| Abstract
| - An in vitro selection system was developed for the selectionof RNA molecules catalyzing bimolecularreactions between small reactants. The system is based on thedirect selection protocol and involveslibraries of multifunctional RNA conjugates rather than unmodified RNAtranscripts. For thepreparation of RNA conjugate libraries, a dinucleotide analog has beendesigned and synthesizedcontaining a poly(ethylene glycol) linker with an embeddedphotocleavage site and a terminalattachment site for coupling potential reactants. Reactants arefirst coupled to the dinucleotide analogby activated ester chemistry and then ligated to the 3‘-ends ofenzymatically prepared RNA poolmolecules, giving libraries of complex conjugates. Species thatbecome attached to biotin on incubationwith a biotinylated partner are isolated using streptavidin-derivatizedmatrices and then subjectedto a photocleavage step. Selective cleavage of the linker releasesonly those RNA species in whichreaction has taken place at the linker-coupled reactant, while productswith the biotin attached tointernal positions of the RNA part remain immobilized. Efficientphotocleavage is achieved by laserirradiation at 355 nm, and the released RNAs are intact and amplifiableby reverse transcription.All steps are shown to be compatible with the overall selectionprocedure, as was shown by performinga model selection cycle. Besides allowing a broader scope ofreaction types to be selected for, thestrategy relieves the RNA from the requirement to possess substrateproperties as well as catalyticactivity, and the use of a cleavable linker will suppress the selectionof catalysts for side reactions.
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