title: Enzymatic incorporation and utilization of an emissive 6-azauridine.
abstract: To display favorable fluorescent properties, the non-emissive native nucleosides need to be modified. Here we present a motif that relies on conjugating 5-membered aromatic heterocycles (e.g., thiophene) to a 6-azapyrimidine (1,2,4-triazine) core. Synthetic accessibility and desirable photophysical properties make these nucleosides attractive candidates for enzymatic incorporation and biochemical assays. While 6-azauridine triphosphate is known to be poorly tolerated by polymerases in RNA synthesis, we illustrate that conjugating a thiophene ring at position 5 overcomes such limitations, facilitating its T7 RNA polymerase-mediated in vitro transcription incorporation into RNA constructs. We further show that the modified transcripts can be ligated to longer oligonucleotides to form singly modified RNAs, as illustrated for an A-site hairpin model RNA construct, which was employed to visualize aminoglycoside antibiotics binding.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093, USA. ytor@ucsd.edu.
authors.forename: Patrycja A
authors.initials: PA
authors.lastname: Hopkins
authors.affiliation: Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093, USA. ytor@ucsd.edu.
authors.forename: Lisa S
authors.initials: LS
authors.lastname: McCoy
authors.affiliation: Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093, USA. ytor@ucsd.edu.
authors.forename: Yitzhak
authors.identifier: http://orcid.org/0000-0003-3726-7799
authors.initials: Y
authors.lastname: Tor
mesh_terms: D001380:Azauridine / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000378:metabolism*; D005453:Fluorescence; D012313:RNA / Q000096:biosynthesis* / Q000737:chemistry; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D014764:Viral Proteins; D012313:RNA; D001380:Azauridine; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases