BRANCHED AND MULTI-CHAIN NUCLEIC ACID SWITCHES FOR SENSING AND SCREENING

• US 20110059555A1
• Filed: 10/11/2010
• Published: 03/10/2011
• Est. Priority Date: 08/03/2004
• Status: Active Grant

• Current Assignee: Syracuse University
• Original Assignee: Syracuse University
• Inventors: Borer, Philip N., Hudson, Bruce S.
• Granted Patent: US 8,080,379 B2
• Time in Patent Office: Days
• US Class Current: 436/501
• 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 method of chemical screening comprising:

providing a bistable branched or multichain switch, comprising DNA, RNA, modified nucleic acid, or combinations thereof, wherein the switch comprises a 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.

2. The method of claim 1, wherein the signaling apparatus comprises a lumiphore and a quencher of the lumiphore.

3. The method of claim 1, wherein the ligand binding domain comprises RNA, the molecular framework comprises DNA and the ligand is a viral protein.

4. The method of claim 1, 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.

5. The method of claim 1, 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 one;

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.

6. The method of claim 1, further comprising equilibrating the molecular switch and the ligand prior to adding the chemical entity.

9. The method of claim 1, wherein the branched or multichain switch is 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), which is partially but not completely complementary to P;

a tether that holds P and C together while the switch changes between the first and second conformations without dissociating the branched or multichain nucleic acid thereby creating a rapidly reversible switch;

a toggle strand (T) tethered to the P-strand and the C-strand by covalent linkage; and

a signaling apparatus comprising a combination of signaling entities, wherein a ligand for the ligand binding domain is selected from the group consisting of proteins, cell-surface features, and small molecules, and wherein the ligand binding domain is sequestered in the first conformation.

10. The branched or multichain switch of claim 9, wherein P, C and T are joined together at a vertex.

11. The method of claim 1, wherein the branched or multichain switch is adapted to switch from a first conformation to a second conformation upon ligand binding, and wherein said switch comprises:

a first strand comprising a ligand binding domain;

a second strand; and

a fastener to couple the two strands together without dissociating the branched or multichain switch, thereby creating a rapidly reversible switch;

wherein the first strand and second strand hybridize to each other in the first conformation which sequesters the ligand binding domain and the ligand binding domain is free in the second conformation, wherein a ligand for the ligand binding domain is selected from the group consisting of proteins, cell-surface features, and small molecules.

12. The method of claim 1, wherein the branched or multichain switch is 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), which is partially but not completely complementary to P;

a tether that holds P and C together while the switch changes between the first and second conformations without dissociating the branched or multichain nucleic acid thereby creating a rapidly reversible switch; and

a signaling apparatus comprising a combination of signaling entities, wherein a ligand for the ligand binding domain is selected from the group consisting of proteins, cell-surface features, and small molecules, and wherein the ligand binding domain is sequestered in the first conformation,

wherein tethering occurs via a fastener duplex, F, to couple two covalently linked nucleic acid strands together by a non-covalent interaction, wherein the first strand comprises C and the second strand comprises P when the branched or multichain switch is in the second conformation.

13. The method of claim 12, wherein the fastener duplex is a base-pair stem that does not vary between the first and second conformations of said switch.

7. 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.

8. A method of using 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, said method 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.