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Abstract

We have previously reported the in vitro selection of a ribozyme that catalyzes an aldol reaction between a levulinic amide aldol donor and a benzaldehyde substrate. The selection scheme involved the priming of the RNA library with a levulinic amide aldol donor group that was introduced via transcription priming in the presence of a modified guanosine mononucleotide derivative. Here we provide a detailed description of the synthesis of the ribozyme substrates and the substrate oligonucleotides used for its isolation and characterization. The aldol donor group was attached to the phosphate moiety of guanosine monophosphate via a photocleavable linker molecule. This initiator nucleotide was efficiently incorporated into RNA molecules of differing sizes and composition by transcription priming with T7 RNA polymerase. With this method modified RNA oligonucleotides as small as a 6-mer sequence can be generated. A temperature profile of the intermolecular reaction indicates that the modified RNA hexamer binds the ribozyme largely by Watson-Crick pairing and only to a minor extent via the non-RNA moiety, whereas the ribozyme appears to have evolved a specific binding site for the aldehyde substrate.