Binding of Mg2+, Ca2+ and Co(NH3)6(3+) ions to the HIV-1 TAR RNA in solution was analysed by 19F NMR spectroscopy, metal ion-induced RNA cleavages and Brownian dynamics (BD) simulations. Chemically synthesised 29mer oligoribonucleotides of the TAR sequence labelled with 5-fluorouridine (FU) were used for 19F NMR-monitored metal ion titration. The chemical shift changes of fluorine resonances FU-23, FU-25 and FU-40 upon titration with Mg2+ and Ca2+ ions indicated specific, although weak, binding at the bulge region with the dissociation constants (K(d)) of 0.9 +/- 0.6 and 2.7 +/- 1.7 mM, respectively. Argininamide, inducing largest (19)F chemical shifts changes at FU-23, was used as a reference ligand (K(d) = 0.3 +/- 0.1 mM). In the Pb2+-induced TAR RNA cleavage experiment, strong and selective cleavage of the C24-U25 phosphodiester bond was observed, while Mg2+ and Ca2+ induced cuts at all 3-nt residues of the bulge. The inhibition of Pb2+-specific TAR cleavage by di- and trivalent metal ions revealed a binding specificity [in the order Co(NH3)6(3+) > Mg2+ > Ca2+] at the bulge site. A BD simulation search of potential magnesium ion sites within the NMR structure of HIV-1 TAR RNA was conducted on a set of 20 conformers (PDB code 1ANR). For most cases, the bulge region was targeted by magnesium cations.
SummaryThe synthesis of ribonucleotide blocks multiply labelled with 2 H, 13 C and 15 N for solid support synthesis of sequence specifically labelled RNA is described. Labels were introduced in the ribose ring ( 13 C), C5 position of pyrimidine nucleobases ( 2 H) and exocyclic amino groups ( 15 N) and serve as multiple probes for studying the various physicochemical consequences of physiologically important RNA folding by high-resolution multi-nuclear NMR spectroscopy.
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