Modified siRNA Constructs for Detecting RISCs
The present teachings provide novel methods, compositions, and kits for detecting siRNA-containing RISCs. In some embodiments, modified siRNA constructs are employed that contain an anti-sense strand and a sense strand, wherein the anti-sense strand comprises a 3′ end, wherein the 3′ end comprises a fluorophore, and wherein the sense strand comprises a 5′ end, wherein the 5′ end comprises a quencher. Following transfection, uptake of the anti-sense strand by RISC liberates the fluorescent signal, allowing for detection of siRNA-containing RISCs.
1 . A method for detecting the formation of an siRNA-containing RISC comprising;
providing a modified siRNA construct, wherein the modified siRNA construct comprises an anti-sense strand and a sense strand, wherein the anti-sense strand comprises a 3′ end, wherein the 3′ end comprises a fluorophore, and wherein the sense strand comprises a 5′ end, wherein the 5′ end comprises a quencher;
transfecting the modified siRNA construct into a sample; and,
measuring fluorescence to detect the formation of the siRNA-containing RISC.
2 . The method according to claim 1 wherein the 3′ end of the sense strand further comprises a cell uptake element.
3 . The method according to claim 2 wherein the cell uptake element is an HIV tat transduction domain.
4 . The method according to claim 2 wherein the cell uptake element is a G-PNA.
5 . A method for detecting the formation of an siRNA-containing RISC comprising;
providing a modified siRNA construct, wherein the modified siRNA construct comprises an anti-sense strand and a sense strand, wherein the anti-sense strand comprises a 3′ end, wherein the 3′ end comprises a quencher, and wherein the sense strand comprises a 5′ end, wherein the 5′ end comprises a fluorophore;
transfecting the modified siRNA construct into a sample; and,
measuring fluorescence to detect the formation of the siRNA-containing RISC.
6 . The method according to claim 5 wherein the 3′ end of the sense strand further comprises a cell uptake element.
7 . The method according to claim 6 wherein the cell uptake element is an HIV tat transduction domain.
8 . The method according to claim 6 wherein the cell uptake element is a G-PNA.
9 . A composition comprising;
a modified siRNA construct, wherein the modified siRNA construct comprises an anti-sense strand and a sense strand, wherein the anti-sense strand comprises a 3′ end, wherein the 3′ end comprises a fluorophore, and wherein the sense strand comprises a 5′ end, wherein the 5′ end comprises a quencher.
10 . The composition according to claim 9 wherein the 3′ end of the sense strand further comprises a cell uptake element.
11 . The composition according to claim 10 wherein the cell uptake element is an HIV tat transduction domain.
12 . The composition according to claim 10 wherein the cell uptake element is a G-PNA.
13 . A composition comprising;
a modified siRNA construct, wherein the modified siRNA construct comprises an anti-sense strand and a sense strand, wherein the anti-sense strand comprises a 3′ end, wherein the 3′ end comprises a quencher, and wherein the sense strand comprises a 5′ end, wherein the 5′ end comprises a fluorophore.
14 . The composition according to claim 13 wherein the 3′ end of the sense strand further comprises a cell uptake element.
15 . The composition according to claim 14 wherein the cell uptake element is an HIV tat transduction domain.
16 . The composition according to claim 14 wherein the cell uptake element is a G-PNA.
17 . A composition comprising a RISC and an anti-sense strand of a modified siRNA construct, wherein the anti-sense strand comprises a 3′ end, wherein the 3′ end comprises a fluorophore.
18 . A composition comprising a RISC and an anti-sense strand of a modified siRNA construct, wherein the anti-sense strand comprises a 3′ end, wherein the 3′ end comprises a quencher.
19 . A kit for detecting the formation of an siRNA-containing RISC, comprising;
a modified siRNA construct, wherein the modified siRNA construct comprises an anti-sense strand and a sense strand, wherein the anti-sense strand comprises a 3′ end, wherein the 3′ end comprises a fluorophore, and wherein the sense strand comprises a 5′ end, wherein the 5′ end comprises a quencher; and,
transfection reagents.
20 . A kit for detecting the formation of an siRNA-containing RISC, comprising;
a modified siRNA construct, wherein the modified siRNA construct comprises an anti-sense strand and a sense strand, wherein the anti-sense strand comprises a 3′ end, wherein the 3′ end comprises a quencher, and wherein the sense strand comprises a 5′ end, wherein the 5′ end comprises a fluorophore; and,
transfection reagents.
21 . The kit according to claim 19 or 20 , wherein the transfection reagents are selected from the group consisting of CaCl 2 , or liposomes.