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Nucleic Acids Research, 2000, Vol. 28, No. 17 3316-3322
© 2000 Oxford University Press

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

Narendra K. Vaish1,2, Andrew W. Fraley1, Jack W. Szostak2 and Larry W. McLaughlin1,*

1Department of Chemistry, Boston College, Chestnut Hill, MA 02467, USA and 2Howard Hughes Medical Institute, Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA

Two analog uridine triphosphates tethering additional functionality, one a primary amino group and the second a mercapto group, were prepared and tested for their compatibility with in vitro RNA selection procedures. 5-(3-Aminopropyl)uridine triphosphate (UNH2) as a uridine substitute was a more effective substrate for T7 RNA polymerase than 5-(2-mercaptoethyl)uridine triphosphate (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 transcribed RNA containing roughly 25% UNH2 residues exhibited better substrate properties for SuperScriptTM II RNase H reverse transcriptase than did RNA transcripts containing ~25% of the USH analog. With either analog, both transcription and reverse transcription proceeded with high fidelity for insertion of the analog residue.

* To whom correspondence should be addressed. Tel: +1 617 552 3622; Fax: +1 617 552 2705; Email: larry.mclaughlin@bc.edu


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