Design, synthesis, and analysis of yeast tRNA(Phe) analogs possessing intra- and interhelical disulfide cross-links

Disulfide cross-links have been site-specifically incorporated into unmodified yeast tRNA(Phe) by total chemical synthesis. One cross-link is between positions 1 and 72 in the amino-acid acceptor stem, and it was prepared by replacing G1 and C72 with N-3-(thioethyl)uridine. A second cross-link is in the central D-region of yeast tRNA(Phe) between 11 and 25, and it was synthesized by replacing C11 and C25 with 2'-O-alkylthiol modified cytosine residues. Air oxidation to form the cross-link at both sites occurs in 12 h and is nearly quantitative. Analysis of the crosslinked products by native and denaturing PAGE along with Pb(II) cleavage experiments demonstrates that the crosslinked molecules are monomeric and suggests that the disulfide bridges do not significantly alter the structure of the modified tRNAs relative to the parent sequence. The finding that cross-link formation between thiol-derivatized residues correlates with the position of these groups in the crystal structure of native yeast tRNA(Phe) that the modifications apparently do not perturb native structure suggests that this methodology should be applicable to the study of RNA structure, dynamics, and folding.