title: Polymerase-Mediated Site-Specific Incorporation of a Synthetic Fluorescent Isomorphic G Surrogate into RNA.
abstract: An enzyme-mediated approach for the assembly of singly modified RNA constructs in which specific G residues are replaced withthG, an emissive isomorphic G surrogate, is reported. Transcription in the presence ofthG and native nucleoside triphosphates enforces initiation with the unnatural analogue, yielding 5'-end modified transcripts that can be mono-phosphorylated and ligated to provide longer site-specifically modified RNA constructs. The scope of this unprecedented enzymatic approach to non-canonical purine-containing RNAs is explored via the assembly of several altered hammerhead (HH) ribozymes and a singly modified HH substrate. By strategically modifying key positions, a mechanistic insight into the ribozyme-mediated cleavage is gained. Additionally, the emissive features of the modified nucleoside and its responsiveness to environmental changes can be used to monitor cleavage in real time by steady state fluorescence spectroscopy.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0358, USA.
authors.forename: Yao
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0358, USA.
authors.forename: Andrea
authors.initials: A
authors.lastname: Fin
authors.affiliation: Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0358, USA.
authors.forename: Lisa
authors.initials: L
authors.lastname: McCoy
authors.affiliation: Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0358, USA.
authors.forename: Yitzhak
authors.initials: Y
authors.lastname: Tor
mesh_terms: D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006151:Guanosine / Q000737:chemistry / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D012313:RNA / Q000378:metabolism*; D016337:RNA, Catalytic / Q000378:metabolism; D013379:Substrate Specificity; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D016337:RNA, Catalytic; D014764:Viral Proteins; C427204:hammerhead ribozyme; D006151:Guanosine; D012313:RNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases
keywords: RNA; fluorescence; hammerhead ribozyme; nucleosides; transcription initiation