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 tested for their compatibility with in vitro RNA selection procedures. 5-(3-Aminopropyl)uridine triphosphate (UNH(2)) 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 UNH(2) 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% UNH(2) residues exhibited better substrate properties for SuperScript(TM) II RNase H reverse transcriptase than did RNA transcripts containing approximately 25% of the USH analog. With either analog, both transcription and reverse transcription proceeded with high fidelity for insertion of the analog residue.
Vaish
N K
NK
Department of Chemistry, Boston College, Chestnut Hill, MA 02467, USA.
Fraley
A W
AW
Szostak
J W
JW
McLaughlin
L W
LW
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D012313:RNA / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism; D013379:Substrate Specificity; D013698:Templates, Genetic; D014158:Transcription, Genetic / Q000235:genetics; D014529:Uridine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D014544:Uridine Triphosphate / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D014764:Viral Proteins
C414761:5-(2-mercaptoethyl)uridine triphosphate; C414760:5-(3-aminopropyl)uridine triphosphate; D014764:Viral Proteins; D012313:RNA; C062210:bacteriophage T7 RNA polymerase; D012194:RNA-Directed DNA Polymerase; D012321:DNA-Directed RNA Polymerases; D016914:Ribonuclease H; D014544:Uridine Triphosphate; D014529:Uridine