TIME RESOLVED SYNCHROTRON XRAY FOOTPRINTING OF NUCLEIC ACIDS

Yeshiva University, New York NY

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Abstract

Hydroxyl radicals (*OH) can cleave the phosphodiester backbone of nucleic acids and are valuable reagents in the study of nucleic acid structure and protein-nucleic acid interactions through "footprinting" studies. Irradiation of solutions by high flux "white light" x-ray beams based on bending magnet beamlines at the National Synchrotron Light Source (NSLS) yields sufficient concentrations of *OH so that quantitative nuclease protection ("foot-printing") studies of DNA and RNA can be conducted with 10 - 20 msec x-ray exposure. The utility of this timescale for nucleic acid cleavage is demonstrated by synchrotron x-ray time-resolved "footprinting" analyses of the Mg2+ -dependent folding of the Tetrahymena thermo-philia L-21 Sca I ribozyme RNA. Since RNA folding progress curves over timescales from msec to minutes have been obtained for 25 regions of the ribozyme, this research provides an unprecedented structural kinetic picture of the folding of a large macromolecule. Synchro-tron x-ray "footprinting" is a new approach of general applicability for the study of time-re-solved structural changes of nucleic acid conformation and protein-nucleic acid complexes.