Direct quantitation of biotin-labeled nucleotide analogs in RNA transcripts

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 [alpha 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 32P 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 T7 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 greater than 90% binding efficiency on a streptavidin/biotin-cellulose affinity column.