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Synthesis and enzymatic incorporation of a fluorescent pyrimidine ribonucleotide

Nature Protocols volume 2, pages 1547–1555 (2007)Cite this article

Abstract

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 ∼6 days, and that for the incorporation of the nucleoside triphosphate and purification of the fluorescently labeled RNA ∼40 h.

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Figure 1: Scheme for the synthesis of furan-conjugated triphosphate 3.
Figure 2: Scheme for the enzymatic incorporation of furan-modified triphosphate 3.
Figure 3: Absorption (50 μM, red) and fluorescence (5 μM, blue) spectra of nucleoside 2 in water.
Figure 4: Structure and fluorescence spectra of furan-labeled A-site duplex.

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Acknowledgements

This work is supported by the US National Institutes of Health (GM 069773 and AI 47673).

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  1. Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, MC 0358, La Jolla, 92093-0358, California, USA

    Seergazhi G Srivatsan & Yitzhak Tor

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  1. Seergazhi G Srivatsan
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  2. Yitzhak Tor
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Correspondence to Yitzhak Tor.

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Srivatsan, S., Tor, Y. Synthesis and enzymatic incorporation of a fluorescent pyrimidine ribonucleotide. Nat Protoc 2, 1547–1555 (2007). https://doi.org/10.1038/nprot.2007.222

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