Nucleic acid multiplex formation
Heteropolymeric triplexes and quadruplexes and methods for making them; the use of accelerator agents such as cations to create them; the use of fluorescent intercalators and fluorescent probe-bound non-intercalators to detect them.
1. A method of creating a nucleic acid multiplex, said method comprising the steps of:
1) creating a mixture comprising water, a Watson-Crick duplex, a sufficient number of single-stranded mixed base sequence molecules to form the multiplex including the Watson-Crick duplex, and an accelerator agent that increases a rate or amount of multiplex formation, said multiplex being a triplex or quadruplex; and
2) incubating said mixture to allow the multiplex to form, each strand of said multiplex related to all other strands of the multiplex by adherence to Watson-Crick base-pairing rules or homologous binding base-pairing rules;
provided that, within the multiplex, the Watson-Crick duplex added in step (1) is heteropolymeric with a G-C content between 10% and 90% and a combined frequency therein of purine-pyrimidine dimers and pyrimidine-purine dimers exceeds 25%.
2. A method of claim 1 wherein the multiplex created is a triplex, in step (1) the sufficient number of single-stranded molecules is 1, and in step (2) the triplex is formed.
3. A method of claim 1 wherein the duplex substantially retains its double-helical structure and the single-stranded molecule resides in a groove of that double-helical structure.
4. A method of claim 1 wherein the single-stranded molecule is related to one strand of the duplex by Watson-Crick base-pairing rules and to the second strand of the duplex by homologous binding base-pairing rules.
5. A method of claim 4 wherein the duplex substantially retains its double-helical structure and the single-stranded molecule resides in a groove of that double-helical structure.
6. A method of claim 1 wherein steps (1) and (2) are performed with at least one of the nucleic acid strands and the duplexes not in a cell.
7. A method of claim 1 wherein step (2) is performed without assistance of a protein.
8. A method of claim 1 wherein in step (1), the water is added so that it accounts, on a volume basis, for at least 50 percent of a final volume of the mixture.
9. A method of claim 1 wherein in step (1), the water is added so that it accounts, on a volume basis, for at least 80 percent of a final volume of the mixture.
10. A method of claim 1 wherein in step (1), the water is added so that it accounts, on a volume basis, for all of the liquid added to the mixture.
11. A method of claim 1 wherein step (2) is performed at a temperature or temperatures above a freezing temperature of the mixture and at not more than 85° C.
12. A method of claim 11 wherein the temperature or temperatures is/are 5° C. to 30° C.
13. A method of claim 12 wherein the temperature or temperatures is/are 15° C. to 25° C.
14. A method of claim 1 wherein in step (1), a cation is added as the accelerator agent.
15. A method of claim 14 wherein said cation is Na + provided at a concentration of 50 mM to 125 mM.
16. A method of claim 14 wherein said cation is selected from the group consisting of Mn +2 provided at a concentration of 10 mM to 45 mM, Mg +2 provided at a concentration of 10 mM to 45 mM, and Ni +2 provided at a concentration of 20 mM.
17. A method of claim 1 wherein in step (1) an intercalator is added as an accelerator agent.
18. A method of claim 17 wherein the intercalator is a fluorescent intercalator.
19. A method of claim 18 wherein the fluorescent intercalator is selected from the group consisting of YOYO-1, TOTO-1, YOYO-3, TOTO-3, POPO-1, BOBO-1, POPO-3, BOBO-3, LOLO-1, JOJO-1, cyanine dimers, YO-PRO-1, TO-PRO-1, YO-PRO-3, TO-PRO-3, TO-PRO-5, PO-PRO-1, BO-PRO-1, PO-PRO-3, BO-PRO-3, LO-PRO-1, JO-PRO-1, cyanine monomers, ethidium bromide, ethidium homodimer-1, ethidium homodimer-2, ethidium derivatives, acridine, acridine orange, acridine derivatives, ethidium-acridine heterodimer, ethidium monoazide, propidium iodide, SYTO dyes, SYBR Green 1, SYBR dyes, Pico Green, SYTOX dyes, and 7-aminoactinomycin D.
20. The method of claim 1 wherein the accelerator agent is a non-intercalating fluorophore.
21. A method of claim 20 wherein the non-intercalating fluorophore is selected from the group consisting of biotin, rhodamine, Alexa dyes, BODIPY dyes, biotin conjugates, thiol-reactive probes, fluorescein and derivatives including but not limited to the caged probes, Oregon Green, Rhodamine Green, QSY dyes.
22. A method of claim 1 wherein in step (1) the accelerator agent is an intercalator that binds to at least one of the minor groove and the major groove of the Watson-Crick duplex.
23. The method of claim 1 wherein in step (1) the accelerator agent at 25° C. is a liquid.
24. The method of claim 23 wherein in step (1) the accelerator agent is an organic liquid soluble in water.
25. The method of claim 1 wherein in step (1) an accelerator agent that is a condensation agent as regards the Watson-Crick duplex is added.
26. The method of claim 1 wherein in step (1) an accelerator agent that is a decondensation agent as regards the Watson-Crick duplex is added.
27. A method of claim 1 wherein the multiplex created is a quadruplex, in step (1) the Watson-Crick duplex is a first Watson-Crick duplex, and in step (1) the sufficient number of single-stranded molecules is 2, those single-stranded molecules are in a second Watson-Crick duplex, and in step (2) the quadruplex is formed from said first and second duplexes.