Branched and multi-chain nucleic acid switches for sensing and screening

• US 20060029933A1
• Filed: 08/02/2005
• Published: 02/09/2006
• Est. Priority Date: 08/03/2004
• Status: Active Grant

• Current Assignee: Syracuse University
• Original Assignee: Syracuse University
• Inventors: Hudson, Bruce S., Borer, Philip N.
• Granted Patent: US 7,521,546 B2
• Time in Patent Office: Days
• US Class Current: 435/5
• 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 2310/3517 Marker; Tag
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/70 involving virus or bacteriophage immunoassay for viruses G01N33/56983
• CPC Class Codes: C12Q 1/701 Specific hybridization probes
• CPC Class Codes: C12Q 2525/205 Aptamer
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2525/313 Branched oligonucleotides
• CPC Class Codes: C12Q 2541/101 Selex
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

Embodiments of the invention relate to a branched or multichain nucleic acid switch adapted to switch from a first conformation to a second conformation upon ligand binding. The switch includes a probe strand, P, which includes the ligand binding domain; a switching framework which includes a cover strand (C), and a tether that holds P and C together and a signaling apparatus. Some embodiments include a toggle strand (T) where now the tether holds P, C, T, and the signaling apparatus together. As the switch changes between the first and second conformations; the signaling apparatus reports the state of the switch. The signaling entity is typically a lumiphore and a quencher located along the switching framework. Nucleic acid switches have applications in real time assays for diverse agents including infectious agents, environmental toxins, and terrorist agents, as well as screening methods for such agents. Further applications are found for nanoelectronics, nanofabrication and nanomachines.

Claims

1. A branched or multichain nucleic acid switch adapted to switch from a first conformation to a second conformation upon ligand binding, said switch comprising:

a probe strand P comprising a ligand binding domain;

a switching framework comprising a cover strand (C);

a tether that holds P and C together while the switch changes between the first and second conformations; and

a signaling apparatus comprising a combination of signaling entities.

2. The branched or multichain nucleic acid switch of claim 1, wherein the probe strand is selected from the group consisting of DNA, RNA, modified nucleic acid, and combinations thereof.

3. The branched or multichain switch of claim 1, wherein said first conformation has P extensively hybridized to C and said second conformation has P not hybridized to C.

4. The branched or multichain switch of claim 1, wherein the signaling entities comprise a lumiphore and a quencher located along the switching framework.

5. The branched or multichain switch of claim 1, further comprising a toggle strand (T).

6. The branched or multichain switch of claim 1, further comprising a fastener, F, to couple two nucleic acid strands together, wherein the first strand comprises C and the second strand comprises P when the branched or multichain switch is in the second conformation.

7. The branched or multichain switch of claim 1, wherein the ligand binding domain comprises a naturally occurring RNA binding site or analog thereof or a naturally occurring DNA binding site or analog thereof or a combinatorially derived sequence or related fragment.

8. The branched or multichain switch of claim 1, wherein the ligand binding domain is adapted to bind a ligand which is selected from the group consisting of:

a disease agent, wherein the disease is 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, or AMV;

a retroviral component which is 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, or onc;

a toxin or other factor derived from bacteria or other microorganisms which are B. anthracis, Burkholderia pseudomallei, Botulinum, Brucellosis, Candida albicans, Cholera, Clostridium perfringins, Kinetoplasts, Malaria, Mycobacteria, Plague, Pneumocystis, Schistosomal parasites, Cryptosporidium, Giardia, Ricin, Saxitoxin, Shiga Toxin, Staphylococcus (including enterotoxin B), Trichothecene mycotoxins, Tularemia, or agents causing Toxoplasmosis; and

nerve gas agents, chemical poisons, contaminants of water supplies, contaminants of food and beverages, or contaminants of air.

9. A population of branched or multichain switch molecules according to claim 1, wherein there are more molecules with C hybridized to P than with P not hybridized to C.

10. A population of branched or multichain switch molecules according to claim 5, wherein there are more molecules with C hybridized to P than with T hybridized to C.

11. The branched or multichain switch of claim 5, wherein P, C and T are joined together at a vertex.

12. A method to generate the branched or multichain switches of claim 1 which comprises adjusting an equilibrium constant, K₁, for the switch from the first conformation to the second conformation.

13. The method of claim 12, wherein a sequence of the C strand is altered, and a free energy difference between the two conformations, is used to estimate K₁.

14. The method of claim 12, wherein K₁ is set to favor conformation 1.

15. The method of claim 12, wherein a value of K₁ is experimentally verified.

16. The method of claim 15, wherein a signal of a candidate switch is used to determine said candidate'"'"'s value of KI by interpolation between that for sequence (a), which favors conformation 1 over conformation 2 by a factor of 100 or more, and sequence (b), which favors conformation 2 over conformation 1 by a factor of 100 or more.

17. The method of claim 15, wherein the value of K₁ for switch candidates is estimated by comparison with known competitors, X, of the ligand, L, designed to interact with conformation 2 of the switch.

18. A method to generate the branched or multichain switches of claim 5 which comprises:

adjusting an equilibrium constant, K₁, for the switch from the first conformation to the second conformation;

altering sequences of C and T; and

estimating K1, based upon the free energy difference between the two conformations.

19. A kit comprising the branched or multichain switch of claim 1 for real time detection of a selected ligand.

20. A kit comprising the branched or multichain switch of claim 5 for real time detection of a selected ligand.

28. A method of using the molecular switch of claim 1, comprising:

binding a ligand to the ligand binding domain in P, whereby the branched or multichain switch changes from the first conformation to the second conformation upon ligand binding; and

measuring a luminescence change as a result of the conformational change.

21. A method of chemical screening comprising:

providing a bistable branched or multichain switch, comprising an analog of an RNA or DNA, wherein the analog comprises the ligand binding domain, a molecular framework and a signaling apparatus;

contacting the branched or multichain switch with the ligand in the absence of a screened chemical entity;

monitoring a signal produced from the signaling apparatus in the absence of the chemical entity;

contacting the branched or multichain switch with the ligand in the presence of the chemical entity;

monitoring a signal produced from the signaling apparatus in the presence of the chemical entity; and

comparing the signal produced in the absence of the chemical entity with the signal produced in the presence of the chemical entity to determine the effect of the chemical entity on the ligand binding.

22. The method of claim 21, wherein the signaling apparatus comprises a lumiphore and a quencher of the lumiphore.

23. The method of claim 21, wherein the ligand binding domain comprises RNA, the molecular framework comprises DNA and the ligand is a viral protein.

24. The method of claim 21, wherein the ligand binding domain comprises RNA, the molecular framework comprises DNA and the ligand binding domain comprises a naturally occurring RNA binding site or analog thereof or a naturally occurring DNA binding site or analog thereof or a combinatorially derived sequence or related fragment.

25. The method of claim 21, wherein the ligand binding domain comprises RNA, the molecular framework comprises DNA and the ligand is selected from the group consisting of:

a disease agent wherein the disease is 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, or AMV;

a retroviral component which is 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, or onc;

a toxin or other factor derived from bacteria or other microorganisms which are B. anthracis, Burkholderia pseudomallei, Botulinum, Brucellosis, Candida albicans, Cholera, Clostridium perfringins, Kinetoplasts, Malaria, Mycobacteria, Plague, Pneumocystis, Schistosomal parasites, Cryptosporidium, Giardia, Ricin, Saxitoxin, Shiga Toxin, Staphylococcus (including enterotoxin B), Trichothecene mycotoxins, Tularemia, or agents causing Toxoplasmosis; and

nerve gas agents, chemical poisons, contaminants of water supplies, contaminants of food and beverages, or contaminants of air.

26. The method of claim 21, further comprising equilibrating the molecular switch and the ligand prior to adding the chemical entity.

27. A molecular switch adapted to switch from a first energy state to a second energy state upon application of triggering photons, said switch comprising:

a photosensitive ligand binding domain comprising a combinatorially-derived sequence;

a molecular framework comprising the photosensitive ligand binding domain adapted to switch from a first stable conformation to a second stable conformation upon binding of the photosensitive ligand by the ligand binding domain; and

a signaling apparatus along the molecular framework comprising a combination of signaling entities that vary upon application of the triggering photons when the molecular framework switches between first and second stable conformations.

29. A branched or multichain switch adapted to switch from a first conformation to a second conformation upon ligand binding, said switch comprising:

a first strand comprising a ligand binding domain;

a second strand; and

a fastener to couple the two strands together;

wherein the first strand and second strand hybridize to each other in the first conformation and the ligand binding domain is free in the second conformation.