Dataset Information

The fluorescent aptamer Squash extensively repurposes the adenine riboswitch fold.

ABSTRACT: Squash is an RNA aptamer that strongly activates the fluorescence of small-molecule analogs of the fluorophore of green fluorescent protein (GFP). Unlike other fluorogenic aptamers, isolated de novo from random-sequence RNA, Squash was evolved from the bacterial adenine riboswitch to leverage its optimized in vivo folding and stability. We now report the 2.7-Å resolution cocrystal structure of fluorophore-bound Squash, revealing that while the overall fold of the riboswitch is preserved, the architecture of the ligand-binding core is dramatically transformed. Unlike previously characterized aptamers that activate GFP-derived fluorophores, Squash does not harbor a G-quadruplex, sandwiching its fluorophore between a base triple and a noncanonical base quadruple in a largely apolar pocket. The expanded structural core of Squash allows it to recognize unnatural fluorophores that are larger than the simple purine ligand of the parental adenine riboswitch, and suggests that stable RNA scaffolds can tolerate larger variation than has hitherto been appreciated.

SUBMITTER: Truong L

PROVIDER: S-EPMC9812287 | biostudies-literature | 2022 Feb

REPOSITORIES: biostudies-literature

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The fluorescent aptamer Squash extensively repurposes the adenine riboswitch fold.

Truong Lynda L Kooshapur Hamed H Dey Sourav Kumar SK Li Xing X Tjandra Nico N Jaffrey Samie R SR Ferré-D'Amaré Adrian R AR

Nature chemical biology 20211222 2

Squash is an RNA aptamer that strongly activates the fluorescence of small-molecule analogs of the fluorophore of green fluorescent protein (GFP). Unlike other fluorogenic aptamers, isolated de novo from random-sequence RNA, Squash was evolved from the bacterial adenine riboswitch to leverage its optimized in vivo folding and stability. We now report the 2.7-Å resolution cocrystal structure of fluorophore-bound Squash, revealing that while the overall fold of the riboswitch is preserved, the arc ...[more]

PMID: 34937911

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Project description:Expression of a large fraction of genes in bacteria is controlled by riboswitches, which are found in the untranslated region of mRNA. Structurally riboswitches have a conserved aptamer domain to which a metabolite binds, resulting in a conformational change in the downstream expression platform. Prediction of the functions of riboswitches requires a quantitative description of the folding landscape so that the barriers and time scales for the conformational change in the switching region in the aptamer can be estimated. Using a combination of all atom molecular dynamics (MD) and coarse-grained model simulations we studied the response of adenine (A) binding add and pbuE A-riboswitches to mechanical force. The two riboswitches contain a structurally similar three-way junction formed by three paired helices, P1, P2, and P3, but carry out different functions. Using pulling simulations, with structures generated in MD simulations, we show that after P1 rips the dominant unfolding pathway in the add A-riboswitch is the rupture of P2 followed by unraveling of P3. In the pbuE A-riboswitch, after P1 unfolds P3 ruptures ahead of P2. The order of unfolding of the helices, which is in accord with single molecule pulling experiments, is determined by the relative stabilities of the individual helices. Our results show that the stability of isolated helices determines the order of assembly and response to force in these non-coding regions. We use the simulated free energy profile for the pbuE A-riboswitch to estimate the time scale for allosteric switching, which shows that this riboswitch is under kinetic control lending additional support to the conclusion based on single molecule pulling experiments. A consequence of the stability hypothesis is that a single point mutation (U28C) in the P2 helix of the add A-riboswitch, which increases the stability of P2, would make the folding landscapes of the two riboswitches similar. This prediction can be tested in single molecule pulling experiments.

Dataset Information

The fluorescent aptamer Squash extensively repurposes the adenine riboswitch fold.

Publications

The fluorescent aptamer Squash extensively repurposes the adenine riboswitch fold.

Similar Datasets

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