TARGET VALIDATION, BINDING SITE IDENTIFICATION, AND PROFILING OF RNA TARGETS
The invention is directed to a method of identifying the interactions of RNA such as miRNA with small molecules interacting with RNA (SMIRNAs). A candidate SMIRNA group is associated with a photoaffinity diazirene group that can form a covalent complex with an RNA target site and with an alkyne group that can be used in subsequent “click chemistry’ reactions such as a “CuAAC” reaction, a copper-catalyzed alkyne-azide cy-cloaddition to yield a stable triazole ring. By this means, the RNA binding site of the small molecule can be identified via isolation of the RNA-targetSMIRNA covalent complex and reverse transcription of the RNA followed by DNA sequencing of the reverse transcription product. Sites on the RNA blocked during reverse transcription by the covalently bound SMIRNA are identified as terminations in the sequence compared to the native RNA.
1 . A method of determining the binding site on an RNA molecule to which a small molecule interacting with RNA (SMIRNA) associates, comprising
forming a derivative of the SMIRNA comprising the small molecule covalently coupled with a moiety comprising a diazirine group and a moiety comprising an alkyne group; then,
contacting the RNA with the SMIRNA derivative and exposing to UV light to activate the diazirene and bring about covalent coupling of the SMIRNA derivative to the RNA forming a SMIRNA-RNA covalent complex; then,
contacting the SMIRNA-RNA covalent complex with an azide-bearing group further comprising a pull-down group comprising a biotin moiety linked to the azide by a linker group comprising a disulfide bond, or alternatively with a bead bearing an azide group bonded thereto by a linker comprising a disulfide bond, under conditions comprising the presence of copper catalyst to bring about a CuAAC reaction; then,
contacting the SMIRNA-RNA covalently bonded to the biotin moiety and a complementary pull-down entity comprising streptavidin immobilized on a bead to isolate the SMIRNA-RNA covalent complex; then,
cleaving the SMIRNA-RNA covalent complex from the bead by reduction of the disulfide bond in the respective linker, to free the complex from the bead; then,
subjecting the RNA thus freed to reverse transcription and sequencing of the DNA from the reverse transcription process, to indicate the SMIRNA binding site on the RNA through the presence of a transcription stop.
2 . The method of claim 1 wherein the SMIRNA is SMIRNA-1
or SMIRNA-2
3 . The method of claim 1 wherein the SMIRNA derivative is TGP-9 ono-diaz
4 . The method of claim 1 wherein the SMIRNA derivative is TGP-96-dirner-diaz
5 . The method of claim 1 wherein the azide group of the CuAAC reaction is linked to a biotin group.
6 . The method of claim 1 wherein the azide group of the CuAAC reaction is linked directly to a bead.
7 . The method of claim 1 , wherein the identifying comprises
(1) reverse transcribing the RNA from the purified complex with a primer to create cDNA;
(2) amplifying the cDNA with a primer set suitable for RT-qPCR or high-throughput sequencings (RNA-seq); and
(3) analyzing the cDNA to identify reverse transcriptase sequence stops to thereby determine the sequence of the binding site of the RNA-binding module on the RNA.
8 . The method of claim 1 , wherein the RNA cross-linking module comprises a diazirine group.
9 . The method of claim 1 , wherein the purification module comprises a biotin group.
10 . The method of claim 1 , wherein the RNA comprises an oncogenic non-coding RNA precursor, and the contacting of step (a) comprises contacting a cell expressing the non-coding RNA precursor and the compound.
11 . The method of claim 1 , wherein the oncogenic non-coding RNA precursor comprises oncogenic primary microRNA-96 (pri-miR-96),
12 . The method of claim 1 , wherein the RNA comprises a transcriptome.
13 . The method of claim 1 , wherein the transcriptome is viral.
14 . The method of claim 1 , wherein the transcriptome is mammalian.
15 . The method of claim 1 , wherein the transcriptome is bacterial.
16 . The method of claim 1 , wherein the RNA comprises one or more of synthetic, semi-synthetic, or natural RNA.
17 . The method of claim 1 , wherein the RNA comprises the genome of an RNA virus.
18 . The method of claim 1 carried out in vitro.
19 . The method of claim 1 carried out in living cells.
20 . The method of claim 1 carried out in a preclinical animal model.
21 . The method of claim 1 , wherein the living cells are virally- or bacterially-infected cells.
22 . The method of claim 1 , wherein the method is performed in a parallel array for a plurality of RNA sequences and candidate RNA-binding small modules.