Switchable nucleic acids for diagnostics, screening and molecular electronics

• US 7,718,784 B2
• Filed: 02/03/2004
• Issued: 05/18/2010
• Est. Priority Date: 02/04/2003
• Status: Active Grant

• Current Assignee: Syracuse University
• Original Assignee: Syracuse University
• Inventors: DeCiantis, Christopher L., Hudson, Bruce S., Borer, Philip N.
• Primary Examiner(s): Strzelecka; Teresa E
• Application Number:
  US10/544,591
• Publication Number:
  US 20060216692A1
• Time in Patent Office: 2,296 Days
• Field of Search: None
• US Class Current: 536/23.1
• 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: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/635 Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 2525/205 Aptamer
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2565/133 conformational analysis

Abstract

In one embodiment, the present invention relates to fluorescent nucleic acid constructs and methods of using these switchable constructs to rapidly screen for target molecule interactions. More particularly, an RNA/DNA chimera comprising a fluorophore-quencher pair and a nucleic acid construct is disclosed for the rapid screening of interactions between the HIV-1 nucleocapsid protein, NCp7, and a stem-loop region, SL3, of the HIV-1 RNA, or antagonists thereof. The compositions and methods disclosed herein can be used in preferred aspects of the present invention for diagnosing disease states, distinguishing the presence of infectious or toxic agents, drug discovery and design, and molecular electronic applications.

Claims

1. A unimolecular bistable molecular switch comprising a nucleic acid comprising a binding domain for a ligand, a framework and a signaling apparatus comprising an attached fluorophore and a quencher, and which alters reversibly between a state H, a state O and a state OL according to the reaction scheme,

2. The bistable molecular switch of claim 1, wherein the signaling apparatus comprises a first reporter on the first stem segment and a second reporter on the first loop portion.

3. The bistable molecular switch of claim 2, wherein the first reporter is a fluorophore and the second reporter is a quencher of said fluorophore.

4. The molecular switch of claim 1, wherein said switch includes one or more modified nucleotide monomers.

5. The molecular switch of claim 1, wherein said LBD comprises a naturally-occurring RNA binding site or analog thereof.

6. The molecular switch of claim 1, wherein said switch exhibits a binding affinity for said ligand of Kd<

1 μ

M.

7. The molecular switch of claim 1, wherein said ligand is from HIV-1.

8. The molecular switch of claim 1, wherein said LBD is adapted to bind NCp7.

9. The molecular switch of claim 1, wherein said ligand is involved in the etiology of HIV-1 infection.

10. The molecular switch of claim 1, wherein said ligand is derived from a pathogen.

11. The molecular switch of claim 1, wherein K1=0.002 to 0.1.

12. A diagnostic method for detecting the presence of a ligand molecule in a sample, comprising the steps of:

providing a bistable molecular switch according to claim 1;

contacting said molecular switch with said sample; and

monitoring changes in the signal.

13. The diagnostic method of claim 12, wherein the signaling apparatus comprises a first reporter on the first stem segment and a second reporter on the first loop portion.

14. The diagnostic method of claim 13, wherein the first reporter is a fluorophore and the second reporter is a quencher of said fluorophore.

15. The diagnostic method of claim 12, wherein said switch includes one or more modified nucleotide monomers.

16. The diagnostic method of claim 12, wherein said LBD has an affinity for said ligand of at least Kd<

1 μ

M.

17. The diagnostic method of claim 12, wherein said ligand is an infectious organism.

18. The diagnostic method of claim 17, wherein said method is adapted for use in a field kit for real-time detection of said infectious organism.

19. An assay method for discovering a chemical entity that interferes with a natural RNA or DNA for binding of a ligand, comprising the steps of:

providing a bistable molecular switch according to claim 1;

contacting said molecular switch with said ligand in the absence of the chemical entity, and monitoring the signal;

contacting said molecular switch with said ligand in the presence of the chemical entity, and monitoring the signal; and

comparing the signals generated in the presence and absence of the chemical entity to determine whether the chemical entity interfered with the binding of said ligand.

20. The assay method of claim 19, wherein the signaling apparatus comprises a first reporter on the first stem segment and a second reporter on the first loop portion.

21. The assay method of claim 20, wherein the first reporter is a fluorophore and the second reporter is a quencher of said fluorophore.

22. The assay method of claim 19, wherein said switch includes one or more modified nucleotide monomers.

23. The assay method of claim 19, wherein said ligand is a viral protein.

24. The assay method of claim 19, wherein the step of contacting said molecular switch with said ligand in the presence of the chemical entity, further comprises allowing said molecular switch and said ligand to equilibrate prior to adding the chemical entity.

25. The assay method of claim 24, wherein said molecular switch is adapted to generate a null fluorescent signal upon equilibration with said ligand.

26. The assay method of claim 19, wherein said molecular switch is bound to a solid substrate in a multiwell dish, an array or a microarray.

27. A method of designing a bistable molecular switch according to claim 1 comprising introducing a mismatch of a base pair.

28. The method of claim 27, wherein the mismatch occurs in a stem structure.

29. The method of claim 27, wherein K1=0.002 to 0.1.

30. A molecular switch, comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOS:

1, 2, 3, 4 and 5.