Synthesis of a bioactive fluorescent dye and enzymatic labeling at the 3-termini of RNAs: An application for the characterization of the thermal stability of tRNAs and oligonucleotides using fluorescence anisotropy measurements

Abstract

A novel fluorescent nucleotide analogue, 2′-O-anthraniloylcytidine 3′,5′-diphosphate (Ant-pCp) was synthesized from cytidine 3′,5′-diphosphate and isatoic anhydride. Using T4 RNA ligase, Ant-pCp was easily joined to the 3′ terminus of the various oligonucleotides and tRNAs, for example, (pA) 4, (pA) 6, (pA) 8, yeast tRNA Phe, B. subtilis tRNA Thr, and B. subtilis unfractionated tRNA. It seems that anthraniloyl group does not influence the ligation reaction. Steady-state fluorescence anisotropy (r s) of these labeled oligonucleotides or tRNAs were measured in the presence or absence of Mg 2+ in the temperature range 5 to 60°C. The complex formation between fluorescent labeled oligonucleotides and poly(U) and the thermal denaturation profiles of the complexes were successfully detected by fluorescence anisotropy at low nucleotide concentration (10 −6 m) in the presence of MgCl 2. Fluorescent labeled tRNA showed characteristic thermal denaturation profiles in the absence of Mg 2+, indicating that tRNA has a secondary and/or tertiary structure. The anisotropy values of the fluorescent probe decreased in the order: oligo(A): poly(U) complex > tRNA (+ Mg 2+) > oligo(A). These results indicate that the mobility of the 3′ terminal of tRNA is more restricted than that of the single strand of oligo(A) in solution.