Using sm-FRET and Denaturants to Reveal Folding Landscapes

Shaw, Euan; St-Pierre, Patrick; McCluskey, Kaley; Lafontaine, Daniel A.; Penedo, J. Carlos

doi:10.1016/b978-0-12-801122-5.00014-3

Cited by 13 publications

(9 citation statements)

References 72 publications

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“…To investigate the formation of the add aptamer-ligand complex in further detail, a later report combined sm-FRET with the use of chemical denaturants such as a urea (Dalgarno et al, 2013 ). Although, chemical denaturants are widely employed in protein folding research at single-molecule level, this constituted the first systematic study demonstrating the feasibility of urea-induced denaturation of the RNA tertiary structure to analyze aptamer-ligand interactions (Shaw et al, 2014 ). The study demonstrated that surface-immobilized adenine aptamers labeled as previously at the P2 and P3 stem loops with the Cy3–Cy5 FRET pair can be subjected to Mg 2+ -induced folding and urea-induced unfolding cycles without compromising surface-attachment or RNA folding integrity (Figure 5B ).…”

Section: Fluorescence Methods Based On Doubly Labeled Aptamersmentioning

confidence: 99%

“…Moreover, the study demonstrated that urea-induced disruption of the loop-loop interaction takes place via an intermediate state ( I ) as observed when using Mg 2+ ions to promote folding. Overall, the study showed how the competing interplay between folding agents such as Mg 2+ and ligand and unfolding species such as urea can be harnessed to extract information that otherwise remains hidden (Shaw et al, 2014 ). For instance, aptamers labeled with a FRET pair to monitor the loop-loop interaction between stems P2 and P3 have identical energy transfer efficiency for the ligand-free and the ligand-bound states ( E ~ 0.9).…”

Section: Fluorescence Methods Based On Doubly Labeled Aptamersmentioning

confidence: 99%

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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: Fluorescence Methods Based On Doubly Labeled Aptamersmentioning

confidence: 99%

Section: Fluorescence Methods Based On Doubly Labeled Aptamersmentioning

confidence: 99%

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

2016

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“…The function of a protein is encoded in its three-dimensional (3D) structure. Although deduction of a protein’s tertiary structure (its native conformation) from its primary sequence has been revolutionized by computational techniques (65), smFRET experiments have provided many additional insights into the process of folding—whether into the correct structure or into incorrect structures (“misfolding”)—and characterization of possible folding intermediates (66, 67). Measurements involving a variety of denaturing agents have yielded evidence of a monotonic shift in the mean FRET value of the unfolded subpopulation as a function of denaturant concentration.…”

Section: A Brief Historical Overview Of Single-molecule Fret In Biochmentioning

confidence: 99%

Toward dynamic structural biology: Two decades of single-molecule Förster resonance energy transfer

Lerner

Cordes

Ingargiola

et al. 2018

Science

479

449

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Classical structural biology can only provide static snapshots of biomacromolecules. Single-molecule Förster resonance energy transfer (smFRET) paved the way for studying dynamics in macromolecular structures under biologically relevant conditions. Since its first implementation in 1996, smFRET experiments have confirmed previously hypothesized mechanisms and provided new insights into many fundamental biological processes, such as DNA maintenance and repair, transcription, translation, and membrane transport. We review 22 years of contributions of smFRET to our understanding of basic mechanisms in biochemistry, molecular biology, and structural biology. Additionally, building on current state-of-the-art implementations of smFRET, we highlight possible future directions for smFRET in applications such as biosensing, high-throughput screening, and molecular diagnostics.

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“…16,17 To gather structural information regarding the role of junction core residues, we employed the FRET technique that is unique in terms of giving quantitative information about nucleic acid conformational changes. [35][36][37][38] It was previously reported, using small-angle X-ray scattering (SAXS) and crystal structures, that the lysine aptamer does not experience significant structural changes upon ligand binding. 16,17,39 However, FRET studies showed that Mg 2C and lysine cofactors both have a strong influence on the energy transfer occurring between fluorophores located in stems P1 and P5, indicating that the aptamer global folding is modulated upon binding of either cofactor.…”

Section: Resultsmentioning

confidence: 99%

Role of lysine binding residues in the global folding of thelysCriboswitch

2015

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Riboswitches regulate gene expression by rearranging their structure upon metabolite binding. The lysine-sensing lysC riboswitch is a rare example of an RNA aptamer organized around a 5-way helical junction in which ligand binding is performed exclusively through nucleotides located at the junction core. We have probed whether the nucleotides involved in ligand binding play any role in the global folding of the riboswitch. As predicted, our findings indicate that ligand-binding residues are critical for the lysine-dependent gene regulation mechanism. We also find that these residues are not important for the establishment of key magnesium-dependent tertiary interactions, suggesting that folding and ligand recognition are uncoupled in this riboswitch for the formation of specific interactions. However, FRET assays show that lysine binding results in an additional conformational change, indicating that lysine binding may also participate in a specific folding transition. Thus, in contrast to helical junctions being primary determinants in ribozymes and rRNA folding, we speculate that the helical junction of the lysine-sensing lysC riboswitch is not employed as structural a scaffold to direct global folding, but rather has a different role in establishing RNA-ligand interactions required for riboswitch regulation. Our work suggests that helical junctions may adopt different functions such as the coordination of global architecture or the formation of specific ligand binding site.

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Using sm-FRET and Denaturants to Reveal Folding Landscapes

Cited by 13 publications

References 72 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

Toward dynamic structural biology: Two decades of single-molecule Förster resonance energy transfer

Role of lysine binding residues in the global folding of thelysCriboswitch

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