LIBRARIES OF MULTIFUNCTIONAL RNA CONJUGATES FOR THE SELECTION OF NEW RNA CATALYSTS

An in vitro selection system was developed for the selection of RNA mo lecules catalyzing bimolecular reactions between small reactants. The system is based on the direct selection protocol and involves librarie s of multifunctional RNA conjugates rather than unmodified RNA transcr ipts. For the preparation of RNA conjugate libraries, a dinucleotide a nalog has been designed and synthesized containing a poly(ethylene gly col) linker with an embedded photocleavage site and a terminal attachm ent site for coupling potential reactants. Reactants are first coupled to the dinucleotide analog by activated ester chemistry and then liga ted to the 3'-ends of enzymatically prepared RNA pool molecules, givin g libraries of complex conjugates. Species that become attached to bio tin on incubation with a biotinylated partner are isolated using strep tavidin-derivatized matrices and then subjected to a photocleavage ste p. Selective cleavage of the linker releases only those RNA species in which reaction has taken place at the linker-coupled reactant, while products with the biotin attached to internal positions of the RNA par t remain immobilized. Efficient photocleavage is achieved by laser irr adiation at 355 nm, and the released RNAs are intact and amplifiable b y reverse transcription. All steps are shown to be compatible with the overall selection procedure, as was shown by performing a model selec tion cycle. Besides allowing a broader scope of reaction types to be s elected for, the strategy relieves the RNA from the requirement to pos sess substrate properties as well as catalytic activity, and the use o f a cleavable linker will suppress the selection of catalysts for side reactions.