Structural Analysis of RNA in Living Cells by In Vivo Synchrotron X-Ray Footprinting

Abstract

Chemical footprinting methods are widely used to probe the solution structures of nucleic acids and their complexes. Among the many available modifying reagents, hydroxyl radical is exceptional in its ability to provide nucleotide-level information on the solvent accessibility of the nucleic acid backbone. Until recently, hydroxyl radical footprinting has been limited to in vitro experiments. We describe the use of synchrotron X-radiation to generate hydroxyl radicals within cells for effective footprinting of RNA-protein complexes in vivo. This technique gives results that are consistent with in vitro footprinting experiments, with differences reflecting apparent structural changes to the RNA in vivo.

The RNA structurome: transcriptome-wide structure probing with next-generation sequencing

“…One future challenge is obtaining more detailed structural information on RNA, including tertiary structure. Besides DMS, other chemicals (e.g., CMCT, hydroxyl radicals, and SHAPE reagents) also modify RNA in vivo [4][5][6]32,72,73]. Given that these chemicals target different aspects of the RNA nucleotide, they complement each other and will facilitate more comprehensive RNA structure prediction.…”

Structure and dynamics of protein waters revealed by radiolysis and mass spectrometry

“…Solutions of 5 μM Alexa 488 (Invitrogen), 10 μM cyt c (horse heart) and 30 μM ubiquitin (bovine erythrocyte) were prepared in 10 mM sodium phosphate buffer pH 7.2. Samples were loaded into 200-μL PCR tubes in 5-μL volume and irradiated for 0-40 ms using a millisecond shutter in the standard multiple sample irradiation set up at RT (25°C) (40). The same experiment was performed after snap-freezing of samples in PCR tubes in liquid nitrogen, while maintaining the temperature at −35°C by Peltier cooling coupled to the multiple sample holder following the protocol for the X-ray footprinting of frozen cell samples (40).…”

“…Samples were loaded into 200-μL PCR tubes in 5-μL volume and irradiated for 0-40 ms using a millisecond shutter in the standard multiple sample irradiation set up at RT (25°C) (40). The same experiment was performed after snap-freezing of samples in PCR tubes in liquid nitrogen, while maintaining the temperature at −35°C by Peltier cooling coupled to the multiple sample holder following the protocol for the X-ray footprinting of frozen cell samples (40). For the experiment at cryogenic temperature (−190°C), a cryocooled (liquid N 2 ) sample holder was used to irradiate sample.…”

On the importance of cotranscriptional RNA structure formation

“…Initial experiments primarily studied how already synthesized and fully denatured RNA molecules fold, whereas more recent studies examine cotranscriptional folding pathways in vitro and, most recently, also in vivo (Adilakshmi et al 2009;Woodson 2010). Because any of these experiments are technically sophisticated, our current view derives from a few well-studied test cases such as the hairpin ribozyme (Donahue et al 2000;Fedor 2002Fedor , 2009Mahen et al 2005Mahen et al , 2010 and the Tetrahymena intron (Koduvayur and Woodson 2004;Jackson et al 2006).…”

“…Numerous recent in vitro experiments that replicate specific aspects of the complex in vivo environment and rapid progress regarding in vivo methodologies (Adilakshmi et al 2009;Alexander et al 2011) are likely to change this.…”