Abstract:
We developed a dual-app ribonucleotide probe composed of a trifluoromethyl-benzofuran-modified uridine core (TFBF-UTP) that combines fluorescence and 19F NMR for a detailed analysis of RNA conformation and recognition. Using the probe, we monitored aminoglycoside antibiotic binding to the bacterial ribosomal decoding A-site RNA, including structurally similar aminoglycosides that conventional probes like 2-aminopurine fail to distinguish. These results highlight the utility of TFBF-UTP as a very good addition to the nucleic acid toolbox for studying RNA structures and their interaction with ligands and proteins. In this chapter, we provide step-wise procedures to synthesize the dual-app nucleotide probe TFBF-UTP, its enzymatic incorporation into model RNA transcripts and bacterial ribosomal decoding site (A-site) RNA motif by in vitro transcription reactions using T7 RNA polymerase. Step-wise procedure to monitor the aminoglycoside-induced conformational changes in the A-site motif by fluorescence- and 19F NMR-based assays has been elaborated. This method can be used as a general platform to incorporate TFBF-U into a desired RNA motif by using an appropriate DNA template and use the properties of the probe to study the target RNA construct by complementing biophysical techniques namely, fluorescence and 19F NMR.