RADIATION TARGET ANALYSIS OF RNA

Ribozymes are polynucleotide molecules with intrinsic catalytic activi ty, capable of cleaving nucleic acid substrates. Large RNA molecules w ere synthesized containing a hammerhead ribozyme moiety of 52 nucleoti des linked to an inactive leader sequence, for total lengths of Either 262 or 1226 nucleotides. Frozen RNAs were irradiated with high energy electrons, Surviving ribozyme activity was determined using the abili ty of the irradiated ribozymes to cleave a labeled substrate. The amou nt of intact RNA remaining was determined from the same irradiated sam ples by scanning the RNA band following denaturing gel electrophoresis . Radiation target analyses of these data revealed a structural target size of 80 kDa and a ribozyme activity target size of 15 kDa for the smaller ribozyme, and 319 kDa and 16 kDa, respectively, for the larger ribozyme. The disparity in target size for activity versus structure indicates that, in contrast to proteins, there is no spread of radiati on damage far from the primary site of ionization in RNA molecules, Th e smaller target size for activity indicates that only primary ionizat ions occurring in the specific active region are effective, This is si milar to the ease for oligosaccharides. Wt concluded that the presence of the ribose sugar in the polymer chain restricts radiation damage t o a small region and prevents major energy transfer throughout the mol ecule, Radiation target analysis should be a useful technique for eval uating local RNA:RNA and RNA:protein interactions in vitro.