RNA is an important target for the design of small molecule therapeutics and probes of function. It is anunderutilized target, however, because of the limited information available about how RNA motifs interact withsmall molecules. This proposal describes studies to identify RNA motif-small molecule partners and investigateimportant features in both the RNA and small molecule that govern molecular recognition. The RNA motifsused are small internal and hairpin loops that are commonly found in biologically important RNAs such as pre-microRNAs, underexploited and important RNA drug targets with secondary structures similar to our RNAmotifs and no known tertiary structure. Two synergistic methods are used to enable parallel probing offeatures in the RNA and the small molecule. The first method, 2D Combinatorial Screening (2DCS), screenstwo libraries simultaneously (an array-immobilized small molecule library and an RNA library) to identify RNAmotif-small molecule partners. RNAs are harvested directly off the array from ligand-functionalized positions,cloned, and sequenced. Selected RNAs and RNA motif-small molecule complexes can be studied by NMRand optical melting experiments to interrogate their structures, flexibilities, and thermodynamic stabilities. Thesecond method, Structure Activity Relationships Through Sequencing (StARTS), assigns relative bindingaffinities from the output of 2DCS via the occurrence of RNA loops in sequencing data. Multiple selectedsequences are ligated together and cloned, yielding more sequence data per single sequencing reaction. If information about RNA-small molecule interactions were available, an RNA target's secondary structurecould be searched for one or several motifs to which a small molecule partner was identified, and the smallmolecule 'modules' custom-linked to accommodate each site in the target RNA. This may eliminate the needto subject each new RNA target to a high throughput screening assay.
The specific aims for this study are: 1.)Characterize the molecular recognition of 6'-N-5-hexynoate kanamycin A by internal loops with ACpairs, the consensus loop determined from a previously completely selection. AC loops are present asmutations in tRNAs that cause disease. 2.) Identify and study the RNA internal loops selected to bindmembers of an aminoglycoside library. 3.) Use StARTS and 2DCS to streamline identification andstatistical analysis of the internal loops that bind 6'-N-5-hexynoate neamine. 4.) Expand the results inAims 2 and 3 to identify RNA hairpin loop-aminoglycoside interactions. 5.) Identify interactionsbetween a peptoid library and RNA internal loops and hairpin loops via StARTS and 2DCS. PROJECT NARRATIVERNA is an important biomolecule that is associated with diseased states, however, veryfew drugs elicit their effects by interacting with RNA. The goal of the proposed researchis to understand how drug-like molecules interact with RNA to design therapeutics orprobes of RNA function.
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