Synthesis and enzymatic incorporation of a fluorescent pyrimidine ribonucleotide.
A detailed protocol for the synthesis of a fluorescent pyrimidine ribonucleoside analogue and its enzymatic incorporation into an RNA strand by transcription reactions is described. Furan-modified ribonucleoside triphosphate is synthesized in two steps with an overall yield of 33%. Incorporation of the triphosphate into an RNA oligomer occurs with nearly 225-fold amplification over the amount of the DNA template. Bacterial rRNA decoding site (known as the A-site) derived from this fluorescently modified ssRNA positively signals a binding event upon interaction with aminoglycoside antibiotics, its cognate ligands. The total time for the synthesis of ribonucleoside triphosphate is approximately 6 days, and that for the incorporation of the nucleoside triphosphate and purification of the fluorescently labeled RNA approximately 40 h.
Srivatsan
Seergazhi G
SG
Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, MC 0358, La Jolla, California 92093-0358, USA.
Tor
Yitzhak
Y
D005453:Fluorescence; D005456:Fluorescent Dyes; D011742:Pyrimidine Nucleotides / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000378:metabolism*; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics; D012335:RNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D014158:Transcription, Genetic
D005456:Fluorescent Dyes; D011742:Pyrimidine Nucleotides; D012329:RNA, Bacterial; D012335:RNA, Ribosomal