Selection of nucleic acid-based sensor domains within nucleic acid switch platform

• US 20090082217A1
• Filed: 07/16/2008
• Published: 03/26/2009
• Est. Priority Date: 07/16/2007
• Status: Abandoned Application

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Greenwood-Goodwin, Midori, Chen, Yvonne, Brown, Arwen, Smolke, Christina D.
• Application Number:
  US12/218,628
• Publication Number:
  US 20090082217A1
• Time in Patent Office: Days
• US Class Current: 506/9
• 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 2310/3519 Fusion with another nucleic acid
• CPC Class Codes: C12N 2320/10 in screening processes

Abstract

The invention relates to a method (preferably a high throughput method) for screening for functional aptamer-regulated, ligand-responsive nucleic acids, or “ampliSwitches,” and uses thereof. The subject method not only applies to large molecules, such as proteins, but also applies to relatively small ligands, such as those with molecular weight of no more than 5 kDa, 3 kDa, or 1 kDa.

Claims

1. A method of screening a library of nucleic acids for a nucleic acid that binds a ligand, wherein each member of said library comprises:

(a) an aptamer that potentially binds the ligand; and

,

(b) a functional domain,

the method comprising;

(1) contacting the library of nucleic acids with the ligand, under conditions that allow binding of the ligand to the aptamer of one or more members of the library in solution;

(2) isolating nucleic acids that form complexes with the ligand; and

,

(3) determining, for each nucleic acids isolated in (2), if any, whether binding of the ligand to said aptamer favors a conformational change in the functional domain from a first ligand-free conformation to a second ligand-bound conformation,

wherein the functional domain is not a ribozyme or a catalytic RNA, or wherein step (2) is not effectuated by denaturing polyacrylamide gel electrophoresis (PAGE) or a chromatography-based selection system, or both.

2. The method of claim 1, further comprising repeating once or more times steps (1)-(2) before step (3), or repeating once or more times steps (1)-(3), each time using any nucleic acids isolated in step (2) of the previous iteration or an amplification product thereof as the library in the immediate subsequent round of screening.

3. The method of claim 1, wherein said conformational change is caused by a strand displacement mechanism, wherein in the first conformation, a complementary strand base pairs with a competing strand, and in the second conformation, an aptamer switching stem displaces the competing strand to base pair with the complementary strand.

4. The method of claim 1, wherein each member of said library of nucleic acids comprises:

(i) the aptamer,

(ii) a complementary strand,

(iii) an aptamer switching stem,

(iv) a competing strand,

(v) an antisense stem, and,

wherein, in the first conformation, the aptamer unbound by the ligand allows said competing strand to base pair with said complementary strand, and said antisense stem to form a double-stranded stem-loop structure;

wherein, in the second conformation, the aptamer bound by the ligand allows said aptamer switching stem to displace said competing strand and base pair with said complementary strand, and disrupts the stem-loop structure formed from the antisense stem.

5. The method of claim 4, wherein the aptamer is flanked by the complementary strand and the aptamer switching stem.

6. The method of claim 5, wherein the aptamer is 3′

to the complementary strand.

7. The method of claim 4, wherein, in the second conformation, the antisense stem is without the stem-loop structure and is capable of hybridizing with a second polynucleotide.

8. The method of claim 1, wherein step (2) is carried out based on the mass-to-charge (m/z) ratio difference among the complexes, the unbound ligand, and the unbound nucleic acid.

9. The method of claim 3, wherein step (2) is carried out based on the availability of the competing strand for hybridization with a second polynucleotide.

10. The method of claim 1, wherein members of said library of nucleic acids have substantially the same m/z ratio.

11. The method of claim 1, wherein each member of said library of nucleic acids has essentially the same length.

12. The method of claim 1, wherein the ligand is a polypeptide.

13. The method of claim 1, wherein the ligand is a small molecule no more than 5 kDa in molecular weight.

14. The method of claim 13, further comprising, before step (2):

(4) contacting the mixture with a second nucleic acid that binds to the functional domain after but not before said conformational change.

15. The method of claim 14, wherein the second nucleic acid is conjugated to a label.

16. The method of claim 15, wherein the label is biotin or a fluorescent label.

17. The method of claim 16, wherein the second nucleic acid is conjugated to biotin, and the method further comprising, before step (2):

(5) contacting the mixture with Avidin, Streptavidin, or an analog thereof.

18. The method of claim 1, wherein step (2) is carried out by capillary electrophoresis (CE).

19. The method of claim 18, wherein said CE is non-equilibrium CE.

20. The method of claim 1, wherein said aptamer comprises a randomized sequence.

21. The method of claim 20, wherein said randomized sequence is about 30-50 nucleotides in length, or 10-60 nucleotides in length.

22. The method of claim 1, wherein the functional domain comprises a priming sequence capable of hybridizing to a target template to form a primer:

template pair, and wherein binding of the ligand to said aptamer favors a conformational change in the nucleic acid that alters the ability of said priming sequence to hybridize to said target template.

23. The method of claim 22, wherein said primer:

template pair is a substrate for an extrinsic enzymatic activity.

24. The method claim 22, wherein said conformational change produces or removes an intramolecular double-stranded feature, including said priming sequence, which double-stranded feature alters the availability of said priming sequence to hybridize to said target template.

25. The method of claim 1, wherein said functional domain is:

(1) a substrate sequence that can form a substrate for an extrinsic enzyme, and

(2) binding of said ligand to said aptamer favors a conformational change in the nucleic acid that alters the ability of said substrate sequence to form said substrate and/or alters the K_m and/or k_cat of said substrate for the extrinsic enzymatic activity.

26. The method of claim 25, wherein the conformational change produces or removes an intramolecular double-stranded feature, including said substrate sequence, which double-stranded feature is said substrate for said extrinsic enzyme.

27. The method of claim 25, wherein said extrinsic enzyme is Dicer.

28. The method of claim 27, wherein said substrate sequence produces siRNA, miRNA or a precursor or metabolite thereof in an RNA interference pathway, as a product of reaction with Dicer.

29. The method of claim 25, wherein the conformation change alters the ability of the substrate sequence to form an intermolecular double-stranded feature with a second nucleic acid species, which double stranded feature is a substrate for said extrinsic enzyme.

30. The method of claim 29, wherein the second nucleic acid species is an mRNA, and said extrinsic enzyme alters the mRNA in a manner dependent on the formation of said double-stranded feature.

31. The method of claim 25, wherein the extrinsic enzyme is an RNase H enzyme and/or an RNase P enzyme.

32. The method of claim 25, wherein said substrate sequence comprises a hairpin loop.

33. The method of claim 1, wherein said functional domain is a ribozyme, and wherein binding of said ligand to said aptamer favors a conformational change in the nucleic acid that alters the activity of the ribozyme.

34. The method of claim 1, wherein said nucleic acid comprises one or more aptamers or one or more effector domains.

35. The method of claim 1, wherein said nucleic acid interacts with and responds to multiple ligands.

36. The method of claim 1, wherein said nucleic acid is a cooperative ligand controlled nucleic acid wherein multiple ligands sequentially bind to multiple aptamers to allosterically regulate one or more effector domains.