Time-Resolved Hydroxyl Radical Footprinting of RNA with X-Rays

Abstract

RNA footprinting by hydroxyl radical cleavage provides 'snapshots' of RNA tertiary structure or protein interactions that bury the RNA backbone. Generation of hydroxyl radicals with a high-flux synchrotron X-ray beam provides analysis on a short timescale (5-100 msec), which enables the structures of folding intermediates or other transient conformational states to be determined in biochemical solutions or cells. This article provides protocols for using synchrotron beamlines for hydroxyl radical footprinting. © 2018 by John Wiley & Sons, Inc.

Keywords: RNA structure probing; hydroxyl radical footprinting; time-resolved footprinting.

Figure 11.6.1

Multi-sample holder for equilibrium footprinting experiments. Adapted from Adilakshmi et al. (2009) with permission from Harcourt.

Figure 11.6.2

Flow set-up for irradiation of live bacterial culture. Adapted from Hulscher et al. (2016) with permission from Harcourt.

Figure 11.6.3

Modified rapid-quench apparatus for time-resolved footprinting. Adapted from Sclavi et al. (1998b) with permission from Harcourt.

Figure 11.6.4

Dose-response for cleavage of 16S rRNA in E. coli MRE600 cells at XFP. The beam incident on the sample was attenuated with aluminum of different thickness. Inverted triangle, 152 μm; triangle, 203 μm; square, 305 μm; circle, 508 μm. The flow rate ranged from 1 to 5 mL/min.

Figure 11.6.5

Results of X-ray footprinting experiments showing Mg²⁺-dependent folding of the P4-P6 domain of the Tetrahymena ribozyme. Reprinted from Deras et al. (2000) with permission from the American Chemical Society.