# US 10,370,669 B2 · Nucleic acid compounds for binding growth differentiation factor 8

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- Number: US 10,370,669 B2
- Document ID: US10370669B2
- Status: Granted
- Kind: B2
- Application number: 15/545,755
- Filed: 2016-02-05
- Published: 2018-01-18
- Granted: 2019-08-06
- Earliest claimed: 2015-02-09
- Assignees: SomaLogic, Inc.
- Applicants: Somalogic, Inc.
- Inventors: Urs Ochsner; Louis Green; Nebojsa Janjic
- Web page: https://patentgrep.com/patent/US10370669B2

## 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}
- G01N 33/5308: {for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites}
- G01N 33/74: involving hormones {or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors}
- C12N 2310/16: Aptamers
- C12N 2310/321: 2'-O-R Modification
- C12N 2310/322: 2'-R Modification
- G01N 2333/475: Assays involving growth factors
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

Described herein are aptamers capable of binding to growth differentiation factor 8 (GDF8) protein; compositions comprising a GDF8 binding aptamer; and methods of making and using the same.

## Claims

### Claim 1

An aptamer that binds GDF8, wherein the aptamer comprises the sequence:
5′-GWWR1CnR2ZGnGWCnX2CWnWWX1AnR1CnX1KWnAGX2nWCX1ZD-3′ (SEQ ID NO: 1),
wherein:
each W is independently, and for each occurrence, a C-5 modified pyrimidine;
X1 and X2 are each independently selected from W and C;
R1 and R2 are each independently selected from G and A;
each Z is independently selected from A and W;
K is selected from G and W;
D is selected from G, A, and W; and
each n is independently selected from 0 and 1;
or wherein the aptamer comprises the sequence:
5′-X1GWWR1CnR2ZGnGWCnX2CWnWWX1AnR1CnX1KWnAGX2nWCX1ZD-3′ (SEQ ID NO: 2),
wherein:
each W is independently, and for each occurrence, a C-5 modified pyrimidine;
X1 and X2 are each independently selected from W and C;
R1 and R2 are each independently selected from G and A;
each Z is independently selected from A and W;
K is selected from G and W;
D is selected from G, A, and W; and
each n is independently selected from 0 and 1;
or wherein the aptamer comprises the sequence:
5′-GWWR1R2ZGWCnX2CWnWWX1AnR1CnX1KAGWCX1ZD-3′ (SEQ ID NO: 49), wherein:
each W is independently, and for each occurrence, a C-5 modified pyrimidine;
X1 and X2 are each independently selected from W and C;
R1 and R2 are each independently selected from G and A;
each Z is independently selected from A and W;
K is selected from G and W;
D is selected from G, A, and W; and
each n is independently selected from 0 and 1;
or wherein the aptamer comprises the sequence:
5′-WGWWAGZGWX2CWWX1AWGAGWCWAD-3′ (SEQ ID NO: 3), wherein:
each W is independently, and for each occurrence, a C-5 modified pyrimidine;
X1 and X2 are each independently selected from W and C;
Z is selected from A and W; and
D is selected from G, A, and W;
or wherein the aptamer comprises the sequence:
5′-GWWAGAGWCCWWWAWGAGWCWAG-3′ (SEQ ID NO: 4), wherein each W is independently, and for each occurrence, a C-5 modified pyrimidine.

### Claim 2 (depends on 1)

The aptamer of claim 1, wherein the aptamer binds GDF8 with an affinity of less than 10 nM, wherein, under the same conditions, the aptamer binds GDF11 with an affinity that is at least 10-fold weaker than the affinity for GDF8 or does not bind GDF 11.

### Claim 3 (depends on 2)

The aptamer of claim 2, wherein the aptamer binds GDF8 with an affinity of less than 8 nM and binds GDF11 with an affinity greater than 100 nM.

### Claim 4 (depends on 2)

The aptamer of claim 2, wherein affinity is determined using a binding assay comprising a polyanionic inhibitor selected from dextran sulfate, heparin, Z-block, poly-dl/dC, sonicated or sheared salmon sperm DNA, calf thymus DNA, and dNTPs.

### Claim 5 (depends on 1)

The aptamer of claim 1, wherein at least one R1 is A.

### Claim 6 (depends on 1)

The aptamer of claim 1, wherein at least one R2 is G.

### Claim 7 (depends on 1)

The aptamer of claim 1, wherein K is G.

### Claim 8 (depends on 1)

The aptamer of claim 1, wherein at least 1 n is 0.

### Claim 9 (depends on 1)

The aptamer of claim 1, wherein at least one X1 is W.

### Claim 10 (depends on 1)

The aptamer of claim 1, wherein at least one X2 is C.

### Claim 11 (depends on 1)

The aptamer of claim 1, wherein each W is independently, and for each occurrence, selected from:
5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU),
5-(N-benzylcarboxyamide)-2′-O-methyluridine,
5-(N-benzylcarboxyamide)-2′-fluorouridine,
5-(N-phenethylcarboxyamide)-2′-deoxyuridine (PEdU),
5-(N-thiophenylmethylcarboxyamide)-2′-deoxyuridine (ThdU),
5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU),
5-(N-tyrosylcarboxyamide)-2′-deoxyuridine (TyrdU),
5-(N-3,4-methylenedioxybenzylcarboxyamide)-2′-deoxyuridine (MBndU),
5-(N-4-fluorobenzylcarboxyamide)-2′-deoxyuridine (FBndU),
5-(N-3-phenylpropylcarboxyamide)-2′-deoxyuridine (PPdU),
5-(N-imidizolylethylcarboxyamide)-2′-deoxyuridine (ImdU),
5-(N-isobutylcarboxyamide)-2′-O-methyluridine,
5-(N-isobutylcarboxyamide)-2′-fluorouridine,
5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU),
5-(N-R-threoninylcarboxyamide)-2′-deoxyuridine (ThrdU),
5-(N-tryptaminocarboxyamide)-2′-O-methyluridine,
5-(N-tryptaminocarboxyamide)-2′-fluorouridine,
5-(N-[1-(3-trimethylamonium) propyl]carboxyamide)-2′-deoxyuridine chloride,
5-(N-naphthylmethylcarboxyamide)-2′-deoxyuridine (NapdU),
5-(N-naphthylmethylcarboxyamide)-2′-O-methyluridine,
5-(N-naphthylmethylcarboxyamide)-2′-fluorouridine,
5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine),
5-(N-2-naphthylmethylcarboxyamide)-2′-deoxyuridine (2NapdU),
5-(N-2-naphthylmethylcarboxyamide)-2′-O-methyluridine,
5-(N-2-naphthylmethylcarboxyamide)-2′-fluorouridine,
5-(N-1-naphthylethylcarboxyamide)-2′-deoxyuridine (NEdU),
5-(N-1-naphthylethylcarboxyamide)-2′-O-methyluridine,
5-(N-1-naphthylethylcarboxyamide)-2′-fluorouridine,
5-(N-2-naphthylethylcarboxyamide)-2′-deoxyuridine (2NEdU),
5-(N-2-naphthylethylcarboxyamide)-2′-O-methyluridine,
5-(N-2-naphthylethylcarboxyamide)-2′-fluorouridine,
5-(N-3-benzofuranylethylcarboxyamide)-2′-deoxyuridine (BF dU),
5-(N-3-benzofuranylethylcarboxyamide)-2′-O-methyluridine,
5-(N-3-benzofuranylethylcarboxyamide)-2′-fluorouridine,
5-(N-3-benzothiophenylethylcarboxyamide)-2′-deoxyuridine (BTdU),
5-(N-3-benzothiophenylethylcarboxyamide)-2′-O-methyluridine, and
5-(N-3-benzothiophenylethylcarboxyamide)-2′-fluorouridine.

### Claim 12 (depends on 1)

The aptamer of claim 1, wherein at least 1, at least 2, at least 3, at least 4, at least 5, or at least 6 W are each independently selected from 5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU), 5-(N-tryptaminocarboxyamide)-2′-O-methyluridine, and 5-(N-tryptaminocarboxyamide)-2′-fluorouridine.

### Claim 13 (depends on 12)

The aptamer of claim 12, wherein each W is independently selected from 5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU), 5-(N-tryptaminocarboxyamide)-2′-O-methyluridine, and 5-(N-tryptaminocarboxyamide)-2′-fluorouridine.

### Claim 14 (depends on 1)

The aptamer of claim 1, wherein the aptamer consists of 18 to 200 nucleotides, wherein each nucleotide may, independently, be a modified or unmodified nucleotide.

### Claim 15 (depends on 1)

The aptamer of claim 1, wherein the aptamer comprises a detectable label.

### Claim 16 (depends on 2)

The aptamer of claim 2, wherein GDF8 is mature human GDF8 comprising the sequence of SEQ ID NO: 63 and GDF11 is mature human GDF11 comprising the sequence of SEQ ID NO: 50.

### Claim 17 (depends on 1)

A method of detecting GDF8 in a sample, comprising contacting proteins from the sample with an aptamer of claim 1.

### Claim 18

An aptamer that binds GDF 8, wherein the aptamer comprises a sequence selected from SEQ ID NOs: 5 to 48.

### Claim 19 (depends on 1)

The aptamer of claim 1, wherein each R1 is A.

### Claim 20 (depends on 1)

The aptamer of claim 1, wherein each R2 is G.

### Claim 21 (depends on 1)

The aptamer of claim 1, wherein each n is 0.

### Claim 22 (depends on 1)

The aptamer of claim 1, wherein each X1 is W.

### Claim 23 (depends on 1)

The aptamer of claim 1, wherein each X2 is C.