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Abstract

More than two decades of investigating nucleic acids and ribonucleic acids (RNA) using single molecule Förster resonance energy transfer (smFRET) have passed. It turned out that sample heterogeneity in structure and function of RNA molecules as well as folding intermediates, kinetic subpopulations, and interconversion rates of conformational states of RNA biomolecules, all of which are usually hidden in ensemble type experiments, are often observed characteristics. Besides proteins, metal ions play a crucial role in RNA folding and dynamics, as well as RNA/RNA or RNA/DNA interactions. RNA molecules form discrete conformational intermediates before reaching the native three-dimensional fold, whereby metal ions guide the folding pathway by changing the energetic barriers between local and global minima in the energy landscape. Here we review recent advances in the characterization of the role of metal ions in folding and function of nucleic acid structures by means of smFRET. Subsequently, the workflow of smFRET data analysis is described and exemplified by the metal ion-depending folding and dynamics of the group IIB intron from Saccharomyces cerevisiae and RNA–RNA binding kinetics of this ribozyme's 5'-splice site formation.

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... Single-molecule Förster resonance energy transfer (smFRET) allows to observe these conformational transitions for individual molecules, and thus, to get a mechanistic view of the biomolecule in action. FRET is a non-radiative transfer of energy between two nearby fluorophores, a donor and an acceptor, with overlapping emission and absorption spectra [4][5][6]. It is particularly successful on the single-molecule level as it allows to detect short-lived or rare folding intermediates that are usually averaged out in the molecule ensemble [7][8][9]. ...
... This method allows to observe hundreds of single molecules in parallel and over time, with a time resolution defined by the frame rate of the camera and an observation time limited by the fluorophores photobleaching probability [12]. The identification of distinct FRET states as well as the quantification of state transition rate constants from SMV is central to the data analysis in single-molecule kinetic studies [13] [4][5][6]. ...
... All steps can be performed with the MATLAB-based Multifunctional Analysis Software for Handling single-molecule FRET data (MASH-FRET) [4,13,[16][17][18]. For system requirements and installation instructions, please refer to the documentation [19]. 1 Markovian process depicted as a Trellis diagram. ...
Chapter
Single-molecule microscopy is often used to observe and characterize the conformational dynamics of nucleic acids (NA). Due to the large variety of NA structures and the challenges specific to single-molecule observation techniques, the data recorded in such experiments must be processed via multiple statistical treatments to finally yield a reliable mechanistic view of the NA dynamics. In this chapter, we propose a comprehensive protocol to analyze single-molecule trajectories in the scope of single-molecule Förster resonance energy transfer (FRET) microscopy. The suggested protocol yields the conformational states common to all molecules in the investigated sample, together with the associated conformational transition kinetics. The given model resolves states that are indistinguishable by their observed FRET signals and is estimated with 95% confidence using error calculations on FRET states and transition rate constants. In the end, a step-by-step user guide is given to reproduce the protocol with the Multifunctional Analysis Software to Handle single-molecule FRET data (MASH-FRET).
... Since the initial proof of concept, single-molecule fluorescence techniques, in particular single-molecule Förster resonance energy transfer (smFRET), have proven powerful tools in probing biomolecular structures and dynamics [1][2][3]. Single fluorescent molecule sensitivity is predominantly achieved using two experimental configurations: (i) confocal microscopy in conjunction with single-photon detection (avalanche photodiodes or photomultiplier tubes) [4,5] and (ii) total internal reflection fluorescence [6] or wide-field microscopy with intensitybased detection using either an electron multiplying charge-coupled device (EM-CCD) [7,8] or a scientific complementary metal-oxide-semiconductor (sCMOS) camera [9]. Camera a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 detection is characterized by a time resolution in the lower millisecond range and a spatial resolution reaching the diffraction limit of visible light [7]. ...
... Owing to the pronounced distance dependence of the FRET efficiency, FRET is frequently referred to as a spectroscopic ruler within a range of 3 to 10 nm [28]. For further information on FRET, please refer to dedicated reviews [3,6,15,29]. Camera-based smFRET typically involves total internal reflection (TIR) excitation of surface-tethered biomolecules that are fluorophore labeled [7]. ...
... A frequently observed phenomenon in smFRET are molecule-to-molecule variations. For example, variations can be observed with regard to the mean FRET value of a certain conformational state, the total emitted intensity and quantum yields [3,37]. We modeled cross-sample variability assuming a Gaussian distribution of the underlying VSP values characterized by a defined center and standard deviation σ, thus, FRET j and σ FRET,j , I tot,0 and σ Itot,0 , and γ and σ γ . ...
Article
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Single-molecule microscopy has become a widely used technique in (bio)physics and (bio)chemistry. A popular implementation is single-molecule Förster Resonance Energy Transfer (smFRET), for which total internal reflection fluorescence microscopy is frequently combined with camera-based detection of surface-immobilized molecules. Camera-based smFRET experiments generate large and complex datasets and several methods for video processing and analysis have been reported. As these algorithms often address similar aspects in video analysis, there is a growing need for standardized comparison. Here, we present a Matlab-based software (MASH-FRET) that allows for the simulation of camera-based smFRET videos, yielding standardized data sets suitable for benchmarking video processing algorithms. The software permits to vary parameters that are relevant in cameras-based smFRET, such as video quality, and the properties of the system under study. Experimental noise is modeled taking into account photon statistics and camera noise. Finally, we survey how video test sets should be designed to evaluate currently available data analysis strategies in camera-based sm fluorescence experiments. We complement our study by pre-optimizing and evaluating spot detection algorithms using our simulated video test sets.
... As ion coordination is transient though, designated binding pockets are often only partially occupied and exon dissociation is thus kinetically heterogeneous 15 . Particularly, Mg 2+ is known to induce such kinetic partitioning by interacting with RNA directly (inner-sphere coordination) or via a water molecule (outer-sphere coordination) [16][17][18] . ...
... As metal ions direct folding and catalysis, heterogeneity is inherent to many RNAs 16,22 . RNA folds hierarchically into a set of interconnected topological modules. ...
... Here, we have investigated the thermodynamics and kinetics governing the stability of this tertiary contact using single-molecule FRET in combination with molecular dynamic simulations. 15,16 . We find that Mg 2+ association and dissociation from the EBS1*/IBS1* contact are linked to very subtle conformational changes within the duplex, indiscernible by FRET. ...
Article
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The fidelity of group II intron self-splicing and retrohoming relies on long-range tertiary interactions between the intron and its flanking exons. By single-molecule FRET, we explore the binding kinetics of the most important, structurally conserved contact, the exon and intron binding site 1 (EBS1/IBS1). A comparison of RNA-RNA and RNA-DNA hybrid contacts identifies transient metal ion binding as a major source of kinetic heterogeneity which typically appears in the form of degenerate FRET states. Molecular dynamics simulations suggest a structural link between heterogeneity and the sugar conformation at the exon-intron binding interface. While Mg2+ ions lock the exon in place and give rise to long dwell times in the exon bound FRET state, sugar puckering alleviates this structural rigidity and likely promotes exon release. The interplay of sugar puckering and metal ion coordination may be an important mechanism to balance binding affinities of RNA and DNA interactions in general.
... Some typical examples, including receptor and antigen interactions [24,25], vesical fusion [26], and ion channel dynamics, also relate to the conformational changes and equilibrium properties of molecules [27]. smFRET technologies have been put to use to study the folding dynamics of nucleic acids [28][29][30]. A single-stranded region in nucleic acids is likely to fold intramolecularly upon itself to form hairpins, internal loops, bulges, and junctions. ...
... The monovalent and divalent cations play a pivotal role in promoting RNA conformational transformation. For instance, monovalent cations (e.g., K + , Na + ) facilitate the first step of RNA folding to secondary structures, followed by divalent cations (e.g., Mg 2+ ) that further enhance secondary structure interactions and tertiary contacts [28,94]. Previous studies in the role of metal cations in RNA folding and kinetics have concentrated on secondary structure formation (e.g., 3WJs [34,95], kissing hairpins [96], GAAA tetraloop-receptors [97], four-way junctions [98], catalytic ribozyme folding [99], RNA/RNA or RNA/DNA interactions [100]). ...
Article
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Single-molecule Förster resonance energy transfer (smFRET) inherits the strategy of measurement from the effective “spectroscopic ruler” FRET and can be utilized to observe molecular behaviors with relatively high throughput at nanometer scale. The simplicity in principle and configuration of smFRET make it easy to apply and couple with other technologies to comprehensively understand single-molecule dynamics in various application scenarios. Despite its widespread application, smFRET is continuously developing and novel studies based on the advanced platforms have been done. Here, we summarize some representative examples of smFRET research of recent years to exhibit the versatility and note typical strategies to further improve the performance of smFRET measurement on different biomolecules.
... As a molecular ruler, FRET is used as a distance constrain to filter a computationally generated structural ensemble. Performing the experiments on a single-molecule level circumvents ensemble averaging and dissects heterogeneities in structure and dynamics [4][5][6]. ...
... In the presence of monovalent ions, like Na + or K + , RNA folds by forming Watson-Crick (WC) base pairs (A-U, C-G) [16,17]. Tertiary structure motifs like the GAAA tetraloop-receptor are promoted by divalent metal ions [5,[18][19][20][21]. These contacts are stabilized by non-WC interactions among those the A-minor motif, which is highly abundant in the large ribosomal subunit [22]. ...
Conference Paper
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Long-range tertiary interactions between RNA tetraloops and their receptors stabilize the folding of ribosomal RNA and support the maturation of the ribosome. Here, we use FRET-assisted structure prediction to develop a structural model of the GAAA tetraloop receptor (TLR) interaction and its dynamics. We build the docked TLR de novo, label the RNA in silico and compute FRET histograms based on MD simulations. The predicted mean FRET efficiency is remarkably consistent with single-molecule experiments of the docked tetraloop. This hybrid approach of experiment and simulation will promote the elucidation of dynamic RNA tertiary contacts and accelerate the discovery of novel RNA and RNA-protein interactions as potential future drug targets.
... Several studies have reported the interaction between G4 DNA and metal ions [28][29][30][31][32][33][34][35], but less information is available on G4 RNAs [36,37]. The latter are known to be more stable than their DNA counterparts [38], but to the best of our knowledge no systematic studies with metal ions are available so far. ...
... The folding into parallel G4s, as observed in our CD experiments of NRAS and TERRA RNAs in all the cation conditions tested, is consistent with the fact that so far only the parallel G4 fold is known for RNA G-quadruplexes [67]. This is in contrast to G4 DNA, which shows an array of topologies [30,68], exchangeable through changes in temperature and/or cation conditions [10]. A clear example can be found in the comparison between the telomeric sequences htelo and TERRA ( Figure S6b). ...
Article
Full-text available
RNA G-quadruplexes, as their well-studied DNA analogues, require the presence of cations to fold and remain stable. This is the first comprehensive study on the interaction of RNA quadruplexes with metal ions. We investigated the formation and stability of two highly conserved and biologically relevant RNA quadruplex-forming sequences (24nt-TERRA and 18nt-NRAS) in the presence of several monovalent and divalent metal ions, namely Li+, Na+, K+, Rb+, Cs+, NH4+, Mg2+, Ca2+, Sr2+, and Ba2+. Circular dichroism was used to probe the influence of these metal ions on the folded fraction of the parallel G-quadruplexes and UV thermal melting experiments allowed to assess the relative stability of the structures in each cationic condition. Our results show that the RNA quadruplexes are more stable than their DNA counterparts under the same buffer conditions. We have observed that the addition of mainly Na+, K+, Rb+, NH4+, as well as Sr2+ and Ba2+ in water, shifts the equilibrium to the folded quadruplex form, whereby the NRAS sequence responds stronger than TERRA. However, only K+ and Sr2+ lead to a significant increase in the stability of the folded structures, which is consistent with their coordination to the O6 atoms from the G-quartet guanosines. Compared to the respective DNA motives, dNRAS and htelo, the RNA sequences are not stabilized by Na+ ions. Finally, the difference in response between NRAS and TERRA, as well as to the corresponding DNA sequences with respect to different metal ions, could potentially be exploited for selective targeting purposes.
... Single-molecule (sm) spectroscopy along with Förster resonance energy transfer (FRET) has emerged as a versatile tool for probing distance distributions in biomacromolecules on the nanometer scale (1,2). Single-molecule detection can unravel conformational, kinetic, and thermodynamic heterogeneities of biomolecules like DNA, RNA, and proteins to provide information that is lost in ensemble experiments (3)(4)(5)(6). An integral part of fluorescence spectroscopy is the choice and incorporation of suitable extrinsic fluorophores. ...
Article
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Labeling of long RNA molecules in a site-specific yet generally applicable manner is integral to many spectroscopic applications. Here we present a novel covalent labeling approach that is site-specific and scalable to long intricately folded RNAs. In this approach, a custom-designed DNA strand that hybridizes to the RNA guides a reactive group to target a preselected adenine residue. The functionalized nucleotide along with the concomitantly oxidized 3'-terminus can subsequently be conjugated to two different fluorophores via bio-orthogonal chemistry. We validate this modular labeling platform using a regulatory RNA of 275 nucleotides, the btuB riboswitch of Escherichia coli, demonstrate its general applicability by modifying a base within a duplex, and show its site-selectivity in targeting a pair of adjacent adenines. Native folding and function of the RNA is confirmed on the single-molecule level by using FRET as a sensor to visualize and characterize the conformational equilibrium of the riboswitch upon binding of its cofactor adenosylcobalamin. The presented labeling strategy overcomes size and site constraints that have hampered routine production of labeled RNA that are beyond 200 nt in length.
... Since the initial proof of concept, single-molecule fluorescence techniques have become powerful tools to probe biomolecular structure and conformational dynamics. [1][2][3] Single molecule Förster Resonance Energy Transfer (smFRET) uses the distance-dependent energy transfer between a donor and an acceptor fluorescent label to probe interdye distances between 2 and 10 nm. 4 Spectral separation of donor and acceptor photon emission upon selective excitation of the donor allows to calculate the apparent transfer efficiency over time, which corresponds to the fraction of photons emitted by the acceptor. 5 If required, absolute transfer efficiencies are generated upon applying a number of corrections to yield interdye distances according to Förster´s theory. ...
Article
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Single-molecule Förster resonance energy transfer (smFRET) is a powerful technique to probe biomolecular structure and dynamics. A popular implementation of smFRET consists in recording fluorescence intensity time traces of surface-immobilized, chromophore-tagged molecules. This approach generates large and complex datasets, the analysis of which is to date not standardized. Here we address a key challenge in smFRET data analysis: the generation of thermodynamic and kinetic models that describe with statistical rigor the behavior of FRET trajectories recorded from surface-tethered biomolecules in terms of the number of FRET states, the corresponding mean FRET values and the kinetic rates at which they interconvert. For this purpose, we first perform Monte-Carlo simulations to generate single-molecule FRET trajectories, in which a relevant space of experimental parameters is explored. Then, we provide an account on current strategies to achieve such model selection, as well as a quantitative assessment of their performances. Specifically, we evaluate the performance of each algorithm (CPA, STaSI, HaMMy, vbFRET, ebFRET) with respect to accuracy, reproducibility and computing time, which yields a range of algorithm-specific referential benchmarks for various data qualities. Data simulation and analysis were performed with our Multifunctional Analysis Software for Handling smFRET data (MASH-FRET).
... Single-molecule (SM) spectroscopy stands out for revealing cross-sample variability, the distribution of subspecies, heterogeneity of structures and dynamics as well as the separation of reaction intermediates or subpopulations usually averaged out in ensemble measurements (Kowerko et al., 2015;Börner et al., 2016). ...
... Next, signals were filtered and separated using dual-view and then imaged on two halves of a high quantum yield EM-CCD camera chip (Andor). Single-molecule videos were analyzed as described previously (31,32,58,60). ...
Article
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Unlike in vivo conditions, group II intron ribozymes are known to require high magnesium(II) concentrations ([Mg²⁺]) and high temperatures (42 °C) for folding and catalysis in vitro. A possible explanation for this difference is the highly crowded cellular environment, which can be mimicked in vitro by macromolecular crowding agents. Here, we combined bulk activity assays and single-molecule Förster Resonance Energy Transfer (smFRET) to study the influence of polyethylene glycol (PEG) on catalysis and folding of the ribozyme. Our activity studies reveal that PEG reduces the [Mg²⁺] required, and we found an “optimum” [PEG] that yields maximum activity. smFRET experiments show that the most compact state population, the putative active state, increases with increasing [PEG]. Dynamic transitions between folded states also increase. Therefore, this study shows that optimal molecular crowding concentrations help the ribozyme not only to reach the native fold but also to increase its in vitro activity to approach that in physiological conditions.
... Les ARN, comme les ADN, sont capables d'adopter un large spectre de conformations. C'est la forme double-hélice A qui est la plus commune dans les ARN double brin (Salazar et al. 1993;Börner et al. 2016), comme par exemple chez certains virus qui stockent leur génome sous forme d'ARN double brin (Wickner 1996). La forme Z existe également (Herbert 2019) alors que la forme B n'est pas adaptée à l'ARN. ...
Thesis
Les protéines régulent et exécutent l'ensemble des fonctions vitales des organismes en interagissant notamment avec les acides nucléiques (AN), dont l’ADN, support de l’information génétique. Appréhender la nature de ces types d’interactions est central en biologie. Le nucléosome, qui est l’unité élémentaire de la compaction de l’ADN chez les cellules eucaryotes, est formé d’un d’ADN enroulé autour d’un cœur protéique d’histone ; il contrôle l’accessibilité de l’ADN en se formant et en se dissociant le long des génomes. Ici, le nucléosome a été modélisé par dynamique moléculaire en solution. L’ analyse de l’interface ADN-histone par une méthode géométrique innovante a permis de comprendre comment la forte cohésion de ce complexe était assurée. La description de l’interface a aussi servi à interpréter des expériences d’assemblage et de désassemblage du nucléosome qui ont par ailleurs démontré l’effet de la séquence d’ADN sur ces processus. Enfin, j’ai comparé les dynamiques de l’ADN nucléosomal et de l’ADN nu, et montré quelles propriétés structurales étaient conservées au sein du nucléosome et comment elles sont utilisées pour moduler l’efficacité de l’association ADN-histones. Une stratégie semblable a été appliquée à des structures expérimentales de complexes entre ADN ou ARN et NCp7, une protéine du VIH-1 chaperon des AN. Cette dernière étude propose un mécanisme d’association entre les partenaires sur des bases rationnelles. Dans ces deux études, je mets en évidence des mécanismes de formation des complexes en plusieurs étapes et j’illustre les préférences de structure et de séquence des AN chez des protéines dites non-spécifiques.
... SMV processing for TIRF-based smFRET experiments is presented elsewhere [38]. However, a number of mandatory steps are described herein. ...
Chapter
Imaging fluorescently labeled biomolecules on a single-molecule level is a well-established technique to follow intra- and intermolecular processes in time, usually hidden in the ensemble average. The classical approach comprises surface immobilization of the molecule of interest, which increases the risk of restricting the natural behavior due to surface interactions. Encapsulation of such biomolecules into surface-tethered phospholipid vesicles enables to follow one molecule at a time, freely diffusing and without disturbing surface interactions. Further, the encapsulation allows to keep reaction partners (reactants and products) in close proximity and enables higher temperatures otherwise leading to desorption of the direct immobilized biomolecules.
Chapter
Ribosomes are large macromolecular complexes responsible for the translation process. During the course of ribosome biogenesis and protein synthesis, extra-ribosomal factors interact with the ribosome or its subunits to assist in these vital processes. Here we describe a method to isolate and analyze not only bacterial ribosomes but also their associated factors, providing insights into translation regulation. This detailed protocol allows the separation and monitoring of the ribosomal species and their interacting partners along a sucrose density gradient. Simultaneously, fractionation of the gradient allows for the recovery of 70S ribosomes and its subunits enabling a wide range of downstream applications. This protocol can be easily adapted to ribosome-related studies in other species or for separating other macromolecular complexes.
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Riboswitches are cis-acting regulatory RNA biosensors that rival the efficiency of those found in proteins. At the heart of their regulatory function is the formation of a highly specific aptamer-ligand complex. Understanding how these RNAs recognize the ligand to regulate gene expression at physiological concentrations of Mg2+ ions and ligand is critical given their broad impact on bacterial gene expression and their potential as antibiotic targets. In this work, we used single-molecule FRET and biochemical techniques to demonstrate that Mg2+ ions act as fine-tuning elements of the amino acid-sensing lysC aptamer's ligand-free structure in the mesophile Bacillus subtilis. Mg2+ interactions with the aptamer produce encounter complexes with strikingly different sensitivities to the ligand in different, yet equally accessible, physiological ionic conditions. Our results demonstrate that the aptamer adapts its structure and folding landscape on a Mg2+-tunable scale to efficiently respond to changes in intracellular lysine of more than two orders of magnitude. The remarkable tunability of the lysC aptamer by sub-millimolar variations in the physiological concentration of Mg2+ ions suggests that some single-aptamer riboswitches have exploited the coupling of cellular levels of ligand and divalent metal ions to tightly control gene expression.
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We report on atomistic mechanism of elementary hopping processes of Li^+ ions in liquid thiophene obtained from ab initio molecular dynamics simulations. We observe the formation of cage structures which solvate the cation. Besides the actual molecular solvation structure, we provide an analysis of the pathway and timescale of the basic Li^+ diffusion steps in terms of the coordination by sulfur atoms. We compare our results to the situation in a thiophene derivative, namely 3,4‐ethylenedioxythiophene (EDOT). The calculations reveal that in both thiophene and EDOT liquids, a tetrahedral structure is formed around the Li^+ ion. While in the case of the former, the Li cation is coordinated by four sulfur atoms, in the latter case it is surrounded by four oxygens. The tetrahedrons act as cages which accommodate the cation for a considerable duration (of the order of 100ps). The elementary diffusion step occurs through a "permeable edge" of the tetrahedron formed by two sulfur (or oxygen) atoms at a characteristic distance. This finding indicates that Li^+ conduction in thiophene derivatives can be improved by rationally designing the compound in such a way that maximizes the occurrence of sulfur atoms at that particular distance from each other.
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Guanine quadruplex (G-quadruplex) structures play a vital role in stabilizing the DNA genome and in protecting healthy cells from transforming into cancer cells. The structural stability of G-quadruplexes is greatly enhanced by the binding of monovalent cations such as Na+ or K+ into the interior axial channel. We computationally study the free energy of binding of Na+ and K+ ions to two intramolecular G-quadruplexes that differ considerably in their degree of rigidity and the presence or absence of terminal nucleotides. The goal of our study is two-fold. On the one hand, we study the free energy of binding every ion, which complements the experimental findings that report the average free energy for replacing Na+ with K+ ions. On the other hand, we examine the role of the G-quadruplex structure in the binding free energy. In the study, we employ all-atom molecular dynamics simulations and the alchemical transformation method for the computation of the free energies. To compare the cation-dependent contribution to the structural stability of G-quadruplexes, we use a two-step approach to calculate the individual free energy difference ∆G of binding two Na+ and two K+ to two G-quadruplexes: the unimolecular DNA d[T2GT2(G3T)3] (Pro- tein Data Bank ID 2M4P) and the human telomeric DNA d[AGGG(TTAGGG)3] (PDB ID 1KF1). In contrast to the experimental studies that estimate the average free energy of binding, we find a varying difference of approximately 2–9 kcal/mol between the free energy contribution of binding the first and second cation, Na+ or K+. Furthermore, we found that the free energy of binding K+ is not affected by the chemical nature of the two quadruplexes. By contrast, Na+ showed dependency on the G-quadruplex structure; the relatively small size allows Na+ to explore larger configura- tional space than K+. Numerical results presented here may offer reference values for future design of cationic drug-like ligands that replace the metal ions in G-quadruplexes.
Chapter
Labeling of large RNAs with reporting entities, e.g., fluorophores, has significant impact on RNA studies in vitro and in vivo. Here, we describe a minimally invasive RNA labeling method featuring nucleotide and position selectivity, which solves the long-standing challenge of how to achieve accurate site-specific labeling of large RNAs with a least possible influence on folding and/or function. We use a custom-designed reactive DNA strand to hybridize to the RNA and transfer the alkyne group onto the targeted adenine or cytosine. Simultaneously, the 3′-terminus of RNA is converted to a dialdehyde moiety under the experimental condition applied. The incorporated functionalities at the internal and the 3′-terminal sites can then be conjugated with reporting entities via bioorthogonal chemistry. This method is particularly valuable for, but not limited to, single-molecule fluorescence applications. We demonstrate the method on an RNA construct of 275 nucleotides, the btuB riboswitch of Escherichia coli.
Chapter
RNA structure is important for understanding RNA function and stability within a cell. Chemical probing is a well-established and convenient method to evaluate the structure of an RNA. Several structure-sensitive chemicals can differentiate paired and unpaired nucleotides. This chapter specifically addresses the use of DMS and CMCT. Although exhibiting different affinities, the combination of these two chemical reagents enables screening of all four nucleobases. DMS and CMCT are only reactive with exposed unpaired nucleotides. We have used this method to analyze the effect of the RNA chaperone Hfq on the conformation of the 16S rRNA. The strategy here described may be applied for the study of many other RNA-binding proteins and RNAs.
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Single-molecule FRET (smFRET) has become a mainstream technique for studying biomolecular structural dynamics. The rapid and wide adoption of smFRET experiments by an ever-increasing number of groups has generated significant progress in sample preparation, measurement procedures, data analysis, algorithms and documentation. Several labs that employ smFRET approaches have joined forces to inform the smFRET community about streamlining how to perform experiments and analyze results for obtaining quantitative information on biomolecular structure and dynamics. The recent efforts include blind tests to assess the accuracy and the precision of smFRET experiments among different labs using various procedures. These multi-lab studies have led to the development of smFRET procedures and documentation, which are important when submitting entries into the archiving system for integrative structure models, PDB-Dev. This position paper describes the current ‘state of the art’ from different perspectives, points to unresolved methodological issues for quantitative structural studies, provides a set of ‘soft recommendations’ about which an emerging consensus exists, and lists openly available resources for newcomers and seasoned practitioners. To make further progress, we strongly encourage ‘open science’ practices.
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Carbocyanine dyes have a long-standing tradition in fluorescence imaging and spectroscopy, due to their photostability and large spectral separation between individual dye species. Herein, we explore the versatility of cyanine dyes for probing the dynamics of nucleic acids and we report on the interrelation of fluorophores, RNA, and metal ions, namely K+ and Mg2+. Photophysical parameters including the fluorescence lifetime, quantum yield and dynamic anisotropy are monitored as a function of the nucleic acid composition, conformation, and metal ion abundance. Occasional excursions to a non-fluorescent cis-state hint at the remarkable sensitivity of carbocyanines to their local environment. Comparison of time-correlated single photon experiments with all-atom simulations demonstrate that the propensity of photoisomerization is dictated by sterical constraints imposed on the fluorophore. Structural features in vicinity of the dye play a crucial role in RNA recognition and have far-reaching implications on the mobility of the fluorescent probe. An atomic level description of the mutual interactions will ultimately benefit the quantitative interpretation of single-molecule FRET measurements on large RNA systems.
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The achievable time resolution of camera-based single-molecule detection is often limited by the frame rate of the camera. Especially in experiments utilizing single-molecule Förster resonance energy transfer (smFRET) to probe conformational dynamics of biomolecules, increasing the frame rate by either pixel-binning or cropping the field of view decreases the number of molecules that can be monitored simultaneously. Here, we present a generalised excitation scheme termed stroboscopic alternating-laser excitation (sALEX) that significantly improves the time resolution without sacrificing highly parallelised detection in total internal reflection fluorescence (TIRF) microscopy. In addition, we adapt a technique known from diffusion-based confocal microscopy to analyse the complex shape of FRET efficiency histograms. We apply both sALEX and dynamic probability distribution analysis (dPDA) to resolve conformational dynamics of interconverting DNA hairpins in the millisecond time range.
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In vitro studies on macromolecules, like proteins and nucleic acids, are mostly carried out in highly diluted systems where the molecules are studied under artificial conditions. These experimental conditions are optimized for both the system under investigation and the technique used. However, these conditions often do not reflect the in vivo situation and are therefore inappropriate for a reliable prediction of the native behavior of the molecules and their interactions under in vivo conditions. The intracellular environment is packed with cosolutes (macromolecules, metabolites, etc.) that create 'macromolecular crowding'. The addition of natural or synthetic macromolecules to the sample solution enables crowding to be mimicked. In this surrounding most of the studied biomolecules show a more compact structure, an increased activity, and a decrease of salt requirement for structure formation and function. Herein, we refer to a collection of examples for proteins and nucleic acids and their interactions in crowding environments and present in detail the effect of cosolutes on RNA folding and activity using a group II intron ribozyme as an example.
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Methods based on single-molecule localization and photophysics have brought nanoscale imaging with visible light into reach. This has enabled single-particle tracking applications for studying the dynamics of molecules and nanoparticles and contributed to the recent revolution in super-resolution localization microscopy techniques. Crucial to the optimization of such methods are the precision and accuracy with which single fluorophores and nanoparticles can be localized. We present a lucid synthesis of the developments on this localization precision and accuracy and their practical implications in order to guide the increasing number of researchers using single-particle tracking and super-resolution localization microscopy.
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A crucial step of the self-splicing reaction of group II intron ribozymes is the recognition of the 5' exon by the intron. This recognition is achieved by two regions in domain 1 of the intron, the exon-binding sites EBS1 and EBS2 forming base pairs with the intron-binding sites IBS1 and IBS2 located at the end of the 5' exon. The complementarity of the EBS1•IBS1 contact is most important for ensuring site-specific cleavage of the phosphodiester bond between the 5' exon and the intron. Here, we present the NMR solution structures of the d3' hairpin including EBS1 free in solution and bound to the IBS1 7-mer. In the unbound state, EBS1 is part of a flexible 11-nucleotide (nt) loop. Binding of IBS1 restructures and freezes the entire loop region. Mg(2+) ions are bound near the termini of the EBS1•IBS1 helix, stabilizing the interaction. Formation of the 7-bp EBS1•IBS1 helix within a loop of only 11 nt forces the loop backbone to form a sharp turn opposite of the splice site, thereby presenting the scissile phosphate in a position that is structurally unique.
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We report the observation of single-molecule colocalization and quantitative fluorescence resonant energy transfer by simultaneously imaging the emission and polarization characteristics of two colocalized fluorophores using a simple optical design. The methodology was tested using the ligand-receptor system streptavidin, fluorescence labeled with the dye Cy5, and biotin labeled with tetramethylrhodamine. Discrimination of the two dyes permitted the observation of single-pair fluorescence resonant energy transfer with an efficiency of 89%. The multidimensional character of our fluorescence microscopy combined with the robustness of our design provides a simple method suitable to study biomolecular interactions on the single molecule level. © 2000 American Institute of Physics.
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Single-molecule fluorescence microscopy experiments on RNA molecules brought to light the highly complex dynamics of key biological processes, including RNA folding, catalysis of ribozymes, ligand sensing of riboswitches and aptamers, and protein synthesis in the ribosome. By using highly advanced biophysical spectroscopy techniques in combination with sophisticated biochemical synthesis approaches, molecular dynamics of individual RNA molecules can be observed in real time and under physiological conditions in unprecedented detail that cannot be achieved with bulk experiments. Here, we review recent advances in RNA folding and functional studies of RNA and RNA-protein complexes addressed by using single-molecule Förster (fluorescence) resonance energy transfer (smFRET) technique.
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Highlights ► Mg2+ is a uniquely suited partner for RNA due to its ability to localize water molecules and pack RNA phosphate groups. ► Current and past work supports the ubiquity of Mg2+, both diffuse and coordinated, in RNA folding and catalysis. ► Review of recent literature reveals inaccuracy in the treatment of RNA-divalent cation interactions in theoretical models. ► A general conceptual framework of cation-RNA interactions is presented, which links thermodynamics and structure. ► Examples of new applications of previous methods and new methods for studying RNA-cation relationships are described.
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Ribozymes, which carry out phosphoryl-transfer reactions, often require Mg 2+ ions for catalytic activity. The correct folding of the active site and ribozyme tertiary structure is also regulated by metal ions in a manner that is not fully understood. Here we employ coarse-grained molecular simulations to show that individual structural elements of the group I ribozyme from the bacterium Azoarcus form spontaneously in the unfolded ribozyme even at very low Mg 2+ concentrations, and are transiently stabilized by the coordination of Mg 2+ ions to specific nucleotides. However, competition for scarce Mg 2+ and topological constraints that arise from chain connectivity prevent the complete folding of the ribozyme. A much higher Mg 2+ concentration is required for complete folding of the ribozyme and stabilization of the active site. When Mg 2+ is replaced by Ca 2+ the ribozyme folds, but the active site remains unstable. Our results suggest that group I ribozymes utilize the same interactions with specific metal ligands for both structural stability and chemical activity.
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1. Introduction 2. Estimation 3. Hypothesis testing 4. Graphical exploration of data 5. Correlation and regression 6. Multiple regression and correlation 7. Design and power analysis 8. Comparing groups or treatments - analysis of variance 9. Multifactor analysis of variance 10. Randomized blocks and simple repeated measures: unreplicated two-factor designs 11. Split plot and repeated measures designs: partly nested anovas 12. Analysis of covariance 13. Generalized linear models and logistic regression 14. Analyzing frequencies 15. Introduction to multivariate analyses 16. Multivariate analysis of variance and discriminant analysis 17. Principal components and correspondence analysis 18. Multidimensional scaling and cluster analysis 19. Presentation of results.
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We describe a set of image processing algorithms for extracting quantitative data from digitized video microscope images of colloidal suspensions. In a typical application, these direct imaging techniques can locate submicrometer spheres to within 10 nn in the focal plane and 150 nn in depth. Combining information from a sequence of video images into single-particle trajectories makes possible measurements of quantities of fundamental and practical interest such as diffusion coefficients and pair-wise interaction potentials, The measurements we describe in detail combine the outstanding resolution of digital imaging with video-synchronized optical trapping to obtain highly accurate and reproducible results very rapidly. (C) 1996 Academic Press,Inc.
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Riboswitches represent a family of highly structured regulatory elements found primarily in the leader sequences of bacterial mRNAs. They function as molecular switches capable of altering gene expression via conformational changes in a regulatory domain that occur as a result of ligand binding in a receptor domain. Numerous studies have investigated the ligand binding process, but little is known about the associated structural changes in the regulatory domain. A mechanistic description of both processes is essential for deeply understanding how riboswitches modulate gene expression. This task is greatly facilitated by studying all aspects of riboswitch structure/dynamics/function in the same model system. To this end, single-molecule fluorescence resonance energy transfer (smFRET) techniques have been used to directly observe the conformational dynamics of a biologically functional, hydroxocobalamin (HyCbl) binding riboswitch (env8HyCbl) with a known crystallographic structure(1). The present single-molecule study reveals that the undocking rate constant associated with the disruption of a long-range kissing-loop (KL) interaction (L5-L13) is substantially decreased when the nearly 1400 Da ligand is bound to the RNA, resulting in a preferential stabilization of the docked conformation. Notably, the formation of this tertiary KL interaction sequesters the ribosome binding site via base-pairing, preventing translation initiation. Quantitatively, these results reveal that the conformational dynamics of this regulatory switch are well described by a four-state model whereby ligand binding facilitates formation of the KL interaction. Lastly, the results of cell-based gene expression experiments conducted in E.coli are strongly correlated with the biophysical results, suggesting that formation of the L5-L13 regulatory switch directly modulates gene expression.
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Specific tertiary structural motifs determine the complete architecture of RNA molecules (see picture for examples). Within the last few years a number of high-resolution crystal structures of complex RNAs have led to new insights into the mechanisms by which these complex folds are attained. In this review the structures of these tertiary motifs and how they influence the folding pathway of biological RNAs are discussed, as well as new developments in modeling RNA structure based upon these findings.
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Super-resolution localization microscopy methods provide powerful new capabilities for probing biology at the nanometer scale via fluorescence. These methods rely on two key innovations: switchable fluorophores (which blink on and off and can be sequentially imaged) and powerful localization algorithms (which estimate the positions of the fluorophores in the images). These techniques have spurred a flurry of innovation in algorithm development over the last several years. In this Review, we survey the fundamental issues for single-fluorophore fitting routines, localization algorithms based on principles other than fitting, three-dimensional imaging, dipole imaging and techniques for estimating fluorophore positions from images of multiple activated fluorophores. We offer practical advice for users and adopters of algorithms, and we identify areas for further development.
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Blinking dynamics of single CdSe/ZnS quantum dots are analyzed by change point analysis, which gives access to intermediate photoluminescence (PL) intensities observed during PL intermittency. The on-times show systematic deviations from a (truncated) power law. This deviation is manifested in variations of the PL intensity distribution and is related with well defined PL intensity jumps. Varying the matrix from polystyrene (PS) to polyvinyl alcohol (PVA) changes the on-time blinking dynamics and reveals coupling of the QDs either to OH-groups of the SiOx interface or to OH-groups of PVA. Analysis of dwell times in respective intensity correlated traps reveals that OH-related traps are strongly stabilized with much longer dwell times as compared to otherwise broadly distributed trap states.
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In this work, the kinetics of short, fully complementary oligonucleotides are investigated at the single-molecule level. Constructs 6-9 bp in length exhibit single exponential kinetics over 2 orders of magnitude time for both forward (kon, association) and reverse (koff, dissociation) processes. Bimolecular rate constants for association are weakly sensitive to the number of basepairs in the duplex, with a 2.5-fold increase between 9 bp (k'on = 2.1(1) × 10(6) M(-1) s(-1)) and 6 bp (k'on = 5.0(1) × 10(6) M(-1) s(-1)) sequences. In sharp contrast, however, dissociation rate constants prove to be exponentially sensitive to sequence length, varying by nearly 600-fold over the same 9 bp (koff = 0.024 s(-1)) to 6 bp (koff = 14 s(-1)) range. The 8 bp sequence is explored in more detail, and the NaCl dependence of kon and koff is measured. Interestingly, konincreases by >40-fold (kon = 0.10(1) s(-1) to 4.0(4) s(-1) between [NaCl] = 25 mM and 1 M), whereas in contrast, koffdecreases by fourfold (0.72(3) s(-1) to 0.17(7) s(-1)) over the same range of conditions. Thus, the equilibrium constant (Keq) increases by ≈160, largely due to changes in the association rate, kon. Finally, temperature-dependent measurements reveal that increased [NaCl] reduces the overall exothermicity (ΔΔH° > 0) of duplex formation, albeit by an amount smaller than the reduction in entropic penalty (-TΔΔS° < 0). This reduced entropic cost is attributed to a cation-facilitated preordering of the two single-stranded species, which lowers the association free-energy barrier and in turn accelerates the rate of duplex formation.
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We present the theoretical rationales for data analysis protocols that afford an efficient extraction of conformational dynamics on a broad range of time scales from single-molecule fluorescence lifetime trajectories. Based on correlation analyses, a photon-by-photon approach on one hand provides the highest time resolution, whereas a minimal-binning method on the other hand is most suitable for experiments experiencing external fluorescence intensity variations. Applications of the two methods are illustrated via computer simulations. In cases where fluorescence quenching is either due to Förster fluorescence resonance energy transfer or due to the excited-state electron transfer, the fluorescence lifetime is dependent on donor-acceptor distance, thereby providing a window through which conformational dynamics are revealed. To assist in interpreting experimental data derived from the new protocols, analytical expressions relating fluorescence lifetime fluctuation correlations to a Brownian diffusion model and to an anomalous diffusion model are discussed.
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Aus eins mach zwei: Der partielle Austausch von Mg2+ gegen Ca2+ fuhrt bei der Faltung des D135-Ribozyms, das auf dem Gruppe-II-Intron Sc.ai5γ basiert, zu einer Aufteilung der RNA-Molekule in zwei deutlich verschiedene Subpopulationen, die sich nicht in einem dynamischen Gleichgewicht befinden. Im Bild ist die Trennung in die beiden Subpopulationen zusammen mit den Einzelmolekul-FRET-Werten gezeigt (smFRET: Einzelmolekul-Forster-Resonanzenergietransfer).
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Single molecule methods have revealed that heterogeneity is common in biological systems. However, interpretations of the complex signals are challenging. By tracking the fluorescence resonance energy transfer (FRET) signals between the A-site tRNA and L27 protein in single ribosomes, we attempt to develop a qualitative method to subtract the inherent patterns of the heterogeneous single molecule FRET data. Seven ribosome subpopulations are identified using this method and spontaneous exchanges among these subpopulations are observed. All of the pre-translocation subpopulations are competent in real-time translocation, but via distinguished pathways. These observations suggest that the ribosome may function through multiple reaction pathways. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.
Article
We present evidence that the photoluminescence intermittency of CdSe∕ZnS core/shell quantum dots is correlated with the dielectric environment surrounding the quantum dots. The statistics of dark state lifetimes in the intermittency is found to be related to the stabilization energy of charges in the local dielectric surrounding of the quantum dot. This supports the model of an ionized quantum dot in the dark state. Charges ejected from the quantum dot are suggested to be self-trapped in mid-bandgap states of the surrounding matrix due to atomic and electronic relaxation processes. These trap states are inherent to disordered materials and proposed to be a general source of power law intermittency of various types of emitters such as quantum dots and dye molecules.
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The kinetics of triplex folding/unfolding is investigated by the single-molecule fluorescence resonance energy transfer (FRET) technique. In neutral pH conditions, the average dwell times in both high-FRET (folded) and low-FRET (unfolded) states are comparable, meaning that the triplex is marginally stable. The dwell-time distributions are qualitatively different: while the dwell-time distribution of the high-FRET state should be fit with at least a double-exponential function, the dwell-time distribution of the low-FRET state can be fit with a single-exponential function. We propose a model where the folding can be trapped in metastable states, which is consistent with the FRET data. Our model also accounts for the fact that the relevant timescales of triplex folding/unfolding are macroscopic.
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Multinuclear and multidimensional nuclear magnetic resonance (NMR) spectroscopy is applied in our groups to gain insights into the role of metal ions for the function and structure of large biomolecules. Specifically, NMR is used i) to investigate how metal ions bind to nucleic acids and thereby control the folding and structure of RNAs, ii) to characterize how metal ions are able to stabilize modified nucleic acids to be used as potential nanowires, and iii) to characterize the formation, structure, and role of the diverse metal clusters within plant metallothioneins. In this review we summarize the various NMR experiments applied and the information obtained, demonstrating the important and fascinating part NMR spectroscopy plays in the field of bioinorganic chemistry.
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A plausible consequence of the rugged folding energy landscapes inherent to biomolecules is that there may be more than one functionally competent folded state. Indeed, molecule-to-molecule variations in the folding dynamics of enzymes and ribozymes have recently been identified in single-molecule experiments, but without systematic quantification or an understanding of their structural origin. Here, using concepts from glass physics and complementary clustering analysis, we provide a quantitative method to analyse single-molecule fluorescence resonance energy transfer (smFRET) data, thereby probing the isomerization dynamics of Holliday junctions, which display such heterogeneous dynamics over a long observation time (T(obs) ≈ 40 s). We show that the ergodicity of Holliday junction dynamics is effectively broken and that their conformational space is partitioned into a folding network of kinetically disconnected clusters. Theory suggests that the persistent heterogeneity of Holliday junction dynamics is a consequence of internal multiloops with varying sizes and flexibilities frozen by Mg(2+) ions. An annealing experiment using Mg(2+) pulses lends support to this idea by explicitly showing that interconversions between trajectories with different patterns can be induced.
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The lysine riboswitch is a cis-acting RNA genetic regulatory element found in the leader sequence of bacterial mRNAs coding for proteins related to biosynthesis or transport of lysine. Structural analysis of the lysine-binding aptamer domain of this RNA has revealed that it completely encapsulates the ligand and therefore must undergo a structural opening/closing upon interaction with lysine. In this work, single-molecule fluorescence resonance energy transfer (FRET) methods are used to monitor these ligand-induced structural transitions that are central to lysine riboswitch function. Specifically, a model FRET system has been developed for characterizing the lysine dissociation constant, as well as the opening/closing rate constants for the Bacillus subtilis lysC aptamer domain. These techniques permit measurement of the dissociation constant (K(D)) for lysine binding of 1.7(5) mM, and opening/closing rate constants of 1.4(3) s(-1) and 0.203(7) s(-1), respectively. These rates predict an apparent dissociation constant for lysine binding (K(D, apparent))of 0.25(9) mM at near physiological ionic strength, which differs markedly from previous reports.
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A new streptocyanine dye has been doped into monolithic silica gels with different porosities, characterized by nitrogen adsorption isotherms. Single-molecule tracking with a wide-field fluorescence microscope is used to determine the diffusivity of the dye in the nanoporous network of the host. The majority of molecules in the gel with wider pores (22 nm) diffuse freely with an average diffusion coefficient of D = 4.7 × 10-9 cm2 s-1. Most of those in the gel with narrower pores (3 nm) are trapped in regions ranging in size from 50 nmthe positioning accuracy of the setupup to 200 nm. Others are alternately trapped and freely diffusing with an average D = 3.5 × 10-10 cm2 s-1. The significance of the distribution of diffusion coefficients measured by single-molecule tracking is discussed. Besides traps, the wide spread of the diffusion coefficients for individual molecules in both gels reveals pronounced microscopic inhomogeneities.
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Human telomeres possess a single-stranded DNA (ssDNA) overhang of TTAGGG repeats, which can self-fold into a G-quadruplex structure. POT1 binds specifically to the telomeric overhang and partners with TPP1 to regulate telomere lengthening and capping, although the mechanism remains elusive. Here, we show that POT1 binds stably to folded telomeric G-quadruplex DNA in a sequential manner, one oligonucleotide/oligosaccharide binding fold at a time. POT1 binds from 3' to 5', thereby unfolding the G-quadruplex in a stepwise manner. In contrast, the POT1-TPP1 complex induces a continuous folding and unfolding of the G-quadruplex. We demonstrate that POT1-TPP1 slides back and forth on telomeric DNA and also on a mutant telomeric DNA to which POT1 cannot bind alone. The sliding motion is specific to POT1-TPP1, as POT1 and ssDNA binding protein gp32 cannot recapitulate this activity. Our results reveal fundamental molecular steps and dynamics involved in telomere structure regulation.
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Förster resonance energy transfer (FRET) is a powerful tool for monitoring molecular distances and interactions at the nanoscale level. The strong dependence of transfer efficiency on probe separation makes FRET perfectly suited for “on/off” experiments. To use FRET to obtain quantitative distances and three-dimensional structures, however, is more challenging. This review summarises recent studies and technological advances that have improved FRET as a quantitative molecular ruler in nucleic acid systems, both at the ensemble and at the single-molecule levels.
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Group II introns are naturally occurring ribozymes in plants, fungi, bacteria, and lower eukaryotes that undergo a fascinating array of reactions. These large molecular machines with a size ranging between 600 and 2500 nucleotides are self-splicing introns also capable of reinserting themselves into RNA or DNA, thus making them mobile genetic elements. The structural information available on group II intron ribozymes is very scarce. So far only one crystal structure and one NMR solution structure of two domains located in the catalytic core are available. For proper folding and function, each intron requires specific concentrations of monovalent and divalent metal ions. Although most of these metal ions are used for charge screening, some are bound to distinct sites as has been shown by hydrolytic cleavage experiments. These specifically bound ions are crucial for tertiary contact formation and catalysis. This review will discuss the different metal ion requirements of self-splicing group II introns, the available structural data and information on the binding location and affinity of metal ions, as well as the methods applied to investigate the metal ion binding properties of these large RNAs. Due to the size of these introns, the richness of local structures, the catalytic versatility and the involvement of metal ions in all of the above mentioned aspects, group II introns are an ideal target to be studied by combined means from the fields of Biochemistry, Molecular Biology, Analytical, and (Bio)Inorganic Chemistry.
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Group II introns are large ribozymes, consisting of six functionally distinct domains that assemble in the presence of Mg2+ to the active structure catalyzing a variety of reactions. The first step of intron splicing is well characterized by a Michaelis–Menten-type cleavage reaction using a two-piece group II intron: the substrate RNA, the 5′-exon covalently linked to domains 1, 2, and 3, is cleaved upon addition of domain 5 acting as a catalyst. Here we investigate the effect of Ca2+, Mn2+, Ni2+, Zn2+, Cd2+, Pb2+, and [Co(NH3)6]3+ on the first step of splicing of the Saccharomyces cerevisiae mitochondrial group II intron Sc.ai5γ. We find that this group II intron is very sensitive to the presence of divalent metal ions other than Mg2+. For example, the presence of only 5% Ca2+ relative to Mg2+ results in a decrease in the maximal turnover rate k cat by 50%. Ca2+ thereby has a twofold effect: this metal ion interferes initially with folding, but then also competes directly with Mg2+ in the folded state, the latter being indicative of at least one specific Ca2+ binding pocket interfering directly with catalysis. Similar results are obtained with Mn2+, Cd2+, and [Co(NH3)6]3+. Ni2+ is a much more powerful inhibitor and the presence of either Zn2+ or Pb2+ leads to rapid degradation of the RNA. These results show a surprising sensitivity of such a large multidomain RNA on trace amounts of cations other than Mg2+ and raises the question of biological relevance at least in the case of Ca2+.
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The fluorescence intermittency of various dye molecules in different environments was studied by wide-field fluorescence microscopy. The present work focuses on the analysis of long dark periods that are not due to triplet states. It is shown that the distributions of the length of dark periods follow power laws for all systems studied here. Furthermore, blinking kinetics is strongly influenced by the gas atmosphere to which the molecules are exposed. The presence of oxygen is of crucial importance.
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Sequence recognition through base-pairing is essential for DNA repair and gene regulation, but the basic rules governing this process remain elusive. In particular, the kinetics of annealing between two imperfectly matched strands is not well characterized, despite its potential importance in nucleic acid-based biotechnologies and gene silencing. Here we use single-molecule fluorescence to visualize the multiple annealing and melting reactions of two untethered strands inside a porous vesicle, allowing us to precisely quantify the annealing and melting rates. The data as a function of mismatch position suggest that seven contiguous base pairs are needed for rapid annealing of DNA and RNA. This phenomenological rule of seven may underlie the requirement for seven nucleotides of complementarity to seed gene silencing by small noncoding RNA and may help guide performance improvement in DNA- and RNA-based bio- and nanotechnologies, in which off-target effects can be detrimental.
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Transfer messenger RNA (tmRNA) and small binding protein B (SmpB) are the main components of the trans-translation rescue machinery that releases stalled ribosomes from defective mRNAs. Little is known about how SmpB binding affects the conformation of the tRNA-like domain (TLD) of tmRNA. It has been previously hypothesized that the absence of a D stem in the TLD provides flexibility in the elbow region of tmRNA, which can be stabilized by its interaction with SmpB. Here, we have used Förster resonance energy transfer to characterize the global structure of the tRNA-like domain of tmRNA in the presence and absence of SmpB and as a function of Mg(2+) concentration. Our results show tight and specific binding of SmpB to tmRNA. Surprisingly, our data show that the global conformation and flexibility of tmRNA do not change upon SmpB binding. However, Mg(2+) ions induce an 11 Å compaction in the tmRNA structure, suggesting that the flexibility in the H2a stem may allow different conformations of tmRNA as the TLD and mRNA-like domain need to be positioned differently while moving through the ribosome.
Article
It is undisputable that the fates of metal ions and nucleic acids are inescapably interwoven. Metal ions are essential for charge compensation of the negatively charged phosphate–sugar backbone, they are instrumental for proper folding, and last but not least they are crucial cofactors for ribozyme catalysis. Considerable progress has been achieved in the past few years on the identification of metal ion binding sites in large DNA and RNA molecules, like in ribozymes including the ribosome. Hereby, most information was gained from crystallography, which fails to explain metal ion binding equilibria in solution as well as the factors that determine the coordination of a metal ion to a specific site. In contrast, such information is readily available for the low-molecular building blocks of large nucleic acids, i.e. for mononucleotides and to some extent also dinucleotides. In this review, we combine and compare for the first time both sets of information. The focus is thereby set on Mg2+, Ca2+, Mn2+, and Cd2+ because these four metal ions are either freely available in cells, have a large impact on the catalytic rate of ribozymes, and/or are often applied in RNA biochemistry. Our comparisons show that results obtained from small molecules can be directly transposed to the findings in large RNA structures like the ribosome. For example, the basic coordination-chemical properties of the different metal ions are reflected in their binding to large nucleic acid structures: macrochelate formation, e.g. the simultaneous intranucleotide coordination of a Mg2+ ion to the phosphate unit and the N7 site of a purine nucleobase (be it inner- or outersphere), is well known for mononucleotides. We show that the frequency of occurrence of this type of coordination is the same for mononucleotides and the ribosome.
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