Journal
NATURE PROTOCOLS
Volume 2, Issue 6, Pages 1547-1555Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2007.222
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Funding
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R01GM069773] Funding Source: NIH RePORTER
- NIAID NIH HHS [AI 47673] Funding Source: Medline
- NIGMS NIH HHS [GM 069773] Funding Source: Medline
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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 similar to 6 days, and that for the incorporation of the nucleoside triphosphate and purification of the fluorescently labeled RNA similar to 40 h.
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