Enzymatic synthesis of 2′-modified nucleic acids: identification of important phosphate and ribose moieties in RNase P substrates

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Abstract
For the first time mosaic nucleic acids composed of 50% RNA and 50% DNA can be obtained as transcripts with T7 RNA polymerase. Two NTPs could be replaced simultaneously in a transcription reaction. This means more than 40 deoxynucleotides were inserted in one transcript. Previously, a maximum of two deoxy-nucleotides could be incorporated and 2′-O-methyl-NTPs were not substrates at all. We obtained reasonable transcript yields with a maximal level of 99% 2′-O-methyl-NTPs, and the products contained up to 58% 2′-O-methylnucleotides at more than 20 positions. Sequence-specific nucleotide Incorporation was monitored by sequence ladders (partial alkali or iodine cleavage). No base misincorporations were detected with 100% dGTP, dCTP and dTTP, and with partial incorporation of dATPαS, 2′-O-methyl-GTPαS and 2′-O-methyl-CTPαS, whereas they were found with dATP, 2′-O-methyl-ATPαS and 2′-O-methyl-UTPαS. Quantitative data allow predetermined modification levels of partially modified transcripts. Highly modified transcripts can be used for structural and functional studies, in modification interference approaches and for In vitro evolution procedures. Modification interference studies revealed a small number of important phosphate and ribose moieties in RNase P substrates. The conversion of 17 RNA polymerase to a DNA polymerase extends the observation that there is no absolute distinction between RNA and DNA polymerases. Accordingly, an adapted concept of a primordial RNA world is presented.