The expansion and application of the RNA--small molecule partner database by two-dimensional combinatorial screening (2DCS)
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RNA has diverse structural elements (motifs) such as hairpins, bulges and internal loops that play crucial functions in biological systems such as regulation of transcription, translation and gene expression. Thus, RNA is an important therapeutic target that can be modulated by small molecules. However, information for the design of small molecules that target RNA is limited. This work aims: (i) to expand the knowledge in targeting RNA using small molecule by identifying RNA motif - small molecule partners using Two - Dimensional Combinatorial Screening (2DCS) and (ii) to identify lead compounds for treatment of RNA associated diseases. 2DCS is a microarray-based screening method that can probe both RNA and chemical space simultaneously. Briefly, a small molecule library is immobilized onto a microarray surface and is probed for binding to RNA libraries under high stringency conditions. Unbound RNAs are washed away from the surface while bound RNAs are identified through harvesting, amplification and sequencing. Importantly, the outcome of 2DCS is RNA motif-small molecule partners that can be used to mine the transcriptomes for RNAs with targetable structural elements, which can be targeted by the identified small molecules. In addition, this information can be used to determine privileged chemical features that allow small molecules to bind specific RNA structural elements. Specifically, using 2DCS we have determined that the aminoglycoside class of antibiotics, which is known to target specifically the ribosomal RNA aminoacyl-tRNA site (A-site), can also bind to other RNAs with higher affinity and selectivity than the A-site RNA. This result explains the toxicities associated with the use of antibiotics such Kanamycin A, Tobramycin, Neamine and Neomycin B. Beyond aminoglycosides, drug-like small molecules have been identified to bind specific RNA structural elements by merging a solution-based high throughput screening with our microarray based screening method to probe for millions of interactions simultaneously. Through screening, drug-like small molecules that contain privileged chemical features such as indole, 2-phenyl indole, 2-phenyl benzimidazole, 2-amino-pyrimidine, 6-amino purine, 1,3-benzodioxole, phenylbenzmide, purine, pyridine, 4-methylpiperazin-1-yl(phenyl)methanone, 1-benzylpiperidine and pyridinium were identified to specifically bind t o RNA. These privileged chemical features can facilitate the design of small molecules targeting RNA. The results of 2DCS are organized in a RNA-small molecule partner database that can facilitate the identification of lead compounds for treatment of RNA associated diseases. For example, fragile X-associated tremor/ataxia syndrome (FXTAS) is caused by an expanded r(CGG) repeats (r(CGG) exp ) located in the 5' untranslated region (UTR) of the fragile X mental retardation 1 ( FMR1 ) mRNA. The structural motif of r(CGG) exp is mined against the RNA-small molecule partner database to identify lead compounds such as Kanamycin A derivative, 6'- N -5-hexynoate kanamycin A ( KanHex ), and Hoechst derivative, N-(3-azidopropyl)-4-(3-(6-(4-methylpiperazin-1-yl)-1H,3'H-2,5'-bibenzo[d]imidazol-2'-yl)phenoxy)butanamide (Hoechst-Azide ( Ht-N 3 )). Lead compounds can be further optimized for treatment of FXTAS.