Nucleotides and aptamers containing boronic acid groups having biased binding to glycosylated proteins, and uses thereof

• US 9,096,856 B2
• Filed: 07/17/2008
• Issued: 08/04/2015
• Est. Priority Date: 07/19/2007
• Status: Active Grant

• Current Assignee: Georgia State University Research Foundation, Inc.
• Original Assignee: Georgia State University Research Foundation, Inc.
• Inventors: Wang, Binghe, Li, Minyong, Huang, Zhen, Lin, Na
• Primary Examiner(s): Woolwine, Samuel
• Assistant Examiner(s): Zhang, Kaijiang
• Application Number:
  US12/669,593
• Publication Number:
  US 20130184160A1
• Time in Patent Office: 2,574 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07H 23/00 Compounds containing boron, silicon, or a metal, e.g. chelates, vitamin B12 esters with inorganic acids C07H11/00; metal salts, see parent compounds
• CPC Class Codes: C12N 15/1048 SELEX
• CPC Class Codes: 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
• CPC Class Codes: C12N 2310/16 Aptamers
• CPC Class Codes: C12N 2310/33 of the base
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 33/6893 related to diseases not provided for elsewhere
• CPC Class Codes: G01N 33/86 involving blood coagulating time or factors, or their receptors

Abstract

The present disclosure encompasses oligonucleotide aptamers selectively binding a target glycosylated polypeptide or protein, and having biased affinity for the glycan through a boronic acid linked to a nucleosidic base of a nucleotide(s). The disclosure further encompasses methods for isolating an aptamer(s) selectively binding a target glycosylated polypeptide, where, from a population of randomized oligonucleotides that have at least one nucleotide having a boronic acid label linked to a base, is selected a first subpopulation of aptamers binding to the target glycosylated polypeptide or protein. This subpopulation is then amplified without using boronic acid-modified TTP, and amplification products not binding to a target glycosylated polypeptide or protein are selected. The second subpopulation of aptamers is then amplified using boronic acid-modified TTP to provide a population of boronic acid-modified aptamers capable of selectively binding to a glycosylation site of a target polypeptide or protein. Other aspects of the disclosure encompass methods for the use of the modified aptamers to detect glycosylated species of a polypeptide or protein.

Claims

1. A nucleotide monomer having the formula:

2. The nucleotide monomer of claim 1, wherein the second alkynyl group is an ethynyl group.

3. The nucleotide monomer of claim 1, wherein the tether has the formula

—

C≡

C—

CH₂—

CH₂—

CO—

NH—

CH₂—

C≡

C—

.

4. The nucleotide monomer of claim 1, wherein R₄ is uracil, R₂ is OH, and R₃ is H.

5. The nucleotide monomer of claim 1, wherein R₅ is a boronic acid selected from the group consisting of:

a substituted phenylboronic acid, a naphthalenylboronic acid, a quinolinylboronic acid, a pyridinylboronic acid, a furanylboronic acid, a thiophenylboronic acid, an indolylboronic acid a 1,8-naphthalimide-based boronic acid, an α

-acetaminoalkylboronic acid, a quinolin-4-ylboronic acid, a quinolin-5-ylboronic acid, a quinolin-8-ylboronic acid, a pyridinylboronic acid, a furan-2-ylboronic acid, and a thiophen-2-ylboronic acid.

6. The nucleotide monomer of claim 5, wherein R₅ is selected from the group consisting of the structures:

7. A nucleotide monomer having the formula:

8. An oligonucleotide comprising at least one nucleotide monomer having the formula:

9. The oligonucleotide of claim 8, wherein R₄ is uracil, R₂ is OH, and R₃ is H.

10. The oligonucleotide of claim 8, wherein R₅ is a boronic acid selected from the group consisting of:

a substituted phenylboronic acid, a naphthalenylboronic acid, a quinolinylboronic acid, a pyridinylboronic acid, a furanylboronic acid, a thiophenylboronic acid, an indolylboronic acid, a 1,8-naphthalimide-based boronic acid, an α

-acetaminoalkylboronic acid, a quinolin-4-ylboronic acid, a quinolin-5-ylboronic acid, a quinolin-8-ylboronic acid, a pyridinylboronic acid, a furan-2-ylboronic acid, and a thiophen-2-ylboronic acid.

11. The oligonucleotide of claim 8, wherein the boronic acid is a fluorescent boronic acid.

12. The oligonucleotide of claim 11, wherein the fluorescent boronic acid is selected from the group consisting of the structures:

13. A method of isolating an aptamer having selective affinity for a target polypeptide and a glycan thereon, comprising:

(a) selecting a first subpopulation of aptamers binding to a target glycosylated polypeptide or protein from a population of randomized oligonucleotides, wherein each oligonucleotide of the population of randomized oligonucleotides includes at least one nucleotide having a boronic acid label linked to a base thereon, wherein said at least one nucleotide monomer has the formula;

14. The method of claim 13, further comprising a counter-selection step, wherein the counter-selection step comprises identifying aptamers capable of selectively binding to a solid support not having a target polypeptide bound thereto, a deglycosylated target polypeptide or protein, or a cross-reacting non-targeted polypeptide or proteins.

15. The method of claim 13, wherein step (a) comprises:

(a1) amplifying a library of randomized oligonucleotides, wherein each oligonucleotide includes at least one nucleotide having a boronic acid label linked to a base thereon;

(a2) contacting the library with a glycosylated target polypeptide or protein under conditions whereby a subpopulation of the library of aptamers selectively binds to a glycosylation site of the target polypeptide;

(a3) isolating target polypeptides or proteins having the subpopulation of aptamers bound thereto, and eluting the subpopulation of aptamers from the target polypeptide;

(a4) amplifying the eluted subpopulation of aptamers; and

(a5) repeating steps (a2)-(a4), thereby isolating a first subpopulation of aptamers from the amplified library from step (a1), wherein the aptamers are capable of binding a glycosylation site of the target polypeptide.

16. The method of claim 13, further comprising:

(i) inserting a population of aptamers isolated in step c into a vector, and isolating clones thereof;

(ii) identifying a plurality of aptamer clones, wherein each aptamer has a nucleotide sequence differing from the sequences of the other aptamers;

(iii) determining the dissociation constants of the individual selected aptamers and the target glycosylated polypeptide;

(iv) comparing the dissociation constants of the aptamer sequences to the dissociation constants of a control aptamer not having a boronic acid thereon; and

(v) selecting one or more aptamers having a lower dissociation constant than the control, whereby the selected aptamers have enhanced selective affinity for a glycosylated site of the target polypeptide compared to control aptamers having an identical nucleotide sequence to that of the selected aptamers but not having a boronic acid group thereon.

17. The method of claim 13, wherein the target polypeptide is immobilized on a solid support.

18. The method of claim 13, wherein R₄ is uracil, R₂ is OH, and R₃ is H.

19. The method of claim 13, wherein R₅ is a boronic acid selected from the group consisting of:

a substituted phenylboronic acid, a naphthalenylboronic acid, a quinolinylboronic acid, a pyridinylboronic acid, a furanylboronic acid, a thiophenylboronic acid, an indolylboronic acid, a 1,8-naphthalimide-based boronic acid, an α

-acetaminoalkylboronic acid, a quinolin-4-ylboronic acid, a quinolin-5-ylboronic acid, a quinolin-8-ylboronic acid, a pyridinylboronic acid, a furan-2-ylboronic acid, and a thiophen-2-ylboronic acid.

20. The method of claim 13, wherein the at least one nucleotide having a boronic acid label modified base thereon comprises a fluorescent boronic acid selected from the group having the formulas:

21. A method of detecting a glycosylated species of a target polypeptide, comprising:

(a) providing a target polypeptide;

(b) contacting the target polypeptide with a first aptamer comprising at least one nucleotide having a boronic acid label linked to a base thereon, wherein the at least one nucleotide monomer has the formula;

22. The method of claim 21, wherein the glycosylation site of the target polypeptide comprises a region of a glycosylation chain and a region of the polypeptide.

23. The method of claim 21, wherein R₄ is uracil, R₂ is OH, and R₃ is H.

24. The method of claim 21, wherein R₅ is a boronic acid selected from the group consisting of:

a substituted phenylboronic acid, a naphthalenylboronic acid, a quinolinylboronic acid, a pyridinylboronic acid, a furanylboronic acid, a thiophenylboronic acid, an indolylboronic acid, a 1,8-naphthalimide-based boronic acid, an a α

-acetaminoalkylboronic acid, a quinolin-4-ylboronic acid, a quinolin-5-ylboronic acid, a quinolin-8-ylboronic acid, a pyridinylboronic acid, a furan-2-ylboronic acid, and a thiophen-2-ylboronic acid.

25. The method of claim 21, wherein the boronic acid is a fluorescent boronic acid selected from the group consisting of the structures:

26. The method of claim 21, wherein the target polypeptide is selected from the group consisting of:

an isolated polypeptide or a fragment thereof, a polypeptide in a cell or tissue of an animal or plant, or a cultured cell.

27. The method of claim 21, wherein the target polypeptide is Prostate Serum Antigen (PSA) or fibrinogen.

28. The method of claim 27, wherein the first aptamer selectively binds to high pl PSA, low pl PSA, or both high and low pl PSA.

29. The method of claim 27, wherein the aptamer selectively binds to a glycosylated species of fibrinogen.