SELEX AND PHOTOSELEX

• US 20090098549A1
• Filed: 07/17/2008
• Published: 04/16/2009
• Est. Priority Date: 07/17/2007
• Status: Active Grant

• Current Assignee: SomaLogic Operating Co., Inc. (Somalogic Incorporated)
• Original Assignee: Somalogic Incorporated
• Inventors: Carter, Jeff, Gold, Larry, Wilcox, Sheri K., Zichi, Dominic, Schneider, Daniel J., Nieuwlandt, Dan
• Granted Patent: US 8,409,795 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12N 15/1048 SELEX
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• 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 2320/13 in a process of directed evolution, e.g. SELEX, acquiring a new function
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2525/205 Aptamer
• CPC Class Codes: C12Q 2541/101 Selex

Abstract

The present disclosure describes improved SELEX methods for generating nucleic acid ligands that are capable of binding to target molecules and improved photoSELEX methods for generating photoreactive nucleic acid ligands that are capable of both binding and covalently crosslinking to target molecules. The disclosure further describes nucleic acid libraries having expanded physical and chemical properties and their use in SELEX and photoSELEX; methods for increasing the crosslinking efficiencies of photoaptamers; methods for producing photoaptamers having selective modifications that enhance functionality and minimize non-specific photoreactions; and methods for generating truncated nucleic acid ligands from nucleic acid ligands of longer length. The disclosure further describes aptamers and photoaptamers obtained by using any of the foregoing.

Claims

1. A method for identifying a photoaptamer, the method comprising:

a) preparing a candidate mixture of nucleic acids, wherein each nucleic acid comprises;

i) at least one non-photoreactive placeholding pyrimidine; and

ii) at least one modified pyrimidine independently selected from the group consisting of the modified pyrimidines shown in FIG. 2;

b) contacting the candidate mixture with a target, wherein nucleic acids having an increased affinity to the target relative to the candidate mixture may be partitioned from the remainder of the candidate mixture;

c) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture;

d) amplifying the increased affinity nucleic acids to yield a nucleic acid ligand-enriched mixture of nucleic acids;

e) repeating (b)-(d) as desired;

f) producing from said nucleic acid ligand-enriched mixture of nucleic acids a candidate photoaptamer or a mixture of candidate photoaptamers by replacing in each nucleic acid of the ligand-enriched mixture of nucleic acids one or more non-photoreactive placeholding pyrimidines with a photoreactive pyrimidine;

g) contacting said candidate photoaptamer(s) with said target wherein a candidate photoaptamer-target complex is formed;

h) irradiating said candidate photoaptamer-target complex;

i) determining whether said candidate photoaptamer-target complex has photocrosslinked;

j) repeating (f)-(i) as desired; and

k) identifying at least one photoaptamer to the target.

2. The method of claim 1 wherein said non-photoreactive placeholding pyrimidine comprises a methyl group at the C-5 position and said photoreactive pyrimidine comprises a Br or I at the C-5 position.

3. The method of claim 1 wherein said non-photoreactive placeholding pyrimidine is 5-methyl-cytosine.

4. The method of claim 1 wherein said non-photoreactive placeholding pyrimidine is thymine.

5. The method of claim 1 wherein said photoreactive pyrimidine is 5-Br-cytosine.

6. The method of claim 1 wherein said photoreactive pyrimidine is 5-Br-uracil.

7. A photoaptamer identified by the method of claim 1.

8. A method for identifying a photoaptamer, the method comprising:

a) preparing a candidate mixture of nucleic acids comprising at least one non-photoreactive placeholding pyrimidine;

b) contacting the candidate mixture with a target, wherein nucleic acids having an increased affinity to the target relative to the candidate mixture may be partitioned from the remainder of the candidate mixture;

c) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture;

d) amplifying the increased affinity nucleic acids to yield a nucleic acid ligand-enriched mixture of nucleic acids;

e) repeating (b)-(d) as desired;

f) producing from said nucleic acid ligand-enriched mixture of nucleic acids a candidate photoaptamer or a mixture of candidate photoaptamers by replacing in each nucleic acid of the ligand-enriched mixture of nucleic acids one or more non-photoreactive placeholding pyrimidines with a photoreactive pyrimidine;

g) contacting said candidate photoaptamer(s) with said target wherein a candidate photoaptamer-target complex is formed;

h) irradiating said candidate photoaptamer-target complex;

i) determining whether said candidate photoaptamer-target complex has photocrosslinked;

j) repeating (f)-(i) as desired; and

k) identifying at least one photoaptamer to the target.

9. A method for identifying a photoaptamer that comprises a reduced number of photoreactive nucleotides, said method comprising:

a) preparing a candidate mixture of nucleic acids, wherein each member of said candidate mixture comprises a plurality of photoreactive bases, and contacting said candidate mixture with said target, wherein nucleic acids having an increased affinity to the target relative to the candidate mixture form complexes with the target;

b) irradiating said complexes, wherein said nucleic acid and said target photocrosslink;

c) partitioning the photocrosslinked nucleic acid and said target complexes from the remainder of the candidate mixture;

d) identifying a candidate photoaptamer that photocrosslinked to the target;

e) determining which photoreactive bases in said candidate photoaptamer photocrosslink to said target; and

f) replacing all photoreactive bases in said candidate photoaptamer other than the photoreactive bases identified in (e) with non-photoreactive cognates of the photoreactive bases.

10. The method of claim 9, wherein step (e) is performed by screening synthetic nucleotide substitutions or by gel mobility shift-based photocrosslinked site identification.

11. A method for identifying a photoaptamer, the method comprising:

a) preparing a candidate mixture of nucleic acids, wherein each nucleic acid comprises a 5′

fixed region comprising at least one photoreactive functional group;

b) contacting the candidate mixture with a target molecule, wherein nucleic acids having an increased affinity to the target molecule relative to other nucleic acids in the candidate mixture bind the target molecule, forming nucleic acid-target molecule complexes;

c) irradiating the nucleic acid-target molecule complexes;

d) partitioning from the remainder of the candidate mixture nucleic acid-target molecule complexes in which the nucleic acid has photocrosslinked to the target molecule;

e) amplifying the nucleic acid of the photocrosslinked nucleic acid-target molecule complexes to yield a mixture of nucleic acids enriched in sequences that are capable of binding and photocrosslinking to the target molecule;

f) repeating (b) through (e) as desired; and

g) identifying at least one photoaptamer to the target molecule, wherein the photoaptamer comprises at least one photoreactive functional group.

12. The method of claim 11 wherein the photoreactive functional group is selected from the group consisting of anthraquinone, psoralen, and 4-azido-2-nitro-aniline.

13. The method of claim 11 wherein said candidate mixture of nucleic acids contain at least one modified nucleotide.

14. A method for identifying an aptamer, the method comprising:

a) preparing a candidate mixture of nucleic acids, wherein each nucleic acid comprises a 3′

fixed region;

b) contacting the candidate mixture with an oligonucleotide comprising a sequence that is complementary to at least a portion of the 3′

fixed region, wherein the oligonucleotide is permitted to hybridize to the 3′

fixed region;

c) contacting the candidate mixture with a target molecule, wherein nucleic acids having an increased affinity to the target molecule relative to other nucleic acids in the candidate mixture bind the target molecule, forming nucleic acid-target molecule complexes;

d) partitioning the nucleic acid-target molecule complexes from the candidate mixture;

e) dissociating the nucleic acid-target molecule complexes to form free nucleic acids;

f) amplifying the free nucleic acids to yield a mixture of nucleic acids enriched with nucleic acids that are capable of binding to the target molecule with increased affinity;

g) repeating (b) through (f) as desired; and

h) identifying at least one aptamer to the target molecule.

15. The method of claim 14, further comprising removing the 3′

fixed region from the aptamer that has been identified.

16. The method of claim 14 wherein said candidate mixture of nucleic acids contain at least one modified nucleotide.

17. A method for identifying an aptamer, the method comprising:

a) preparing a candidate mixture of nucleic acids, wherein each nucleic acid comprises a 5′

fixed region;

b) contacting the candidate mixture with an oligonucleotide that is complementary to at least a portion of the 5′

fixed region, wherein the oligonucleotide is permitted to hybridize to the 5′

fixed region;

c) contacting the candidate mixture with a target molecule, wherein nucleic acids having an increased affinity to the target molecule relative to other nucleic acids in the candidate mixture bind the target molecule, forming nucleic acid-target molecule complexes;

d) partitioning the nucleic acid-target molecule complexes from the candidate mixture;

e) dissociating the nucleic acid-target molecule complexes to form free nucleic acids;

f) amplifying the free nucleic acids to yield a mixture of nucleic acids enriched with nucleic acids that are capable of binding to the target molecule with increased affinity;

g) repeating (b) through (f) as desired; and

h) identifying at least one aptamer to the target molecule.

18. The method of claim 17, further comprising removing the 5′

fixed region from the aptamer that has been identified.

19. The method of claim 17 wherein said candidate mixture of nucleic acids contain at least one modified nucleotide.

20. A method for identifying an aptamer, the method comprising:

a) preparing a candidate mixture of nucleic acids, wherein each nucleic acid comprises a 3′

fixed region and a 5′

fixed region;

b) contacting the candidate mixture with a first oligonucleotide that is complementary to at least a portion of the 3′

fixed region, wherein the first oligonucleotide is permitted to hybridize to the 3′

fixed region;

c) contacting the candidate mixture with a second oligonucleotide that is complementary to at least a portion of the 5′

fixed region, wherein the second oligonucleotide is permitted to hybridize to the 5′

fixed region;

d) contacting the candidate mixture with a target molecule, wherein nucleic acids having an increased affinity to the target molecule relative to other nucleic acids in the candidate mixture bind the target molecule, forming nucleic acid-target molecule complexes;

e) partitioning the nucleic acid-target molecule complexes from the candidate mixture;

f) dissociating the nucleic acid-target molecule complexes to form free nucleic acids;

g) amplifying the free nucleic acids to yield a mixture of nucleic acids enriched with nucleic acids that are capable of binding to the target molecule with increased affinity;

h) repeating (b) through (g) as desired; and

i) identifying at least one aptamer to the target molecule

21. The method of claim 20, further comprising removing the 3′

fixed region, the 5′

fixed region or both the 3′

fixed region and the 5′

fixed region from the aptamer that has been identified.

22. The method of claim 20 wherein said candidate mixture of nucleic acids contain at least one modified nucleotide.

23. A method for identifying an aptamer, the method comprising:

a) preparing a candidate mixture of nucleic acids;

b) contacting the candidate mixture with a biotinylated target molecule, wherein nucleic acids having an increased affinity to the target molecule relative to other nucleic acids in the candidate mixture bind the target molecule, forming nucleic acid-target molecule complexes;

c) partitioning the nucleic acid-target molecule complexes from the candidate mixture by contacting the candidate mixture with a solid support having streptavidin bound to the solid support and washing the solid support with a solution containing biotin;

d) dissociating the nucleic acid-target molecule complexes to form free nucleic acids;

e) amplifying the free nucleic acids to yield a mixture of nucleic acids enriched with nucleic acids that are capable of binding to the target molecule with increased affinity;

f) repeating (b) through (e) as desired; and

g) identifying at least one aptamer to the target molecule

24. A method for reducing the attachment of nucleic acids to an avidin column during an aptamer selection process, the method comprising:

a) preparing a candidate mixture of nucleic acids and contacting said candidate mixture with a biotinylated target, wherein nucleic acids having an increased affinity to the target relative to the candidate mixture form nucleic acid-target complexes with the target;

b) partitioning the nucleic acid-target complexes from the candidate mixture with an avidin column;

c) washing the column with a solution containing biotin to displace nucleic acids bound to the avidin column; and

d) identifying a candidate aptamer to the target.

25. The method of claim 24 wherein said avidin column contains streptavidin, neutravidin, or ExtrAvidin.

26. The method of claim 24 wherein said avidin column contains neutravidin.