Poster abstracts

Poster number 42 submitted by Jeff Joyner

Mechanisms of Oxidative Scission of HIV RRE RNA by Rev-Coupled Metal Chelates

Jeff C. Joyner (Ohio State Biochemistry Program, The Ohio State University), Kevin D. Keuper (Chemistry Department, The Ohio State University), James A. Cowan (Chemistry Department, The Ohio State University)

Abstract:
A method of analysis and resulting data are presented that utilize matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) to monitor the kinetics and mechanisms of RNA cleavage. The method is illustrated through application to the study of targeted oxidation of a RNA stem loops from HIV-1 Rev Response Element mRNA (RRE RNA) and ribosomal 16S A-site RNA (16S RNA) by catalytic artificial ribonucleases. Following incubation of each RNA with catalysts and/or redox coreactants, reaction mixtures were desalted, and MALDI-TOF MS was used to monitor both time-resolved formation of cleavage products and disappearance of full length RNA. A unique list was generated for each RNA that contained the predicted masses of both the full length RNA and all possible RNA cleavage fragments that resulted from the combination of all possible cleavage sites and each of six expected overhangs formed at nascent termini adjacent to cleavage sites. The overhangs monitored at each position were 2’,3’-cyclic phosphates, 3’-phosphates, 3’-phosphoglycolates, 5’- hydroxyls, and 5’- phosphates, which corresponded to differing oxidative, hydrolytic, and/or 2’-OH-mediated-endonucleolytic modes of scission. Each mass spectrum was compared with a corresponding list of predicted masses, and peaks were rapidly assigned by use of a Perl script, with a user-input mass matching tolerance of 3 amu. The resulting data allowed a semi-quantitative assessment of the rate of formation of each overhang, at each nucleotide position. Both time-dependent cleavage mediated by artificial ribonucleases and artifactual fragmentation induced by MALDI-TOF were observed and were distinguished by use of time-dependent incubations. The method provides a rapid, accurate, highly-detailed, and semi-quantitative analysis of RNA cleavage that may be readily applied to other systems. Additionally, the sites and mechanisms of oxidative, hydrolytic, and/or 2'-OH-mediated scission of HIV RRE RNA promoted by each of 22 catalysts are addressed in detail. Moreover, a novel colorimetric assay for H-4 abstraction from RNA has been developed; unique H-4 abstraction products are detected by reaction with thiobarbituric acid to form a fluorescent adduct.

Keywords: RNA cleavage, mass spectrometry, mechanisms