Nucleic acid fragmentation on the millisecond timescale using a conventional X-ray rotating anode source: application to protein-DNA footprinting.
Nucleic acid fragmentation (footprinting) by *OH radicals is used often as a tool to probe nucleic acid structure and nucleic acid-protein interactions. This method has proven valuable because it provides structural information with single base pair resolution. Recent developments in the field introduced the 'synchrotron X-ray footprinting' method, which uses a high-flux X-ray source to produce single base pair fragmentation of nucleic acid in tens of milliseconds. We developed a complementary method that utilizes X-rays generated from a conventional rotating anode machine in which nucleic acid footprints can be generated by X-ray exposures as short as 100-300 ms. Our theoretical and experimental studies indicate that efficient cleavage of nucleic acids by X-rays depends upon sample preparation, energy of the X-ray source and the beam intensity. In addition, using this experimental set up, we demonstrated the feasibility of conducting X-ray footprinting to produce protein-DNA protection portraits at sub-second timescales.
Henn
Arnon
A
Department of Structural Biology, The Weizmann Institute of Science, Rehovot 76100, Israel.
Halfon
J
J
Kela
I
I
Orion
I
I
Sagi
I
I
D001426:Bacterial Proteins / Q000737:chemistry; D018983:DNA Footprinting / Q000379:methods*; D004269:DNA, Bacterial / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D034982:Integration Host Factors; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D013997:Time Factors; D014965:X-Rays
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D034982:Integration Host Factors