Mechanisms for differentiation between cognate and near-cognate ligands by purine riboswitches

Wacker, Anna; Buck, Janina; Richter, Christian; Schwalbe, Harald; Wöhnert, Jens

doi:10.4161/rna.20106

Cited by 9 publications

(11 citation statements)

References 29 publications

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“…[14] Ap opulars ystem for real-time NMR are purine riboswitches, where the reactioni ss tarted with addition of the ligand. [23,[33][34][35] Similarly,L ee et al followed adenine-induced folding of an adenine-sensing riboswitch. [30] They identified distinct steps associated with the ligand-induced folding of the riboswitch:r ecognition of the ligand,f ormation of the long range loop-loop interactions followed by stabilization of long-range interactions that result in af ormation of as table complex ( Figure 5).…”

Section: Real-time(rt) Nmr Spectroscopymentioning

confidence: 99%

RNA Dynamics by NMR Spectroscopy

2019

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An ever‐increasing number of functional RNAs require a mechanistic understanding. RNA function relies on changes in its structure, so‐called dynamics. To reveal dynamic processes and higher energy structures, new NMR methods have been developed to elucidate these dynamics in RNA with atomic resolution. In this Review, we provide an introduction to dynamics novices and an overview of methods that access most dynamic timescales, from picoseconds to hours. Examples are provided as well as insight into theory, data acquisition and analysis for these different methods. Using this broad spectrum of methodology, unprecedented detail and invisible structures have been obtained and are reviewed here. RNA, though often more complicated and therefore neglected, also provides a great system to study structural changes, as these RNA structural changes are more easily defined—Lego like—than in proteins, hence the numerous revelations of RNA excited states.

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Section: Real-time(rt) Nmr Spectroscopymentioning

confidence: 99%

RNA Dynamics by NMR Spectroscopy

2019

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“…Upon ligand and Mg 2? binding, the imino proton of U34 (Wacker et al 2012), which forms a reverse Hoogsteen base pair to A65 (Serganov et al 2004), becomes protected from exchange and is therefore visible in the spectrum. In comparison to the unlabeled RNA, the imino proton signal of U34 is not shifted in the spectrum of the 19 F-labeled Gsw apt .…”

Section: Ligand Binding To 19 F-labeled Gsw Aptmentioning

confidence: 99%

“…Ligand binding to the RNA is shown by the H1 signal of hypoxanthine (Fig. 6b), which is protected from exchange when bound, and by the reporter signals from the binding pocket U22 and U47 (Wacker et al 2012). In the ligand unbound state, U22 is Watson-Crick paired to A52 (Serganov et al 2004).…”

Section: Ligand Binding To 19 F-labeled Gsw Aptmentioning

confidence: 99%

19F-labeling of the adenine H2-site to study large RNAs by NMR spectroscopy

et al. 2015

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In comparison to proteins and protein complexes, the size of RNA amenable to NMR studies is limited despite the development of new isotopic labeling strategies including deuteration and ligation of differentially labeled RNAs. Due to the restricted chemical shift dispersion in only four different nucleotides spectral resolution remains limited in larger RNAs. Labeling RNAs with the NMR-active nucleus 19

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“…Metabolite-responsive RNA regulators that respond to changing conditions through molecular interactions are widespread in biology 1 . In many of these systems, kinetic control mechanisms coordinate co-transcriptional RNA folding with metabolite binding and enable responses that are highly-sensitive and highly-selective to target ligands [2][3][4][5][6] . Although synthetic riboswitches exhibiting kinetic control have been identified by chance 7,8 , it has not been possible to intentionally engineer kinetically-controlled RNA aptamer devices 9 .…”

Section: Introductionmentioning

confidence: 99%

“…For example, the E. coli thiamine pyrophosphate (TPP) riboswitches have binding windows as large as 12 seconds, conferring 10-fold increases in ligand sensitivity 5 . By a similar rationale, binding windows that allow molecular discrimination on the basis of RNA-ligand association rates can dramatically increase the selectivity for a target even when the equilibrium binding affinities among cognate molecules are very similar 6 .…”

Section: Introductionmentioning

confidence: 99%

Multi-state design of kinetically-controlled RNA aptamer ribosensors

Burke

Sparkman-Yager

Carothers

2017

Preprint

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Metabolite-responsive RNA regulators with kinetically-controlled responses are widespread in nature. By comparison, very limited success has been achieved creating kinetic control mechanisms for synthetic RNA aptamer devices. Here, we show that kinetically-controlled RNA aptamer ribosensors can be engineered using a novel approach for multi-state, cotranscriptional folding design. The design approach was developed through investigation of 29 candidate p-aminophenylalanine-responsive ribosensors. We show that ribosensors can be transcribed in situ and used to analyze metabolic production directly from engineered microbial cultures, establishing a new class of cell-free biosensors. We found that kinetically-controlled ribosensors exhibited 5-10 fold greater ligand sensitivity than a thermodynamically-controlled device. And, we further demonstrated that a second aptamer, promiscuous for aromatic amino acid binding, could be assembled into kinetic ribosensors with 45-fold improvements in ligand selectivity. These results have broad implications for engineering RNA aptamer devices and overcoming thermodynamic constraints on molecular recognition through the design of kinetically-controlled responses.

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Mechanisms for differentiation between cognate and near-cognate ligands by purine riboswitches

Cited by 9 publications

References 29 publications

RNA Dynamics by NMR Spectroscopy

RNA Dynamics by NMR Spectroscopy

19F-labeling of the adenine H2-site to study large RNAs by NMR spectroscopy

Multi-state design of kinetically-controlled RNA aptamer ribosensors

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