# US 12,180,482 B2 · Oligonucleotides comprising modified nucleosides

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- Number: US 12,180,482 B2
- Document ID: US12180482B2
- Status: Granted
- Kind: B2
- Application number: 18/153,428
- Filed: 2023-01-12
- Published: 2023-10-05
- Granted: 2024-12-31
- Earliest claimed: 2016-07-01
- Assignees: SomaLogic Operating Co., Inc.
- Applicants: Somalogic Operating Co., Inc.
- Inventors: Jeff Carter; Bharat Gawande; Nebojsa Janjic; Daniel Schneider
- Web page: https://patentgrep.com/patent/US12180482B2

## CPC

- C12N 15/115: Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith {; Nucleic acids binding to non-nucleic acids, e.g. aptamers}
- A61P 3/06: Antihyperlipidemics
- A61P 9/10: for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
- C12N 15/1048: {SELEX}
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12N 2310/16: Aptamers
- C12N 2310/334: Modified C
- C12N 2310/3341: 5-Methylcytosine
- C12N 2310/335: Modified T or U
- C12N 2330/31: Libraries, arrays
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

Polynucleotides, such as aptamers, comprising at least first one 5-position modified pyrimidine and at least one second 5-position modified pyrimidine are provided, wherein the first and second 5-position modified pyrimidines are different. Methods of selecting and using such polynucleotides, such as aptamers, are also provided.

## Claims

### Claim 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;
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 cytidine; or
wherein the first 5-position modified pyrimidine is a 5-position modified cytidine and wherein the second 5-position modified pyrimidine is a 5-position modified uridine;
wherein the 5-position modified cytidine is PPdC and the 5-position modified uridine is TyrdU; or
wherein the 5-position modified cytidine is PPdC and the 5-position modified uridine is PPdU; or
wherein the 5-position modified cytidine is PPdC and the 5-position modified uridine is NapdU; or
wherein the 5-position modified cytidine is PPdC and the 5-position modified uridine is ThrdU; or
wherein the 5-position modified cytidine is NapdC and the 5-position modified uridine is NapdU; or
wherein the 5-position modified cytidine is NapdC and the 5-position modified uridine is PPdU; or
wherein the 5-position modified cytidine is NapdC and the 5-position modified uridine is ThrdU; or
wherein the 5-position modified cytidine is NapdC and the 5-position modified uridine is MOEdU.

### Claim 2 (depends on 1)

The aptamer of claim 1, wherein the moiety of the 5-position modified uridine is covalently linked via a linker comprising a group selected from an amide linker, a carbonyl linker, a propynyl linker, an alkyne linker, an ester linker, a urea linker, a carbamate linker, a guanidine linker, an amidine linker, a sulfoxide linker, and a sulfone linker.

### Claim 3 (depends on 1)

The aptamer of claim 1, wherein the moiety of the 5-position modified cytidine is covalently linked via a linker comprising a group selected from an amide linker, a carbonyl linker, a propynyl linker, an alkyne linker, an ester linker, a urea linker, a carbamate linker, a guanidine linker, an amidine linker, a sulfoxide linker, and a sulfone linker.

### Claim 4 (depends on 1)

The aptamer of claim 1, 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.

### Claim 5

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;
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 cytidine; or
wherein the first 5-position modified pyrimidine is a 5-position modified cytidine and wherein the second 5-position modified pyrimidine is a 5-position modified uridine;
wherein the 5-position modified cytidine is PPdC and the 5-position modified uridine is TyrdU; or
wherein the 5-position modified cytidine is PPdC and the 5-position modified uridine is PPdU; or
wherein the 5-position modified cytidine is PPdC and the 5-position modified uridine is NapdU; or
wherein the 5-position modified cytidine is PPdC and the 5-position modified uridine is ThrdU; or
wherein the 5-position modified cytidine is NapdC and the 5-position modified uridine is NapdU; or
wherein the 5-position modified cytidine is NapdC and the 5-position modified uridine is PPdU; or
wherein the 5-position modified cytidine is NapdC and the 5-position modified uridine is ThrdU; or
wherein the 5-position modified cytidine is NapdC and the 5-position modified uridine is MOEdU.

### Claim 6 (depends on 5)

The composition of claim 5, wherein each polynucleotide comprises a fixed region at the 5′ end of the polynucleotide and/or the 3′ end of the polynucleotide.

### Claim 7 (depends on 6)

The composition of claim 6, 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.

### Claim 8 (depends on 6)

The composition of claim 6, 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.

### Claim 9 (depends on 5)

The composition of claim 5, wherein each polynucleotide comprises a random region.

### Claim 10 (depends on 5)

The composition of claim 5, 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.

### Claim 11 (depends on 1)

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 an aptamer of claim 1.

### Claim 12 (depends on 1)

A composition 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, second aptamer and the target are capable of forming a trimer complex; and
wherein the first 5-position modified pyrimidine and the second 5-position modified pyrimidine are different 5-position modified pyrimidines; and
wherein the first aptamer is an aptamer of claim 1.

### Claim 13 (depends on 12)

The composition of claim 12, wherein the target is selected from a protein, a peptide, a carbohydrate, a small molecule, a cell and a tissue.

### Claim 14 (depends on 1)

A method comprising:
(a) contacting an aptamer capable of binding to a target molecule with a sample;
(b) incubating the aptamer with the sample to allow an aptamer-target complex to form;
(c) enriching for the aptamer-target complex in the sample and
(d) detecting for the presence of the aptamer, aptamer-target complex or target molecule, wherein the detection of the aptamer, aptamer-target complex or target molecule indicates that the target molecule is present in the sample, and wherein the lack of detection of the aptamer, aptamer-target complex or target molecule indicates that the target molecule is not present in the sample;
wherein the aptamer is an aptamer of claim 1.

### Claim 15 (depends on 14)

The method of claim 14, wherein the sample is selected from whole blood, leukocytes, peripheral blood mononuclear cells, plasma, serum, sputum, breath, urine, semen, saliva, meningial fluid, amniotic fluid, glandular fluid, lymph fluid, nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate, cells, a cellular extract, stool, tissue, a tissue biopsy, and cerebrospinal fluid.

### Claim 16 (depends on 1)

A method for detecting a target in a sample comprising
a) contacting the sample with a first aptamer to form a mixture, wherein the first aptamer is capable of binding to the target to form a first complex;
b) incubating the mixture under conditions that allow for the first complex to form;
c) contacting the mixture with a second aptamer, wherein the second aptamer is capable of binding the first complex to form a second complex;
d) incubating the mixture under conditions that allow for the second complex to form;
e) detecting for 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;
wherein the first aptamer is an aptamer of claim 1;
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.

### Claim 17 (depends on 16)

The method of claim 16, wherein the first aptamer, second aptamer and the target are capable of forming a trimer complex.

### Claim 18

A method for identifying one or more aptamers capable of binding to a target molecule comprising:
(a) contacting a library of aptamers with the target molecule to form a mixture, and allowing for the formation of an aptamer-target complex, wherein the aptamer-target complex forms when an aptamer has affinity for the target molecule;
(b) partitioning the aptamer-target complex from the remainder of the mixture (or enriching for the aptamer-target complex);
(c) dissociating the aptamer-target complex; and
(d) identifying the one or more aptamers capable of binding to the target molecule;
wherein the library of aptamers comprises 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;
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 cytidine; or
wherein the first 5-position modified pyrimidine is a 5-position modified cytidine and wherein the second 5-position modified pyrimidine is a 5-position modified uridine;
wherein the 5-position modified cytidine is PPdC and the 5-position modified uridine is TyrdU; or
wherein the 5-position modified cytidine is PPdC and the 5-position modified uridine is PPdU; or
wherein the 5-position modified cytidine is PPdC and the 5-position modified uridine is NapdU; or
wherein the 5-position modified cytidine is PPdC and the 5-position modified uridine is ThrdU; or
wherein the 5-position modified cytidine is NapdC and the 5-position modified uridine is NapdU; or
wherein the 5-position modified cytidine is NapdC and the 5-position modified uridine is PPdU; or
wherein the 5-position modified cytidine is NapdC and the 5-position modified uridine is ThrdU; or
wherein the 5-position modified cytidine is NapdC and the 5-position modified uridine is MOEdU.

### Claim 19 (depends on 1)

The aptamer of claim 1, wherein the aptamer has improved nuclease stability and/or a longer half-life in human serum compared to an aptamer of the same length and nucleobase sequence that comprises an unmodified pyrimidine in place of each of the first 5-position modified pyrimidines or an unmodified pyrimidine in place of each of the second 5-position modified pyrimidine.