of the aptamers, a broad set of sequences was selected, representing not only high-copy-unique sequences but also low-copy sequences from different families (data not shown). All aptamers were chemically synthesized using modified/unmodified phosphoramidite reagents via standard solid-phase phosphoramidite chemistry. All aptamers were initially screened in solution by radiolabeled filter binding assays to truncated 40-nucleotide polymers containing a 30-nucleotide random region and an additional 5 nucleotides from the 5' and 3' ends of the fixed primer regions for their PCSK9 binding affinity. The truncated aptamers (40-nucleotide polymers) contained 10 nucleotides from the fixed regions. Unmodified control DNA libraries (dC/dT) did not produce any active sequences ( _Kd < 32 nM), which was expected because pool affinity for this library was monotonic (data not shown) and additionally, deep sequencing data did not produce any enriched multicopy sequences (data not shown). Single-modification libraries with Nap (naphthyl) modification on dC or dU produced aptamers with affinity for the target; however, the aptamers with the highest affinity for the target were obtained from dC with Nap (naphthyl) or PP (benzyl) modification on dU with Tyr (tyrosine moiety) modification (Figure 2). Replacing Tyr-dU with dT canceled binding to the target, indicating the importance of the tyrosine moiety in the binding interaction with the target surface of PCSK9 (data not shown). Affinity data demonstrate that aptamers with two modified nucleotides generally have greater affinity than aptamers with a single modified nucleotide, and also provide a greater number of aptamers binding PCSK9 compared to aptamers with a single modified nucleotide (Figure 3). Furthermore, aptamers with high copy numbers of a single modified nucleotide have average affinity between 0.1 and 100 nM, while aptamers with high copy numbers of two modified nucleotides have average affinity ≤0.1 nM.

A summary of the data comparing the single-modified aptamer (40-mer) and the dual-modified aptamer (40-mer) for PCSK9 is shown in Table 3 below.

Table 3. Overview of binding data for single and dual-modified aptamers for PCSK9

Based on the information in Table 3, the percentage of all determined single-modification aptamers that did not show binding was 62%. Non-binding was defined as an aptamer with a Kd of 320 nM or greater. The percentage of all single-modification aptamers with a Kd ≤ 10 nM was less than 21%, and the average Kd of all single-modification aptamers was 5.2 nM. In contrast, the percentage of all determined double-modification aptamers that did not show binding was 43%. Furthermore, the percentage of all double-modification aptamers with a Kd ≤ 10 nM was 47%, and the average Kd of all double-modification aptamers was 0.12 nM.

Example 2: Truncation of Dual-Modified Aptamers

The study investigated the effect of further truncation on binding to high-affinity ( _Kd < 1 nM) aptamers. Truncation of the aptamers to 30-mers resulted in a 25% length reduction. The PP-dC/Tyr-dU combination exhibited the highest number of aptamers that could be truncated to 30-mers while maintaining high affinity (Figure 4A). Single-base modified aptamers showed 21.5% truncation (blue bar, 3/14), two-base modified Nap-dC aptamers with modified dU showed approximately 23% truncation (red bar, 11/48), while two-base modified PP-dC aptamers with other modified dU showed 60% enhanced truncation (green bar, 27/45). For two-base modified combinations of PP-dC with PP-dU, Nap-dU, or Tyr-dU, the percentage and number of 40-mers that could be truncated to 30-mers were also higher compared to other libraries (Figure 4B). Fewer aptamers were tested from libraries with single-base modifications, as only 14 aptamers across the three libraries exhibited an affinity ≤1 nM (40-mers, gray area in Figure 4B for single modifications). In contrast, the number of aptamers from libraries with two base modifications exhibiting high affinity was 93 (40-mers, gray area in Figure 4B for both modifications), 48 for Nap-dC with modified dU, and 45 for PP-dC with modified dU. The black horizontal lines on each library indicate the median for all aptamers in that library. Without intending to be bound by any particular theory, it is possible that the extended carbon chains in PP-modified bases (compared to other modifications) facilitate access to epitopes that are difficult to access on the target surface and eliminate the need for fixed primer regions for structural folding and effective protein-protein binding interactions.

The specificity of the PCSK9 aptamer for several other protoprotein convertases (PCs) was also evaluated. Three highest-affinity aptamers (n = 33, 40-mers; none from the unmodified DNA control library, only two aptamers from the dC/Tyr-dU library and one aptamer from the PP-dC/MOE-dU library) were selected from each library, and their specificity for other PCs was tested. The results demonstrated that the aptamers were specific for PCSK9, and no detectable binding was observed with other PCs at a concentration of 100 nM (Figure 5).

Cross-species reactivity of truncated aptamers (n = 41, 30-mers) with Kd values ≤1 nM was tested for PCSK9 in rodents (mice and rats) and rhesus monkeys (see Table 4). The percentage of identity among PCSK9 from multiple species is shown at the top of the graph in Figure 6. Mouse/rat PCSK9 is approximately 76% identical to monkey and human proteins. Most aptamers bind rhesus monkey PCSK9 with similar affinity (96.4% identity); however, fewer aptamers from the two modified libraries (PP-dC/Nap-dU and PP-dC/Tyr-dU) bind rat and mouse PCSK9 (approximately 76% identity). These results demonstrate that certain two-base-modified libraries (e.g., PP-dC/Nap-dU and PP-dC/Tyr-dU) produce aptamers that can bind rodent and human/monkey PCSK9 with similar affinity (Figure 6).

Table 4. Cross-species binding activity of single and dual-modified aptamers

A "-" indicates that no binding was detected in the assay.

Example 3: Aptamer binding in sandwich assay

The SELEX method sometimes yields aptamers that preferentially bind to dominant "aptagenic" epitopes on target surfaces. Therefore, reports on aptamer sandwich pairs are limited to the literature. Modifications to the selection method can be used to search for aptamers that can bind to different epitopes on target proteins, such as Multivalent Aptamer Separation (MAI-SELEX) and Array-Based Discovery Platform for Multivalent Aptamers (AD-MAP) sandwich selection, where primary aptamers are overused to block the first epitope, thereby attempting to discover a second aptamer that binds to a non-competitive signaling epitope. To demonstrate whether the chemical diversity resulting from the multiplicity of modifications via dC and dU combinations expands when selecting aptamers that can bind to different epitopes on target surfaces, a bead-based sandwich pair screening assay was developed, in which avidin-coupled magnetic beads are used to capture biotinylated primary aptamers (Figure 7A). Capture beads containing individual aptamers are mixed together and used to search for second binding pairs in multiple pair combinations (Figure 7A). For this experiment, 40-mer aptamers (n = 96, 9216 pairs) with affinity K_{_d} ≤ 1 nM were used from single and two base-modified libraries. In short, individual aptamers (0.05 pmol per sample) were captured onto a single MagPlex avidin bead type and mixed together (24 beads in one experiment, 1000 beads per sample) and captured at room temperature with shaking at 1850 rpm for 20 min. The beads were washed with 1X SBT for 2 min, followed by washing with 0.5 mM free biotin in 1X SBT for 5 min, and then washed three times with 1X SBT for 2 min each time. The beads were blocked with free streptavidin for 5 min and washed again with 1X SBT for 2 min. The 24 different bead types with individual aptamers were mixed together to screen sandwich pairs for each captured aptamer. The detection or secondary aptamer was diluted to 500 nM in 1X SBT, thermally cooled, and mixed with PCSK9 (final 10 nM), then incubated at 25 °C for 1 h. 1000 capture beads were added and incubated further with shaking for 1 h. The beads were then captured on a magnet, washed three times with 1X SBT for 2 min each time, and resuspended in 75 μL of 1X SBT containing 0.1% BSA and 100 μM x SO4. 75 μL of the antibiotic streptavidin phycoerythrin (final 5 μg/mL) was added, and the mixture was incubated at 25 °C with shaking for 20 min. The beads were finally washed again with 1X SBT for 2 min and read on a Luminex 3D xMAP instrument.

The single-base modified library produces a small number of sandwich pairs (three), while adding aptamers from two base modified libraries in combination with the single-base modified aptamer produces a larger number of sandwich pairs (22 pairs). Furthermore, when the two pairing (capture and detection) aptamers are from two base modified libraries, the number of sandwich pairs in each library increases dramatically (45 pairs, Fig. 7C, Fig. 7B).

Multiple epitope binding results from sandwich screening indicate that increased chemical diversity in the initial random library leads to modified aptamers that can bind to non-competitive sites on the target surface. Subsequently, the PCSK9 concentration-dependent responses of the sandwich pairs producing the highest signals (10 nM PCSK9 concentration; 0.75% of all pairs tested, 70 out of 9216 pairs; Fig. 7C) were measured, with subsets of the results shown in Fig. 8A (showing the concentration-dependent signals for the single-base modified primary aptamer dC/PP-dU and the optimal secondary aptamer Nap-dC/Nap-dU with good function [triangle]) and 8B (showing the concentration-dependent signals for the two-base modified secondary aptamer Nap-dC/Nap-dU and the optimal primary aptamer with good function [closed square]). Interestingly, a specific pair constituting a single-base-modified primary aptamer (PP-dU, affinity Kd 175 pM) and two-base-modified secondary aptamers (Nap-dU/Nap-dC, affinity Kd 531 pM) produced a much stronger stable signal in one orientation than any other pair (Fig. 8C). However, when this single-base-modified primary aptamer converted to the secondary aptamer, the signal was lost, indicating that the orientation of the aptamer was important for this sandwich pair. This aptamer sandwich pair can also be used to measure the activity of the gain-of-function mutant protein PCSK9D374Y (Fig. 8D), which has a higher affinity for LDL-R than wild-type PCSK9 and has been reported to be overexpressed in patients with severe form of familial hypercholesterolemia (FH). The sensitivity and MFU value of the mutant PCSK9 D374Y are higher than those of the wild-type protein.

The specificity of aptamer sandwich pairs was also measured by the lack of endogenous signal compared to human plasma (data not shown) when recombinant human PCSK9 was incorporated into newborn calf serum (NBCS).

This sandwich assay was further characterized to develop an aptamer sandwich assay for detecting circulating concentrations of plasma PCSK9 in human clinical samples. The performance of the sandwich assay was evaluated through studies such as sensitivity (Figures 9A and 9B and Tables 5 and 6), precision (Tables 7 and 8), accuracy (Table 9), and plasma dilution linearity measurements (Figure 10), all of which demonstrated a robust analytical window. To assess the sample dilution linearity of the assay, the sandwich assay was performed using samples containing and/or doped with high concentrations of PCSK9. Plasma samples (n = 5) were continuously diluted with assay buffer to fit values within the dynamic range of the assay.

The limit of detection (LLoD), shown in Table 5, is defined as the PCSK9 concentration (40 pg/mL) that produces an RFU value higher than the average RFU of the blank (dilution buffer) plus 3 standard deviations. The lower limit of quantification (LLoQ) and upper limit of quantification (ULoQ), shown in Table 6, are defined as the lowest and highest PCSK9 concentrations that can be quantified using a 4-parameter logic (4PL) applied to the standard curve, resulting in 80%–120% recovery of the known target concentration. To determine intra-assay variability, five plasma samples at known concentrations were tested 16 times in a single plate. See Table 7. The coefficient of variation (CV) within the assays ranged from 4.3% to 6%. To determine inter-analytical variability, five plasma samples at known concentrations were measured in five independent assays. See Table 8. The CV between assays ranged from 2.3% to 9.8%. Finally, to determine the accuracy of the target measurements, five plasma samples were infused with different amounts of PCSK9 and measured. The recovery of PCSK9 levels infused within the ranges of the assays was evaluated. See Table 9. The recovery percentage of the samples ranged from an average of 83.1% to 137.5% of the doped target.

The collection of plasma samples obtained from two groups of individuals (a control group (n=42) and another study group (n=42, via self-reporting) in which subjects received statine therapy) was evaluated to determine whether the assay was statistically distinguishable between the two groups, as statine use is known to increase plasma PCSK9 concentrations. The sandwich assay was developed using a capture or primary SOMA polymer (11723-5) as a single-base modified aptamer (PP-dU/dC) and a secondary or detection aptamer (11727-20) as two-base modified aptamers (Nap-dC/Nap-dU).

These results indicate that the aptamer sandwich assay can be statistically distinguished between the two groups by Mann-Whitney analysis with a p-value of 0.0044 (Figure 11), and this assay may be used to identify individuals who may benefit from anti-PCSK9 therapy due to their high plasma concentrations of PCSK9.

Aptamer sandwich assays were also used to measure the concentration of PCSK9 in cell-free supernatant from PCSK9-overexpressing HepG2 cells to identify overexpressing clones and demonstrate the research utility of the assay (Figure 12). PCSK9 was overexpressed in HepG2 cells using the SBISystem Biosciences LentiViral system (LV300A-1). The HepG2 cell lines were transduced with lentiviral expression clones derived from Origene (RC220000L1) for wild-type human PCSK9 to generate stable cell lines. A total of 96 individual clones were screened for their PCSK9 secretion capacity. The relative amount of PCSK9 secreted in the culture medium was measured by aptamer sandwich assays for each clone and compared with expression from wild-type HepG2 cells. The number of cells used to produce the recombinant protein was normalized using a Cell luminescence cell viability assay. Clones number 45 secreted approximately 100 times more PCSK9 than wild-type HepG2 cells.

Table 5: Sensitivity (Lower Limit of Detection) of Sandwich Assay

Table 6: Sensitivity (Lower Limit of Quantification) of Sandwich Assay

Table 7: Accuracy within the Measurement

Table 8: Accuracy between measurements

Table 9: Accuracy of Target Measurements

Example 4: Target activity inhibition via dual-modified aptamers

To identify PCSK9 inhibitors that block the binding of PCSK9 to LDL-R, 41 truncated 30-mer aptamers with Kd ≤ 1 nM were screened for biotinylated PCSK9 binding using a chemiluminescent reagent in a plate-based assay. The plates were coated with LDL-R, and the binding of biotinylated PCSK9 was detected using an antibiotic streptavidin-HRP conjugate (Figure 13). Recombinant LDL-R (Acro Biosystems) was plated on ELISA plates (2.5 μg/mL) overnight at 4 °C, followed by washing of the wells at room temperature and blocking with Super Block (Invitrogen) for 1 h. Biotinylated PCSK9 (Acro Biosystems, Avi-labeled) and the aptamer were mixed and incubated at room temperature for 1 h, then added to the ELISA plates and further incubated at room temperature with shaking for 2 h. The highest PCSK9 concentration was 0.5 nM and the highest aptamer concentration was 100 nM. These concentrations were then serially diluted with a 1/2 log value to the inhibition curve. HRP (Invitrogen, 1 μg/ml) conjugated with streptavidin was added to each well and incubated at room temperature with shaking for 30 min. Pico-sensitive chemiluminescent substrate (Invitrogen) was then added, and luminescence was measured using a Hidex Plate Chameleon spectrophotometer. The data were plotted using Graph Pad Prism 6.0 software to calculate the _EC50 value. A PCSK9 neutralizing antibody (BPS Bioscience) was used as a control.

Results from the inhibition assays (test concentrations of aptamers at 100 nM and PCSK9 at 1 nM) showed that 70% of the aptamers were inhibitors (over 90% inhibition) and some of the two modified aptamers potentially inhibited the interaction between PCSK9 and LDL-R (data not shown). The aptamers were further evaluated against dose-response curves to determine their _EC50 values for inhibition. The results indicated that many inhibitors potentially inhibited the interaction between PCSK9 and LDL-R with _EC50 in the range of 0.1–1 nM (Figure 14). To demonstrate the potential therapeutic value of the two modified aptamers, a species-cross-reactive PCSK9 aptamer (30-mer, Seq ID. 11733-915(11733-198)) was selected to measure the affinity of the target for PCSK9 from multiple species. This aptamer exhibits affinities of 14.7, 11.3, 5.2, 77, and 165 pM for PCSK9 in humans (wild-type), rhesus monkeys, humans (GOF mutant D374Y), mice, and rats, respectively (Fig. 15A). This aptamer also blocks the interaction of wild-type human PCSK9 LDL-R with an _EC50 of 2.1 nM and the interaction of GOF mutant PCSK9 D374Y LDL-R with an _EC50 of 3.6 nM (Fig. 15B). The specificity of this aptamer for PCSK9 was evaluated compared to other PCs, and the results showed that this aptamer binds only to PCSK9 and not to other PCs (data not shown).

To test the neutralizing effect of the PCSK9 aptamer in blocking LDLR degradation, the PP-dC/Nap-dU aptamer was tested in an LDL uptake reversal assay, in which wild-type HepG2 cells were incubated with recombinant PCSK9 for 16 h, followed by cell washing and the addition of fluorescently labeled LDL for 3 h. The results showed that the aptamer could reverse LDL uptake at 159 nM _EC50 (Figs. 16 and 17). Furthermore, aptamer treatment increased LDL-R levels in PCSK9-overexpressing HepG2 cells, as measured by FACS (Fig. 17). These results, obtained from species-cross-reactivity, high affinity, truncation, specificity, and high efficiency aptamers, indicate the potential therapeutic value of both base-modified aptamers, which can be further optimized for length and biostability through SELEX post-modification.

Example 5: Serum stability of dual-modified aptamers

To determine the serum stability of the dual-modified aptamer in human serum, 1 μM gel-purified aptamer was incubated at 37°C in 90% combined human serum in PBS buffer at a total volume of 200 μL. At multiple time points, 20 μL aliquots were collected and an equal volume of an EDTA/formamide/dye mixture (formamide 87.7%, SDS 0.03%, sodium EDTA 20 mM, xylene blue 0.05%, bromophenol blue 0.05%, Orange G 0.1%) was added. The aliquot mixtures were then stored at -20°C. Prior to analysis, 40 μL aliquot mixtures were diluted with 100 μL _H₂O and extracted with 150 μL of 25:24:1 phenol:chloroform:isoamyl alcohol. The samples were centrifuged at 16, 100 x g for 15 min, and the aqueous phase containing the aptamer was removed and stored at -20°C until gel analysis.

The aptamer samples were loaded onto 15% TBE PAGE denaturing gel (8M urea) and stained with 1X (approximately 2 μM) SYBR gold for 10 minutes. The amount of full-length aptamers at each time point was quantified using FluorChemQ analysis software (AlphaInnotech).

The results of this experiment are shown in Table 10 and Figure 18. Table 10 shows the composition of the aptamers tested in this experiment, the percentage of full-length aptamers remaining in 90% human serum after 96 hours, and the half-life of each aptamer, calculated using GraphPad Prism 7 software with gel quantification data fitted by linear regression. Figure 18 shows the percentage of full-length aptamers remaining over time. In general, dual-modified aptamers demonstrated greater stability over time in human serum than single-modified aptamers.

Table 10: Aptamer composition.

Example 6: Aptamers containing two modified bases

The libraries described in Example 1 were used to select aptamers that bind to ErbB2, ErbB3, and PSMA. The selection was essentially performed target-specific as described in Example 1. For ErbB2 and ErbB3, single-modified Nap-dC/dT, PP-dC/dT, and dC/Tyr-dU libraries, as well as dual-modified Nap-dC/Tyr-dU and PP-dC/Tyr-dU libraries, were used. For PSMA, use unmodified dC/dT libraries; single-modified Nap-dC/dT; PP-dC/dT; dC/Nap-dU, dC-PP-dU, dC-MOE-dU, and dC/Tyr-dU libraries; and double-modified Nap-dC/Nap-dU, Nap-dC/PP-dU, Nap-dC/MOE-dU, Nap-dC/Tyr-dU, PP-dC/PP-dU, PP-dC/Nap-dU, and PP-dC/Tyr-dU libraries.

As before, the unmodified control DNA library (dC/dT) for PSMA did not produce any aptamers that bind to PSMA. Single-modified libraries with Nap modification (naphthyl moiety) on dC or dU produced binding agents for all three targets; however, dual-modified libraries provided aptamers with greater affinity than single-modified libraries (Figures 19A-19C).

A summary of the data comparing the single-modified aptamer (40-mer) and dual-modified aptamer (40-mer) of each of PSMA, ErbB2, and ErbB3 is shown in Tables 11, 12, and 13, respectively.

Table 11. Overview of binding data for single and dual-modified aptamers of PSMA

N.T. means "Untested"; N/A means "Not Applicable" or "No Data Available".

Based on the information in Table 11, 71% of all determined single-modified aptamers did not show binding. Non-binding was defined as an aptamer with a Kd of 320 nM or greater. 12% of all single-modified aptamers had a Kd ≤ 10 nM, and the average Kd for all single-modified aptamers was 12.3 nM. In contrast, 54% of all determined double-modified (dual-modified) aptamers did not show binding. Furthermore, 20% of all double-modified aptamers had a Kd ≤ 10 nM, and the average Kd for all double-modified aptamers was 6.6 nM.

Table 12. Overview of binding data for single and dual-modified aptamers of ERBB2

Based on the information in Table 12, 70% of all determined single-modified aptamers did not show binding. Non-binding was defined as an aptamer with a Kd of 320 nM or greater. The percentage of all single-modified aptamers with a Kd ≤ 10 nM was less than 2%, and the average Kd of all single-modified aptamers was 15.1 nM. In contrast, 44% of all determined double-modified (dual-modified) aptamers did not show binding. Furthermore, 25% of all double-modified aptamers had a Kd ≤ 10 nM, and the average Kd of all double-modified aptamers was 0.7 nM.

Table 13. Overview of binding data for single and dual-modified aptamers of ERBB3

Based on the information in Table 13, the percentage of all determined single-modified aptamers that did not show binding was 38%. Non-binding was defined as an aptamer with a Kd of 320 nM or greater. The percentage of all single-modified aptamers with a Kd ≤ 10 nM was 40%, and the average Kd of all single-modified aptamers was 6 nM. In contrast, the percentage of all determined double-modified (dual-modified) aptamers that did not show binding was 24%. Furthermore, the percentage of all double-modified aptamers with a Kd ≤ 10 nM was 69%, and the average Kd of all double-modified aptamers was 0.02 nM.

Example 7: Other aptamers containing two modified bases

Libraries comprising the modification pairs shown in Table 14 are prepared as follows. In some embodiments, each library contains 40 or more randomized nucleotides. In some embodiments, each library contains 30 randomized nucleotides, thereby allowing ^≥10¹⁵ different sequences. The libraries can be synthesized using exonuclease KOD DNA polymerase using natural and/or modified nucleotide triphosphates. In some embodiments, the random regions are flanked by fixed sequences for hybridization PCR amplification primers, with or without additional spacer regions at the 5' and 3' ends. In some cases, a master synthetic template is used to generate modified libraries in which all dU and/or dC positions are uniformly modified in the alternative primer extension reaction. Library synthesis can be performed substantially as described in Example 1.

Table 14: Dual-Modified Adapter Library

One or more libraries in Table 14 can be used to select aptamers that bind to targets, such as protein targets. Libraries containing two modified bases typically produce aptamers with high specificity and/or affinity for the target.

Example 8: Exemplary Dual-Modified Adaptor

PCSK9 binding aptamers from the conserved sequence family of pool 11720 (Nap-dC/dT) are shown in Table 15. Only random regions of each sequence are shown. The copy number of each sequence is indicated in the total of 11,380 sequences (identical or equal to up to 5 mismatches). All aptamers in this family share the conserved sequence element TTppGGpp, where p = Nap-dC. Aptamer 11730-6 (SEQ ID No: 4, Kd = 0.1 nM) is a representative from this pool selected for metabolic stability assays.

Table 15: Aptamers from Pool 11720

PCSK9 binding aptamers from the conserved sequence family of pool 11730 (Nap-dC/Tyr-dU) are shown in Table 16. Only random regions of each sequence are shown. The copy number of each sequence is indicated in the total of 17,695 sequences (identical or equal to up to 5 mismatches). All aptamers in this family share the conserved sequence element yGpppG, where p = Nap-dC and Y = Tyr-dU. Many sequences also contain the conserved sequence element yyAyGpAp. Aptamer 11730-19 (SEQ ID No: 27, Kd = 0.2 nM) is representative from this pool selected for metabolic stability assays.

Table 16: Aptamers from Pool 11730

PCSK9 binding aptamers from the conserved sequence family of pool 11733 (Pp-dC/Nap-dU) are shown in Table 17. Only random regions of each sequence are shown. The copy number of each sequence in a total of 16,118 sequences is indicated (identical or equal to up to 5 mismatches). All aptamers in this family share the conserved sequence element rPPPAAGGrrPAPPG (SEQ ID NO: 83), where r = Pp-dC and P = Nap-dU. Aptamer 11733-44 (SEQ ID NO: 44, SL1063) is the most potent 30-mer inhibitor of wild-type human PCSK9 ( _IC50 = 2.8 nM). Aptamer 11733-198 (SEQ ID No: 46, _Kd = 0.07 nM) is a representative selected from this pool for metabolic stability assays.

Table 17: Aptamers from Pool 11733

Claims (57)

1. An aptamer comprising a first 5-position modified pyrimidine and a second 5-position modified pyrimidine, wherein the first 5-position modified pyrimidine and the second 5-position modified pyrimidine are different 5-position modified pyrimidines; Wherein the first 5-position modified pyrimidine is a 5-position modified uracil, and wherein the second 5-position modified pyrimidine is a 5-position modified cytosine; or Wherein the first 5-position modified pyrimidine is a 5-position modified cytosine and wherein the second 5-position modified pyrimidine is a 5-position modified uracil; The 5-position modified uracil is contained in a tyrosine methyl group at the 5-position; and the 5-position modified cytosine is contained in a naphthyl methyl group at the 5-position; The tyrosine acyl moiety is Tyr, and the naphthyl moiety is Nap, 2Nap, NE, or 2NE. 2. The aptamer of claim 1, wherein the portion of the 5-position modified uracil is covalently linked by a connector comprising a group selected from amide connectors, carbonyl connectors, propynyl connectors, alkyne connectors, ester connectors, urea connectors, carbamate connectors, guanidine connectors, amidine connectors, sulfoxide connectors, and sulfone connectors. 3. The aptamer of claim 1 or claim 2, wherein the portion of the 5-position modified cytosine is covalently linked by a connector, the connector comprising a group selected from amide connectors, carbonyl connectors, propynyl connectors, alkyne connectors, ester connectors, urea connectors, carbamate connectors, guanidine connectors, amidine connectors, sulfoxide connectors, and sulfone connectors. 4. The aptamer according to claim 1 or claim 2, wherein the 5-position modified cytosine is 5-(N-1-naphthylmethylformamide)-2'-deoxycytosine nucleoside (NapdC). 5. The aptamer according to claim 1 or claim 2, wherein the 5-position modified uracil is 5-(N-tyrosinylformamide)-2'-deoxyuracil nucleoside (TyrdU). 6. The aptamer of claim 1, wherein the first 5-position modified pyrimidine is NapdC and the second 5-position modified pyrimidine is TyrdU. 7. The aptamer of claim 1, claim 2 or claim 6, wherein the aptamer comprises at least 10, at least 15, at least 20, at least 25 or at least 30 nucleotides in length, or 5 to 30, 10 to 30, 15 to 30, 5 to 20 or 10 to 20 nucleotides in length at the 5' end of the aptamer, wherein the region at the 5' end of the aptamer lacks a 5-position modified pyrimidine. 8. The aptamer of claim 1, claim 2 or claim 6, wherein the aptamer comprises at least 10, at least 15, at least 20, at least 25 or at least 30 nucleotides in length, or 5 to 30, 10 to 30, 15 to 30, 5 to 20 or 10 to 20 nucleotides in length at the 3' end of the aptamer, wherein the region at the 3' end of the aptamer lacks a 5-position modified pyrimidine. 9. The aptamer of claim 1, claim 2 or claim 6, wherein the aptamer is 20 to 100, or 20 to 90, or 20 to 80, or 20 to 70, or 20 to 60, or 20 to 50, or 30 to 100, or 30 to 90, or 30 to 80, or 30 to 70, or 30 to 60, or 30 to 50, or 40 to 100, or 40 to 90, or 40 to 80, or 40 to 70, or 40 to 60, or 40 to 50 nucleotides in length. 10. A composition comprising multiple polynucleotides, wherein each polynucleotide comprises a first 5-position modified pyrimidine and a second 5-position modified pyrimidine, wherein the first 5-position modified pyrimidine and the second 5-position modified pyrimidine are different 5-position modified pyrimidines; Wherein the first 5-position modified pyrimidine is a 5-position modified uracil, and wherein the second 5-position modified pyrimidine is a 5-position modified cytosine; or Wherein the first 5-position modified pyrimidine is a 5-position modified cytosine and wherein the second 5-position modified pyrimidine is a 5-position modified uracil; The 5-position modified uracil is contained in a tyrosine methyl group at the 5-position; and the 5-position modified cytosine is contained in a naphthyl methyl group at the 5-position; The tyrosine acyl moiety is Tyr, and the naphthyl moiety is Nap, 2Nap, NE, or 2NE. 11. The composition of claim 10, wherein each polynucleotide includes a fixed region at the 5' end of the polynucleotide. 12. The composition of claim 11, wherein the fixed region at the 5' end of each polynucleotide is at least 10, at least 15, at least 20, at least 25, or at least 30 nucleotides long, or 5 to 30, 10 to 30, 15 to 30, 5 to 20, or 10 to 20 nucleotides long. 13. The composition of any one of claims 10 to 12, wherein each polynucleotide includes a fixed region at the 3' end of the polynucleotide. 14. The composition of claim 13, wherein the fixed region at the 3' end of the polynucleotide is at least 10, at least 15, at least 20, at least 25, or at least 30 nucleotides in length, or 5 to 30, 10 to 30, 15 to 30, 5 to 20, or 10 to 20 nucleotides in length. 15. The composition of any one of claims 10 to 12, or claim 14, wherein the portion of the 5-position modified uracil is covalently linked by a connector comprising a group selected from amide connectors, carbonyl connectors, propynyl connectors, alkyne connectors, ester connectors, urea connectors, carbamate connectors, guanidine connectors, amidine connectors, sulfoxide connectors, and sulfone connectors. 16. The composition of any one of claims 10 to 12, or claim 14, wherein the portion of the 5-position modified cytosine is covalently linked by a linker comprising a group selected from amide linkers, carbonyl linkers, propynyl linkers, alkyne linkers, ester linkers, urea linkers, carbamate linkers, guanidine linkers, amidine linkers, sulfoxide linkers, and sulfone linkers. 17. The composition of any one of claims 10 to 12, or claim 14, wherein the 5-position modified cytosine is NapdC. 18. The composition of any one of claims 10 to 12, or claim 14, wherein the 5-position modified uracil is TyrdU. 19. The composition of any one of claims 10 to 12, or claim 14, wherein the first 5-position modified pyrimidine is NapdC and the second 5-position modified pyrimidine is TyrdU. 20. The composition of any one of claims 10 to 12, or claim 14, wherein each polynucleotide comprises a random region. 21. The composition of claim 20, wherein the random regions are 20 to 100, or 20 to 90, or 20 to 80, or 20 to 70, or 20 to 60, or 20 to 50, or 20 to 40, or 30 to 100, or 30 to 90, or 30 to 70, or 30 to 60, or 30 to 50, or 30 to 40 nucleotides in length. 22. The composition of any one of claims 10 to 12, 14, or 21, wherein each polynucleotide is 20 to 100, or 20 to 90, or 20 to 80, or 20 to 70, or 20 to 60, or 20 to 50, or 30 to 100, or 30 to 90, or 30 to 80, or 30 to 70, or 30 to 60, or 30 to 50, or 40 to 100, or 40 to 90, or 40 to 80, or 40 to 70, or 40 to 60, or 40 to 50 nucleotides in length. 23. A composition comprising an aptamer and a target, wherein the aptamer and the target are capable of forming a complex, and wherein the aptamer is the aptamer as claimed in any one of claims 1 to 9. 24. A composition comprising a first aptamer, a second aptamer, and a target, The first aptamer comprises a first 5-position modified pyrimidine and a second 5-position modified pyrimidine; The second aptamer comprises a third 5-position modified pyrimidine or the second aptamer comprises a third 5-position modified pyrimidine and a fourth 5-position modified pyrimidine; The first aptamer, the second aptamer, and the target can form a trimeric complex; and The first 5-position modified pyrimidine and the second 5-position modified pyrimidine are different 5-position modified pyrimidines; and The first aptamer is the aptamer as described in any one of claims 1 to 9. 25. The composition of claim 24, wherein the target is selected from proteins, cells, and tissues. 26. A method comprising: (a) To bring the aptamer, which can bind to the target molecule, into contact with the sample; (b) Incubate the aptamer with the sample to allow aptamer-target complex formation; (c) Enriching the aptamer-target complex in the sample, and (d) Detecting the presence of the aptamer, the aptamer-target complex, or the target molecule, wherein the detection of the aptamer, the aptamer-target complex, or the target molecule indicates that the target molecule is present in the sample, and wherein the absence of the detection of the aptamer, the aptamer-target complex, or the target molecule indicates that the target molecule is not present in the sample; The aptor is the aptor as described in any one of claims 1 to 9. 27. The method of claim 26, wherein the method includes an additional step selected from: adding a competing molecule to the sample; capturing the aptamer-target complex on a solid support; and adding the competing molecule and diluting the sample; wherein the additional step occurs after step (a) or step (b). 28. The method of claim 27, wherein the competing molecule is selected from polyanionic competing agents. 29. The method of claim 28, wherein the polyanionic competitive agent is selected from oligonucleotides, dextran, DNA, heparin, and dNTPs. 30. The method of claim 29, wherein the dextran is sulfated dextran; and the DNA is herring sperm DNA or salmon sperm DNA. 31. The method of any one of claims 26 to 30, wherein the target molecule is selected from proteins, cells, and tissues. 32. The method of any one of claims 26 to 30, wherein the sample is selected from whole blood, white blood cells, peripheral blood mononuclear cells, plasma, serum, sputum, exhaled air, urine, semen, saliva, meningeal fluid, amniotic fluid, glandular fluid, lymph, nipple aspiration fluid, bronchial aspiration fluid, synovial fluid, joint aspiration fluid, cell extract, feces, tissue, tissue biopsy, and cerebrospinal fluid. 33. The method of any one of claims 26 to 30, wherein the sample is cells. 34. A method for detecting a target in a sample, comprising: a) Contact the sample with a first aptamer to form a mixture, wherein the first aptamer is capable of binding the target to form a first complex; b) Incubate the mixture under conditions that allow the formation of the first complex; c) Contact the mixture with a second aptamer, wherein the second aptamer is capable of binding the first complex to form a second complex; d) Incubate the mixture under conditions that allow the formation of the second complex; e) Detect the presence or absence of the first aptamer, the second aptamer, the target, the first complex, or the second complex in the mixture, wherein the presence of the first aptamer, the second aptamer, the target, the first complex, or the second complex indicates that the target is present in the sample; The first aptamer is the aptamer as described in any one of claims 1 to 9; wherein the second aptamer comprises a third 5-position modified pyrimidine, or wherein the second aptamer comprises a third 5-position modified pyrimidine and a fourth 5-position modified pyrimidine. 35. The method of claim 34, wherein the target molecule is selected from proteins, cells, and tissues. 36. The method of claim 34 or claim 35, wherein the first aptamer, the second aptamer, and the target are capable of forming a trimer complex. 37. A method for identifying one or more aptamers capable of binding to a target molecule, the method comprising: (a) Contacting a library of aptamers with the target molecule to form a mixture and allowing the formation of an aptamer-target complex, wherein the aptamer-target complex is formed when the aptamer has an affinity for the target molecule; (b) Dispensing or enriching the aptamer-target complex from the remainder of the mixture; (c) Dissociation of the aptamer-target complex; and (d) Identify one or more aptamers capable of binding the target molecule; The aptamer library contains multiple polynucleotides, each polynucleotide containing a first 5-position modified pyrimidine and a second 5-position modified pyrimidine, wherein the first 5-position modified pyrimidine and the second 5-position modified pyrimidine are different 5-position modified pyrimidines; Wherein the first 5-position modified pyrimidine is a 5-position modified uracil, and wherein the second 5-position modified pyrimidine is a 5-position modified cytosine; or Wherein the first 5-position modified pyrimidine is a 5-position modified cytosine and wherein the second 5-position modified pyrimidine is a 5-position modified uracil; The 5-position modified uracil is contained in a tyrosine methyl group at the 5-position; and the 5-position modified cytosine is contained in a naphthyl methyl group at the 5-position; The tyrosine acyl moiety is Tyr, and the naphthyl moiety is Nap, 2Nap, NE, or 2NE. 38. The method of claim 37, wherein each polynucleotide includes a fixed region at the 5' end of the polynucleotide. 39. The method of claim 38, wherein the fixed region at the 5' end of each polynucleotide is at least 10, at least 15, at least 20, at least 25, or at least 30 nucleotides long, or 5 to 30, 10 to 30, 15 to 30, 5 to 20, or 10 to 20 nucleotides long. 40. The method of any one of claims 37 to 39, wherein each polynucleotide includes a fixed region at the 3' end of the polynucleotide. 41. The method of claim 40, wherein the fixed region at the 3' end of the polynucleotide is at least 10, at least 15, at least 20, at least 25, or at least 30 nucleotides in length, or 5 to 30, 10 to 30, 15 to 30, 5 to 20, or 10 to 20 nucleotides in length. 42. The method of any one of claims 37 to 39 or claim 41, wherein the portion of the 5-position modified uracil is covalently linked by a connector comprising a group selected from amide connectors, carbonyl connectors, propynyl connectors, alkyne connectors, ester connectors, urea connectors, carbamate connectors, guanidine connectors, amidine connectors, sulfoxide connectors, and sulfone connectors. 43. The method of any one of claims 37 to 39 or claim 41, wherein the portion of the 5-position modified cytosine is covalently linked by a linker comprising a group selected from amide linkers, carbonyl linkers, propynyl linkers, alkyne linkers, ester linkers, urea linkers, carbamate linkers, guanidine linkers, amidine linkers, sulfoxide linkers, and sulfone linkers. 44. The method of any one of claims 37 to 39 or claim 41, wherein the 5-position modified cytosine is NapdC. 45. The method of any one of claims 37 to 39 or claim 41, wherein the 5-position modified uracil is TyrdU. 46. The method of any one of claims 37 to 39 or claim 41, wherein the first 5-position modified pyrimidine is NapdC and the second 5-position modified pyrimidine is TrydU. 47. The method of any one of claims 37 to 39 or claim 41, wherein each polynucleotide comprises a random region. 48. The method of claim 47, wherein the random region is 20 to 100, or 20 to 90, or 20 to 80, or 20 to 70, or 20 to 60, or 20 to 50, or 20 to 40, or 30 to 100, or 30 to 90, or 30 to 70, or 30 to 60, or 30 to 50, or 30 to 40 nucleotides in length. 49. The method of any one of claims 37 to 39, 41, or 48, wherein each polynucleotide is 20 to 100, or 20 to 90, or 20 to 80, or 20 to 70, or 20 to 60, or 20 to 50, or 30 to 100, or 30 to 90, or 30 to 80, or 30 to 70, or 30 to 60, or 30 to 50, or 40 to 100, or 40 to 90, or 40 to 80, or 40 to 70, or 40 to 60, or 40 to 50 nucleotides in length. 50. The method of any one of claims 37 to 39, 41 or 48, wherein each polynucleotide is an aptamer that binds to a target, and wherein the library contains at least 1,000 aptamers, wherein each aptamer contains a different nucleotide sequence. 51. The method of any one of claims 37 to 39, 41 or 48, wherein steps (a), (b) and/or (c) are repeated at least once, twice, three times, four times, five times, six times, seven times, eight times, nine times or ten times. 52. The method of any one of claims 37 to 39, 41, or 48, wherein one or more aptamers capable of binding the target molecule are amplified. 53. The method of any one of claims 37 to 39, 41, or 48, wherein the mixture comprises a polyanionic competing molecule. 54. The method of claim 53, wherein the polyanionic competitive agent is selected from oligonucleotides, dextran, DNA, heparin, and dNTPs. 55. The method of claim 54, wherein the dextran is sulfated dextran; and the DNA is herring sperm DNA or salmon sperm DNA. 56. The method of any one of claims 37 to 39, 41, 48, and 53 to 55, wherein the target molecule is selected from proteins, cells, and tissues. 57. The aptamer of any one of claims 1 to 9, wherein the first 5-position modified pyrimidine and the second 5-position modified pyrimidine are capable of being incorporated by polymerase.