G-quadruplex binding assays and compounds therefor
View Patent ↗The present invention provides methods for assaying binding of compounds to G-quadruplex structures. Also provided are methods for screening candidate compounds for use as modulators of G-quadruplex activity, and methods for screening candidate compounds for telomerase inhibitory activity. The invention further provides novel compounds useful in the assays of the invention.
1. A method for determining the binding affinity of a candidate compound for G-quadruplex structures comprising the steps of:
(a) providing a G-quadruplex polynucleotide analog, said G-quadruplex polynucleotide analog having a G-quadruplex structure and at least one intrinsic fluorescent moiety;
(b) contacting said G-quadruplex polynucleotide analog with said candidate compound;
(c) measuring a difference in a fluorescence property of said G-quadruplex polynucleotide analog; and
(d) determining the binding affinity of the candidate compound;
wherein the G-quadruplex analog polynucleotide comprises:
a nucleobase sequence that is an analog of a telomeric repeat sequence; and
at least one nucleotide comprising a 6-methyl-isoxanthopterin nucleobase;
wherein:
said G-quadruplex analog polynucleotide forms a G-quadruplex structure; and
each 6-methyl-isoxanthopterin nucleobase is located within said G-quadruplex structure.
2. The method of claim 1 , wherein the G-quadruplex analog polynucleotide comprises the structure:
(5′-(L 1 ) q (L 2 ) r (L 3 ) s (L 4 ) t (L 5 ) u (L 6 ) v ) x
wherein:
each L 1 , L 2 , L 3 , L 4 , L 5 and L 6 is independently selected from the group consisting of a nucleotide, a nucleotide analog, and a nucleotide comprising a 6-methyl-isoxanthopterin nucleobase;
q, r, s, t, u and v are each independently 0, 1 or 2, provided that the sum of q+r+s+t+u+v is at least 3; and
x is 1 to 8;
provided that:
5′-(L 1 ) q (L 2 ) r (L 3 ) s (L 4 ) t (L 5 ) u (L 6 ) v is an analog of a telomeric repeat sequence.
3. The method of claim 1 , wherein the G-quadruplex analog polynucleotide is an analog of a human telomeric repeat sequence.
4. The method of claim 2 , wherein the G-quadruplex analog polynucleotide is an analog of a human telomeric repeat sequence.
5. The method of claim 2 , wherein q, r, s, t, u and v are each 1.
6. The method of claim 2 , wherein x is from 2 to 6.
7. The method of claim 2 , wherein x is from 3 to 5.
8. The G-quadruplex analog polynucleotide of claim 2 , wherein x is 4.
9. The method of claim 1 , wherein said G-quadruplex analog polynucleotide comprises one nucleotide comprising a 6-methyl-isoxanthopterin nucleobase.
10. The method of claim 1 , wherein said G-quadruplex analog polynucleotide comprises two nucleotides comprising a 6-methyl-isoxanthopterin nucleobase.
11. The method of claim 1 , wherein said G-quadruplex analog polynucleotide comprises any of SEQ ID NOs: 1-8.
12. The method of claim 1 , comprising contacting a plurality of candidate compounds with an array, wherein the array comprises a plurality of said G-quadruplex analogs; and
measuring a difference in a fluorescence property of one or more of said G-quadruplex analogs.
13. A method for identifying a candidate compound as a telomerase inhibitor comprising the steps of:
(a) providing a G-quadruplex analog polynucleotide, said G-quadruplex analog polynucleotide having at least one intrinsic fluorescent moiety;
(b) contacting said G-quadruplex analog with said candidate compound;
(c) measuring a difference in a fluorescence property of said G-quadruplex analog; and
(d) assaying the compound for telomerase inhibitory activity;
wherein the G-quadruplex analog polynucleotide comprises:
a nucleobase sequence that is an analog of a telomeric repeat sequence; and
at least one nucleotide comprising a 6-methyl-isoxanthopterin nucleobase;
wherein:
said G-quadruplex analog polynucleotide forms a G-quadruplex structure; and
each 6-methyl-isoxanthopterin nucleobase is located within said G-quadruplex structure.
14. The method of claim 13 , wherein the G-quadruplex analog polynucleotide comprises the structure:
(5′-(L 1 ) q (L 2 ) r (L 3 ) s (L 4 ) t (L 5 ) u (L 6 ) v ) x
wherein:
each L 1 , L 2 , L 3 , L 4 , L 5 and L 6 is independently selected from the group consisting of a nucleotide, a nucleotide analog, and a nucleotide comprising a 6-methyl-isoxanthopterin nucleobase;
q, r, s, t, u and v are each independently 0, 1 or 2, provided that the sum of q+r+s+t+u+v is at least 3; and
x is 1 to 8;
provided that:
5′-(L 1 ) q (L 2 ) r (L 3 ) s (L 4 ) t (L 5 ) u (L 6 ) v is an analog of a telomeric repeat sequence.
15. The method of claim 13 , wherein the G-quadruplex analog polynucleotide is an analog of a human telomeric repeat sequence.
16. The method of claim 13 , wherein said G-quadruplex analog polynucleotide comprises one nucleotide comprising a 6-methyl-isoxanthopterin nucleobase.
17. The method of claim 13 , wherein said G-quadruplex analog polynucleotide comprises any of SEQ ID NOs: 1-8.