Target validation and profiling of the RNA targets of small molecules
View Patent ↗A method for the precise cellular destruction of an oncogenic non-coding RNA with a RNA-binding small molecule conjugated with bleomycin A5 is described. The method affords reversal of phenotype. Bleomycin A5 was coupled to an RNA-binding molecule that selectively binds the microRNA-96 hairpin precursor (pri-miR-96). By coupling of bleomycin A5's free amine to the RNA-binding molecule, its affinity for binding to pri-miR-96 is >100-fold stronger than to DNA. The conjugate compound selectively cleaves pri-miR-96 in triple negative breast cancer (TNBC) cells. Selective cleavage of pri-miR-96 enhances expression of FOXO1 protein, a pro-apoptotic transcription factor that miR-96 silences, and triggers apoptosis in TNBC cells. No effects were observed in healthy breast epithelial cells. This method provides programmable control for targeting RNA through the selection of an RNA-binding molecule/bleomycin A5 conjugate and provides a facile method of mapping the cellular binding sites of an RNA-binding molecule.
1 . A method comprising
contacting a library of RNA sequences and an effective amount of a compound comprising a conjugate of an RNA-cleaving moiety and an RNA-binding small molecule to cleave the RNA of the library at a binding site of the compound to the RNA to provide a cleaved RNA fragment;
amplifying and sequencing the cleaved RNA fragment, thereby identifying the site of binding of the small RNA-binding molecule to the RNA of the library which is associated with the site of cleavage; and
wherein at least one of the RNA sequences is from an oncogenic non-coding RNA precursor, and the compound binds to the oncogenic non-coding RNA precursor.
2 . The method of claim 1 , wherein the RNA-binding small molecule comprises an N-methyl-piperazinyl-bis-benzimidazole group.
3 . The method of claim 1 , wherein the RNA-cleaving moiety comprises bleomycin.
4 . The method of claim 3 , wherein the bleomycin is bleomycin A5.
5 . The method of claim 2 , wherein the RNA-binding small molecule is Targaprimir-96.
6 . The method of claim 1 , further comprising contacting a cell expressing the non-coding RNA precursor with an effective amount of the compound.
7 . The method of claim 1 , wherein the oncogenic non-coding RNA precursor comprises oncogenic primary microRNA-96.
8 . The method of claim 1 , wherein the compound is a covalent conjugate of Targaprimir-96 and bleomycin A5.
9 . The method of claim 1 , wherein at least one of the RNA sequences is from FOXO1.
10 . The method of claim 9 , further comprising enhancing expression of FOXO1 protein in breast cancer cells by contacting the cells with an effective amount of the compound.
11 . The method of claim 10 , wherein the contacting comprises administering to a human patient.
12 . The method of claim 9 , wherein the compound is a covalent conjugate of Targaprimir-96 and bleomycin A5.
13 . A method of triggering apoptosis in triple negative breast cancer cells, comprising contacting the triple negative breast cancer cells with an effective amount of a compound comprising a conjugate of an RNA-cleaving moiety and an RNA-binding small molecule, wherein the RNA-binding small molecule comprises an N-methyl-piperazinyl-bis-benzimidazole group, and the RNA-cleaving moiety comprises bleomycin.
14 . The method of claim 13 , wherein the contacting comprises administering to a human patient.
15 . The method of claim 13 , wherein the compound is a covalent conjugate of Targaprimir-96 and bleomycin A5.
16 . A method of treating triple negative breast cancer, comprising administering to a patient afflicted therewith an effective dose of a compound comprising a conjugate of an RNA-cleaving moiety and an RNA-binding small molecule, wherein the RNA-binding small molecule comprises an N-methyl-piperazinyl-bis-benzimidazole group, and the RNA-cleaving moiety comprises bleomycin.
17 . The method of claim 16 , wherein the breast cancer comprises expression of oncogenic primary microRNA-96.
18 . The method of claim 16 , wherein the compound is a covalent conjugate of Targaprimir-96 and bleomycin A5.
19 . The method of claim 1 , wherein the RNA sequence library comprises a transcriptome.
20 . The method of claim 19 , wherein the transcriptome is viral.
21 . The method of claim 1 , wherein the RNA sequence library comprises one or more of synthetic, semi-synthetic, or natural RNA.
22 . The method of claim 1 , wherein the RNA sequence library comprises the genome of an RNA virus.
23 . The method of claim 1 carried out in vitro.
24 . The method of claim 1 carried out in living cells.
25 . The method of claim 24 , wherein the cells are virally-or bacterially-infected cells.
26 . The method of claim 1 , wherein a set of RNA sequences and a set of compounds comprising candidate RNA-binding small molecules are assayed in a 2-dimensional parallel array.
27 . A compound comprising a conjugate of an RNA-cleaving moiety and an RNA-binding small molecule, wherein the RNA-binding small molecule is Targaprimir-96.
28 . The compound of claim 27 , wherein the RNA-cleaving moiety comprises bleomycin.
29 . The method of claim 19 , wherein the transcriptome is mammalian.
30 . The method of claim 19 , wherein the transcriptome is bacterial.
31 . The method of claim 13 , wherein the RNA-binding small molecule is Targaprimir-96.
32 . The method of claim 16 , wherein the RNA-binding small molecule is Targaprimir-96.
33 . A method comprising
contacting a library of RNA sequences and an effective amount of a compound comprising a conjugate of an RNA-cleaving moiety and an RNA-binding small molecule to cleave the RNA of the library at a binding site of the compound to the RNA to provide a cleaved RNA fragment;
amplifying and sequencing the cleaved RNA fragment, thereby identifying the site of binding of the small RNA-binding molecule to the RNA of the library which is associated with the site of cleavage; and
wherein the RNA-binding small molecule is Targaprimir-96.