Direct quantitation of biotin-labeled nucleotide analogs in RNA transcripts

Abstract We have developed a method to determine directly the number of biotinylated (Bio) nucleotide analogs incorporated into RNA transcripts. Transcripts synthesized in vitro in the presence of [ α 32 -P]CTP and varying concentrations of Bio-4-UTP were subjected to alkaline hydrolysis and the resulting 2′ and 3′ nucleoside monophosphates separated by reverse-phase HPLC. The amount of 32 P transferred to each monophosphate was indicative of the frequency of their incorporation into the transcript. Transcripts synthesized in the presence of equimolar concentrations of Bio-4-UTP and UTP resulted in 70 out of the 125 possible UTP sites occupied by Bio-4-UMP. This study agrees with kinetic data in suggesting that T 7 RNA polymerase does not significantly discriminate between the natural and the biotinylated nucleotide. Therefore, the number of biotinylated residues that are incorporated into a transcript can be controlled by varying the ratio of Bio-4-UTP to UTP in the transcription reaction. We have shown that as few as 10 Bio-4-UMP residues per 486 nucleotide transcript still results in >90% binding efficiency on a streptavidin/biotin-cellulose affinity column.