Switchable nucleic acids for diagnostics, screening and molecular electronics

• US 20060216692A1
• Filed: 02/03/2004
• Published: 09/28/2006
• Est. Priority Date: 02/04/2003
• Status: Active Grant

• Current Assignee: Syracuse University
• Original Assignee: Syracuse University
• Inventors: Hudson, Bruce S., Borer, Philip N.
• Granted Patent: US 7,718,784 B2
• Time in Patent Office: Days
• US Class Current: 435/5
• 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 bistable molecular switch, comprising a binding domain for a ligand, a chimeric nucleic acid framework comprising both RNA and DNA nucleotide residues, and a signaling apparatus, said molecular switch being adapted to vary between first and second stable conformations upon binding of said ligand, whereupon said signaling apparatus produces a detectable change in a signal.

2. The molecular switch of claim 1, wherein said signaling apparatus comprises a fluorophore and quencher pair located along said framework and having changeable positions relative to one another, such that a difference is detectable in a fluorescent signal upon change in conformation of said switch.

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

4. The molecular switch of claim 1, wherein said framework comprises a double-hairpin construct.

5. The molecular switch of claim 1, wherein said first and second stable conformations further comprise double helical and cruciform structures, respectively.

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

7. The molecular switch of claim 1, wherein said binding domain comprises a naturally-occurring DNA binding site or analog thereof.

8. The molecular switch of claim 1, wherein said binding domain comprises a combinatorially-derived sequence or related fragment, which is empirically chosen to bind said ligand.

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

1 μ

M.

10. The molecular switch of claim 1, further comprising a nucleic acid sequence selected from the group consisting of SEQ ID Nos. 1, 2, 3, 4 and 5.

11. The molecular switch of claim 1, wherein said binding domain is adapted to bind a ligand selected from the group consisting of NC, tat, and rev proteins from HIV-1.

12. The molecular switch of claim 1, wherein said binding domain is adapted to bind NCp7.

13. The molecular switch of claim 1, wherein said ligand is involved in the etiology of a viral infection, which is selected from the group consisting of Hepatitis C, Congo-Crimean hemorrhagic fever, Ebola hemorrhagic fever, Herpes, human cytomegalovirus, human pappiloma virus, influenza, Marburg, Q fever, Rift valley fever, Smallpox, Venezuelan equine encephalitis, HIV-1, MMTV, HIV-2, HTLV-1, SNV, BIV, BLV, EIAV, FIV, MMPV, Mo-MLV, Mo-MSV, M-PMV, RSV, SIV, AMV.

14. The molecular switch of claim 1, wherein said ligand is selected from the group consisting of TAR-tat, RRE-rev, DIS, PBS, RT, PR, IN, SU, TM, vpu, vif, vpr, nef, mos, tax, rex, sag, v-src, v-myc and precursors and protease products of the precursors, gag, gag-pol, env, src, and onc.

15. The molecular switch of claim 1, wherein said ligand is derived from an organism selected from the group consisting of bacteria, fungi, insects, pathogens, and pests to humans, animals, and plants.

16. The molecular switch of claim 1, wherein said ligand is a toxin or other factor derived from bacteria and other microorganisms selected from the group consisting of B. anthracis, Burkholderia pseudomallei, Botulinum, Brucellosis, Candida albicans, Cholera, Clostridium perfringins, Kinetoplasts, Malaria, Mycobacteria, Plague, Pneumocystis, Schistosomal parasites, Cryptosporidium, Giardia, and other environmental contaminants of public and private water supplies, Ricin, Saxitoxin, Shiga Toxin from certain strains of E. coli, Staphylococcus (including enterotoxin B), Trichothecene mycotoxins, Tularemia, and agents causing Toxoplasmosis, and food or beverage contaminants that may be deleterious to human or animal health.

17. The molecular switch of claim 1, wherein said ligand is a small-molecule target selected from the group consisting of nerve gas agents, chemical poisons, contaminants of public and private water supplies, food and beverage contaminants, and contaminants of indoor air that may be deleterious to human or animal health.

18. A method for detecting the presence of a ligand molecule in a sample, comprising contacting the bistable molecular switch of claim 1 with said sample;

and monitoring changes in the signal.

19. The method of claim 18, wherein said ligand is an infectious organism or toxic agent.

20. The method of claim 19, wherein said method is adapted for use in a field kit for real-time detection of said infectious organism or toxic agent.

21. 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, comprising an analog of said RNA or DNA, said analog comprising a binding domain for said ligand, said bistable molecular switch further comprising a molecular framework and a signaling apparatus, said molecular switch being adapted to vary between first and second stable conformations upon binding of said ligand, wherein said signaling apparatus produces a detectable change in a signal;

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.

22. The assay method of claim 21, wherein said signaling apparatus comprises a fluorophore and quencher pair located along the framework and having changeable positions relative to one another, such that a difference is detectable in a fluorescence signal upon the change in conformation of said switch.

23. The assay method of claim 21, wherein said switch comprises one or more modified nucleotide monomers.

24. The assay method of claim 21, wherein said ligand is a viral protein.

25. The assay method of claim 21, 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.

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

27. The assay method of claim 21, wherein said binding domain comprises a combinatorially-derived sequence which has been empirically chosen to bind said ligand.

28. The assay method of claim 21, wherein said molecular switch is bound to a solid substrate in a multiwell dish, an array or a microarray.

29. A bistable molecular switch for use in molecular electronics, comprising:

a binding domain comprising a nucleic acid sequence configured to bind a photosensitive ligand which is adapted to alter between first and second energy states upon application of triggering photons, wherein the binding affinity of said binding domain for said photosensitive ligand is greater in one energy state compared to the other energy state;

a molecular framework encompassing said binding domain, and being adapted to switch from a first stable conformation to a second stable conformation upon binding of said photosensitive ligand by said binding domain;

a signaling apparatus adapted to generate a detectable signal, such that said detectable signal varies upon application of the triggering photons.

30. The bistable molecular switch of claim 29, wherein said signaling apparatus comprises a fluorophore and quencher pair located along the switch framework and having changeable positions relative to one another, such that a difference is detectable in a fluorescence signal upon the change in conformation of said switch.