Site-specific enzymic incorporation of an unnatural base, N^6-(6-aminohexyl)isoguanosine, into RNA

Abstract

An efficient enzymatic method is described for the sequence-specific incorporation of a functionalizable modified base into RNA molecules. A deoxy-5-methylisocytidine (d^(Me)isoC) in the DNA template directs the T7 RNA polymerase incorporation of N^6-(6-aminohexyl)isoguanosine (6-AH-isoG) into the transcribed RNA product. The misincorporation of isoGTP derivatives opposite T is eliminated in the presence of ATP, and the misincorporation of A opposite d^(Me)isoC is negligible in the presence of isoGTP derivatives. The isolated yield of RNA products using modified templates is approximately 50% that for reactions using natural templates. A post-transcriptional modification of the reactive primary amino group with N-hydroxysuccinimide-activated biotin or the dianhydride of ethylenediaminetetraacetic acid affords site-specifically modified RNA sequences suitable for further studies. This method for the generation of RNA molecules containing a primary amine suitable for post-transcriptional modification should be useful for mapping the structure of folded RNA polymers and RNA-protein complexes by affinity cleavage and affinity labeling.

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