Use of SHAPE to Select 2AP Substitution Sites for RNA–Ligand Interactions and Dynamics Studies

Soulière, Marie F.; Micura, Ronald

doi:10.1007/978-1-62703-730-3_17

Cited by 7 publications

(5 citation statements)

References 19 publications

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“…However, in the absence of X-ray data, selecting appropriate positions within the nucleic acid structure to insert a 2AP substitution that can be sensitive to ligand binding or folding events can be a tedious and random task. Here, a strategy recently developed by Souliere et al allows to identify positions to within the RNA sequence where the introduction of a 2AP substitution may act as a sensitive reporter of folding or ligand binding (Soulière et al, 2011 ; Soulière and Micura, 2014 ). The method uses SHAPE reactivity profiles to predict positions where the nucleotides are flexible because they are looped out or partially unstacked and differentiate them from those located in more constrained positions.…”

Section: Biophysical Methods To Investigate Aptamer-ligand Complexesmentioning

confidence: 99%

Fluorescence-Based Strategies to Investigate the Structure and Dynamics of Aptamer-Ligand Complexes

2016

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In addition to the helical nature of double-stranded DNA and RNA, single-stranded oligonucleotides can arrange themselves into tridimensional structures containing loops, bulges, internal hairpins and many other motifs. This ability has been used for more than two decades to generate oligonucleotide sequences, so-called aptamers, that can recognize certain metabolites with high affinity and specificity. More recently, this library of artificially-generated nucleic acid aptamers has been expanded by the discovery that naturally occurring RNA sequences control bacterial gene expression in response to cellular concentration of a given metabolite. The application of fluorescence methods has been pivotal to characterize in detail the structure and dynamics of these aptamer-ligand complexes in solution. This is mostly due to the intrinsic high sensitivity of fluorescence methods and also to significant improvements in solid-phase synthesis, post-synthetic labeling strategies and optical instrumentation that took place during the last decade. In this work, we provide an overview of the most widely employed fluorescence methods to investigate aptamer structure and function by describing the use of aptamers labeled with a single dye in fluorescence quenching and anisotropy assays. The use of 2-aminopurine as a fluorescent analog of adenine to monitor local changes in structure and fluorescence resonance energy transfer (FRET) to follow long-range conformational changes is also covered in detail. The last part of the review is dedicated to the application of fluorescence techniques based on single-molecule microscopy, a technique that has revolutionized our understanding of nucleic acid structure and dynamics. We finally describe the advantages of monitoring ligand-binding and conformational changes, one molecule at a time, to decipher the complexity of regulatory aptamers and summarize the emerging folding and ligand-binding models arising from the application of these single-molecule FRET microscopy techniques.

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Section: Biophysical Methods To Investigate Aptamer-ligand Complexesmentioning

confidence: 99%

Fluorescence-Based Strategies to Investigate the Structure and Dynamics of Aptamer-Ligand Complexes

2016

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“…2b ). This 2-AP assay was originally developed to study the binding of aminoglycosides to bacterial rRNA and the effect of binding on A-site dynamics 84 , but has been extended to other targets 70 , 85 , 86 . Notably, the position of the 2-AP substitution within the RNA should be carefully chosen to ensure a strong signal.…”

Section: Strategies To Identify Small-molecule Rna Bindersmentioning

confidence: 99%

“…For small RNAs, this can be accomplished by simply substituting each adenine residue. For longer RNAs, such as riboswitches, SHAPE can be used to elucidate where large conformational changes occur upon ligand binding, and hence the optimal position (or postions) for 2-AP substitution 85 , 86 .…”

Section: Strategies To Identify Small-molecule Rna Bindersmentioning

confidence: 99%

Targeting RNA structures with small molecules

Childs‐Disney

Yang

Gibaut

et al. 2022

Nat Rev Drug Discov

262

219

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RNA adopts 3D structures that confer varied functional roles in human biology and dysfunction in disease. Approaches to therapeutically target RNA structures with small molecules are being actively pursued, aided by key advances in the field including the development of computational tools that predict evolutionarily conserved RNA structures, as well as strategies that expand mode of action and facilitate interactions with cellular machinery. Existing RNA-targeted small molecules use a range of mechanisms including directing splicing — by acting as molecular glues with cellular proteins (such as branaplam and the FDA-approved risdiplam), inhibition of translation of undruggable proteins and deactivation of functional structures in noncoding RNAs. Here, we describe strategies to identify, validate and optimize small molecules that target the functional transcriptome, laying out a roadmap to advance these agents into the next decade.

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“…This approach relies on synthetic RNAs with a single, strategically positioned 2-aminopurine which substitutes a nucleobase under minimal steric interference and preservation of existing H-bond and stacking patterns. Thereby, the selection of suitable Ap positions is either based on the three-dimensional structure (if available) or on SHAPE probing [3,34,35]. Hence, the conformational rearrangements of the Ap-labeled RNA in response to external stimuli (such as Mg 2+ cations or lowmolecular weight compounds that bind RNA) are analyzed by pursuing the Ap fluorescence intensity signal, revealing the underlying kinetic and thermodynamic parameters.…”

Section: Resultsmentioning

confidence: 99%

Practical synthesis of N-(di-n-butylamino)methylene-protected 2-aminopurine riboside phosphoramidite for RNA solid-phase synthesis

Neuner¹,

Micura²

2019

Monatsh Chem

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2-Aminopurine (Ap) is a fluorescent nucleobase analog that is frequently used as structure-sensitive reporter to study the chemical and biophysical properties of nucleic acids. In particular, thermodynamics and kinetics of RNA folding and RNAligand binding, as well as RNA catalytic activity are addressable by pursuing the Ap fluorescence signal in response to external stimuli. Site-specific incorporation of Ap into RNA is usually achieved by RNA solid-phase synthesis and requires appropriately functionalized Ap riboside building blocks. Here, we introduce a robust synthetic path toward a 2-aminopurine riboside phosphoramidite whose N 2 functionality is masked with the N-(di-n-butylamino)methylene group. This protection is considered advantageous over previously described N-(dimethylamino)methylene or acyl protection patterns needed for the fine-tuned deprotection conditions to achieve large synthetic RNAs.

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Use of SHAPE to Select 2AP Substitution Sites for RNA–Ligand Interactions and Dynamics Studies

Cited by 7 publications

References 19 publications

Fluorescence-Based Strategies to Investigate the Structure and Dynamics of Aptamer-Ligand Complexes

Fluorescence-Based Strategies to Investigate the Structure and Dynamics of Aptamer-Ligand Complexes

Targeting RNA structures with small molecules

Practical synthesis of N-(di-n-butylamino)methylene-protected 2-aminopurine riboside phosphoramidite for RNA solid-phase synthesis

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