Radiation target analysis of RNA
(hammerhead ribozymes/molecular biology/molecular mass analysis)
S. L. BERNSTEIN* AND E. KEMPNERtt
*National Eye Institute and tNational Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892
Communicated by Theodor 0. Diener, University of Maryland, College Park, MD, March 4, 1996 (received for review September 28, 1995)
Ribozymes are polynucleotide molecules with intrinsic catalytic activity, capable of cleaving nucleic acid substrates. Large RNA molecules were synthesized containing a hammerhead ribozyme moiety of 52 nucleotides 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 ability of the irradiated ribozymes to cleave a labeled substrate. The amount of intact RNA remaining was determined from the same irradiated samples 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 radiation damage far from the primary site of ionization in RNA molecules. The smaller target size for activity indicates that only primary ionizations occurring in the specific active region are effective. This is similar to the case for oligosaccharides. We concluded that the presence of the ribose sugar in the polymer chain restricts radiation damage to a small region and prevents major energy transfer throughout the molecule. Radiation target analysis should be a useful technique for evaluating local RNA:RNA and RNA:protein interactions in vitro.
Proc. Natl. Acad. Sci. USA. Vol. 93, pp. 6410-6414, June 1996.
Radiation target analysis is a powerful technique that can be used to measure the molecular mass of proteins either in vivo or in vitro (1). It is based on the fact that the probability of a single electron impact on a protein molecule is directly proportional to the mass of the polypeptide; thus, the larger the protein, the smaller the radiation dose required to inactivate a single polypeptide. Radiation energy from a single electron impact anywhere along a polypeptide chain is transmitted throughout the polypeptide, destroying all function. Subjecting the inactivated molecule to denaturing conditions yields smaller molecular fragments. The rate of disappearance of biological activity with radiation dose yields a radiation target size for the measured function, whereas the disappearance of the intact polypeptide gives a target size for the structure. We wanted to test the feasibility of radiation target analysis of RNA. RNA has been the subject of several radiation target studies almost all of which involved assay of a complex process, the infectivity of RNA viruses. Because infectivity and replication of these viruses requires contributions from the majority, if not all, of viral genes present on the viral genome, interpretation of these studies was unclear. Only a few other radiation studies were performed on RNA molecules. Wohlhieter et al. (2) reported on the physical destruction of tobacco mosaic virus RNA, while Kempner and Pollard (3) performed an early study on target sizes of the protein synthetic mechanism, although at the time it was only speculated that RNA could be the target. Since the structure of RNAs involved in protein synthesis was not yet known, it was unclear whether radiation target analysis could be applied directly to RNA. The discovery of intrinsic RNA-enzyme activity (4, 5) permits a simple, more direct test of the effects of radiation on RNA function and structure.
Hammerhead ribozymes are catalytic RNA molecules that can cleave substrate RNA molecules in a simple in vitro assay (6, 7). These RNA sequences can be well defined in their overall length and substrate specificity. Hammerhead ribozymes can be synthesized of specific size, with cleavage activity determined by the length of the flanking sequences complementary to the substrate cleavage site (8). The optimal in vitro characteristics have been determined for ribozymes cleaving at a specific ‘GUC’ site in the murine interphotoreceptor retinoid binding protein (IRBP) mRNA (S.L.B., unpublished data). The catalytic portion of the transcribed molecule is only 52 nt in length (15.5 kDa). However, the total transcript size can be varied to yield ribozymes of vastly different lengths but with similar substrate specificities. This study was undertaken to determine whether radiation target analysis could be used to estimate the specific molecular mass of RNA species. The results yielded target sizes that accurately correspond to the known sizes of ribozymes and yield new insight into the nature of radiation action in these molecules.