Single-molecule chemical denaturation of riboswitches

Abstract

To date, single-molecule RNA science has been developed almost exclusively around the effect of metal ions as folding promoters and stabilizers of the RNA structure. Here, we introduce a novel strategy that combines single-molecule Förster resonance energy transfer (FRET) and chemical denaturation to observe and manipulate RNA dynamics. We demonstrate that the competing interplay between metal ions and denaturant agents provides a platform to extract information that otherwise will remain hidden with current methods. Using the adenine-sensing riboswitch aptamer as a model, we provide strong evidence for a rate-limiting folding step of the aptamer domain being modulated through ligand binding, a feature that is important for regulation of the controlled gene. In the absence of ligand, the rate-determining step is dominated by the formation of long-range key tertiary contacts between peripheral stem-loop elements. In contrast, when the adenine ligand interacts with partially folded messenger RNAs, the aptamer requires specifically bound Mg2+ ions, as those observed in the crystal structure, to progress further towards the native form. Moreover, despite that the ligand-free and ligand-bound states are indistinguishable by FRET, their different stability against urea-induced denaturation allowed us to discriminate them, even when they coexist within a single FRET trajectory; a feature not accessible by existing methods.

Single-molecule studies of riboswitch folding

“…further explored the add aptamer L2-L3 docking interaction by using urea to selectively disrupt tertiary contacts [58]. Urea is known to destabilize secondary, as well as tertiary, structures in RNA by mechanisms that remain unclear [92], but the authors used sufficiently low urea concentrations that the overall secondary structure content appeared to remain intact, based on circular dichroism measurements.…”

“…Typically, a riboswitch aptamer domain is site-specifically labeled with both donor and acceptor fluorophores for FRET, then attached to a glass (or quartz) surface via linker chemistry (Figure 2) [51]. To date, lysine [52], SAM class I [53], SAM class II [54], guanine [55], adenine [56–58], TPP [59], c-di-GMP class I [60], and preQ 1 class I & II riboswitches [61, 62] have been explored using smFRET methodologies (section 4 ).…”

A Kissing Loop Is Important for btuB Riboswitch Ligand Sensing and Regulatory Control

“…A GCG sequence was added at the beginning of the transcript to minimize the 5Ј heterogeneity of the RNA population (29). Transcription and RNA purifications steps were performed as described previously (30).…”

An integrated perspective on RNA aptamer ligand-recognition models: clearing muddy waters

“…67 Furthermore, the rates are highly sensitive to the environment. Depending on the concentrations of Mg 2+ and denaturants, 82 the overall apparent mechanism can be IF, CS, or a hybrid in the kinetic sense. 28 The adenine riboswitch sheds further light on the potential disjoint between the timing of ligand binding and its role in the folding mechanism.…”