EVIDENCE FOR CROSS-LINKING OF POLYRIBONUCLEOTIDES WITH 4'-AMINOMETHYL-4,5',8-TRIMETHYLPSORALEN HYDROCHLORIDE

Evidence is presented for the formation of cross-links in base-paired regions of polyribonucleotides by long-wave ultraviolet irradiation in the presence of 4‘-aminomethyl-4, 5‘,8-trimethylpsoralen hydrochloride. The instantaneous reannealing of base-paired structure following heating and fast cooling was taken as evidence for the presence of covalent bridges. Single-strand-specific nuclease S1 was used as a probe for base-paired structure. This was done with 16S rRNA from Escherichia coli and the double-stranded homopolymer poly(I)-poly(C). The photoreaction with poly(I)-poly(C) suggested that the reagent is not pyrimidine specific, contrary to what was deduced more indirectly by others. In the case of 16S rRNA, a procedure was developed for the demonstration of the instantaneously reannealing structure by (1) predigestion of endogenous unpaired loop regions with nuclease S1; (2) inactivation of the nuclease by diethyl pyrocarbonate; (3) heating and fast cooling; (4) re-challenge of the structure with nuclease S1 The photoreacted 16S rRNA and poly(I)-poly(C) both had altered structural properties by optical parameters. The spectra showed hyperchromicity with a peak at long (peak at 350 nm) and at short (250 nm and below wavelengths and a very small hypochromicity at 270 nm. Melting curves at 260 nm showed no significant change in total thermal hyperchromicity, but a large increase in melting temperature. With high levels of the drug, the melting temperature increased by 18 °C for 16S rRNA and by 12 °C for poly(I)-poly(C). When the reaction was limited by lower concentrations of reagent, significant shifts of the entire melting curve became detectable with the initial ratio of drug in the range of 10-2 to 10-1 mol per base pair for 16S rRNA and in the range of 10-3 to 10-2 mol per base pair for poly(I)-poly(C). The reaction had no effect on the compact structure which we had shown earlier to be resistant to the spreading conditions of the electron microscopy. However, the compact structure thus observed was no longer susceptible to denaturation by heating in formalaldehyde. © 1979, American Chemical Society. All rights reserved.