RNA structures play important roles inside cells. However, measurement of RNA structures inside cells, which are found very different from isolated RNA, is still challenging due to limited tools. Thus chemical probing methods have been developed and several chemicals have been successfully employed to study RNA inside cells, yet more chemical tools are essentially needed. We found nicotinoyl azide (NAz) that probes nucleobase solvent accessibility of RNA under handheld UV lamp. Time-resolved IR spectroscopy, HPLC analysis and DFT calculation imply that NAz modifies C-8 position of Guanosine or Adenosine through a barrierless Friedel-Crafts reaction between purine and nitrenium ion, the latter of which is generated in situ upon UV excitation of NAz. This new RNA structure probing method, light-activated structure examination of RNA (LASER), is first tested on SAM-I riboswitch. Ligand binding on SAM-I RNA rearranges ribonucleotides adjacent to the binding site in a concentration-dependent manner, which is unambiguously resolved by LASER probing. Notably, a ligand binding induced tertiary structural change involving a pseudoknot is detected. Next, LASER probing is applied to measure RNA structures inside living cells. The residues of 18S rRNA and U1 snRNP that are protected by protein as shown in reported cryo-EM structures obtain no or little LASER reactivity, whereas, those exposed to solvent generate relatively stronger LASER reactivity. Furthermore, protein protected RNA regions show little LASER reactivity inside cells gain substantial modifications on G and A residues when LASER probing is performed on purified total RNA. In contrast, unprotected regions show indistinguishable NAz modifications between probing experiments inside and outside cells. These results demonstrate that LASER probing can be applied to footprinting of RNA-protein complexes. Consequently, LASER probing can be used to examine purine solvent accessibility of RNA both in vitro and in vivo.

Light-activated chemical probing of nucleobase solvent accessibility inside cells

256th National Meeting (2018)

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