Methods and kits for discovery of RNA-binding compounds
AssigneeRibotargets, Ltd.
InventorsJonathan Karn, Catherine Denise Prescott
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Abstract
The invention relates to a solution-based assay for identifying RNA binding compounds, based on competition. The assay comprises a reporter molecule carrying a fluorescent or chromogenic group that can form a one-to-one complex with an RNA target molecule carrying a second fluorescent or chromogenic group in such a way that the two groups are in sufficient proximity for fluorescence resonance energy transfer and/or quenching to take place. Addition of a compound-to-be-tested prevents formation of the complex and thereby increases the fluorescence of the RNA target and/or reporter molecules relative to the signal obtained in the absence of the test compound. The invention also provides for quantitative screening methods and kits are also included.
Claims25
A method for determining whether a test compound binds to a target RNA, the Sethod comprising the steps of:
- (a) contacting the test compound with a pair of indicator molecules comprising a reporter labelled with a donor group or an acceptor group and the target RNA labelled with a complementary acceptor or donor group, wherein said reporter is not an oligonucleotide that has a sequence which is able to hybridize via Watson-Crick base-pairing under stringent conditions to an unfolded target RNA, the pair being capable of binding to each other in an orientation that pennits the donor group to come into sufficient proximity to the acceptor group to permit fluorescent resonance energy transfer and/or quenching to take place;
- and
- (b) measuring the fluorescence of the target RNA and the reporter molecule in the presence of the test compound and comparing this value to the fluorescence of a standard;
- thereby determining whether said test compound binds said target RNA.
A method for determining whether a test compound binds to a target RNA that is a viral RNA from a region of the TAR of HIV, the method comprising the steps of:
- (a) contacting the test compound with a pair of indicator molecules comprising a reporter labelled with a donor group or an-acceptor group and the target RNA labelled with a complementary acceptor or donor group, wherein said target RNA is selected from the group consisting of:
- (a) SEQ ID NO:
- 1 and FAM-labelled SEQ ID NO:
- 2;
- (b) SEQ ID NO:
- 3 and FAM-labelled SEQ ID NO:
- 4;
- (c) SEQ ID NO:
- 5, labelled with Cy3, FAM or DABCYL;
- and the reporter is SEQ ID NO:
- 7, labelled with FAM, DABCYL or TAMRA, the pair being capable of binding to each other in an orientation that permits the donor group to come into sufficient proximity to the acceptor group to permit fluorescent resonance energy transfer and/or quenching to take place;
- and
- (b) measuring the fluorescence of the target RNA and the reporter molecule in the presence of the test compound and comparing this value to the fluorescence of a standard;
- thereby determining whether said test compound binds said target RNA.
A kit for determining whether a test compound binds to a target RNA, the kit comprising (a) a target RNA labelled with a donor group or an acceptor group and (b) a reporter labelled with a complementary acceptor or donor group, wherein said reporter is not an oligonucleotide that has a sequence which is able to hybridize via Watson-Crick base-pairing under stringent conditions to an unfolded target RNA, and wherein the reporter and the target RNA are capable of binding to each other in an orientation that permits the donor group to come into sufficient proximity to the acceptor group to permit fluorescent resonance energy transfer and/or quenching.
A method for determining the presence in a biological sample of a compound that binds to a target RNA molecule, comprising
- (a) contacting the sample with a pair of indicator molecules comprising a reporter labelled with a donor group or an acceptor group and the target RNA labelled with a complementary acceptor or donor group, wherein said reporter is not an oligonucleotide that has a sequence which is able to hybridize via Watson-Crick base-pairing under stringent conditions to an unfolded target RNA, the pair being capable of binding to each other in an orientation that permits the donor group to come into sufficient proximity to the acceptor group to permit fluorescent resonance energy transfer and/or quenching to take place;
- and
- (b) measuring the fluorescence of the target RNA and the reporter molecule in said step (a) to obtain a fluorescence value and comparing this value to the fluorescence of a standard wherein a difference in said value and the fluorescence of said standard is an indication of the presence in a biological sample of a compound that binds to said target RNA.
Dependencies are inferred from the claim text.