Expanding the structural and functional diversity of RNA: analog uridine triphosphates as candidates for in vitro selection of nucleic acids

Two analog uridine triphosphates tethering additional functionality, one a primary amino group and the second a mercapto group, were prepared and test ed for their compatibility with in vitro RNA selection procedures. 5-(3-Ami nopropyl)uridine triphosphate (UNH2) as a uridine substitute was a more eff ective substrate for T7 RNA polymerase than 5-(2-mercaptoethyl)uridine trip hosphate (USH), However, both functioned in transcription assays of 100 nt templates to generate RNA transcripts in quantities sufficient to initiate RNA selection procedures. Transcription of RNA pools with T7 RNA polymerase and UNH2 or USH occurred with efficiencies of 43 and 29%, respectively, of the values obtained for native UTP transcription. In addition, the transcr ibed RNA containing roughly 25% UNH2 residues exhibited better substrate pr operties for SuperScript(TM) II RNase H reverse transcriptase than did RNA transcripts containing similar to 25% of the USH analog, With either analog , both transcription and reverse transcription proceeded with high fidelity for insertion of the analog residue.