Encoding multiple virtual signals in DNA barcodes with single-molecule FRET

ABSTRACTDNA barcoding provides a way to label a huge number of different biological molecules using the extreme programmability in DNA sequence synthesis. Fluorescence imaging is an easy-to-access method to detect individual DNA barcodes, which can be scaled up to a massively high-throughput format. Large overlaps between emission spectra of fluorescence dyes, however, severely limit the numbers of DNA barcodes–and thus its signal space–that can be detected in a simultaneous manner. We here demonstrate the use of single-molecule fluorescence resonance energy transfer (FRET) to encode virtual signals in DNA barcodes using conventional two-color fluorescence microscopy. By optimizing imaging and biochemistry conditions for weak hybridization events for DNA barcodes, we markedly enhanced accuracy in our determination of the efficiency by which single-molecule FRET occurred. This allowed us to unambiguously differentiate six DNA barcodes exhibiting different FRET values without involving probe sequence exchanges. Our method can be directly incorporated with previous DNA barcode techniques, and may thus be widely adopted to expand the signal space of the DNA barcode techniques.

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Single-molecule FRET for Ultrasensitive Detection of Biomolecules

NanoBioImaging ◽

10.2478/nbi-2013-0002 ◽

2014 ◽

Vol 1 (1) ◽

Cited By ~ 5

Author(s):

Kun Yang ◽

Yong Yang ◽

Chun-yang Zhang

Keyword(s):

Single Molecule ◽

New Technologies ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Molecular Probes ◽

Ultrasensitive Detection ◽

Single Molecule Fret ◽

Biological Reaction ◽

Spatio Temporal ◽

Detection Of Biomolecules

AbstractSingle-molecule Förster resonance energy transfer (sm- FRET) has been widely employed to detect biomarkers and to probe the structure and dynamics of biomolecules. By monitoring the biological reaction in a spatio-temporal manner, smFRET can reveal the transient intermediates of biological processes that cannot be obtained by conventional ensemble measurements. This review provides an overview of singlemolecule FRET and its applications in ultrasensitive detection of biomolecules, including the major techniques and the molecular probes used for smFRET as well as the biomedical applications of smFRET. Especially, the combination of sm- FRET with new technologies might expand its applications in clinical diagnosis and biomedical research

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Single-molecule FRET monitors CLC transporter conformation and subunit independence

10.1101/2020.09.07.286831 ◽

2020 ◽

Author(s):

Ricky C. Cheng ◽

Ayush Krishnamoorti ◽

Vladimir Berka ◽

Ryan J Durham ◽

Vasanthi Jayaraman ◽

...

Keyword(s):

Conformational Change ◽

Single Molecule ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Chloride Ions ◽

Conformational State ◽

Triple Mutant ◽

Transport Pathways ◽

Single Molecule Fret ◽

Extracellular Side

Abstract“CLC” transporters catalyze the exchange of chloride ions for protons across cellular membranes. As secondary active transporters, CLCs must alternately allow ion access to and from the extracellular and intracellular sides of the membrane, adopting outward-facing and inward-facing conformational states. Here, we use single-molecule Förster resonance energy transfer (smFRET) to monitor the conformational state of CLC-ec1, an E. coli homolog for which high-resolution structures of occluded and outward-facing states are known. Since each subunit within the CLC homodimer contains its own transport pathways for chloride and protons, we developed a labeling strategy to follow conformational change within a subunit, without crosstalk from the second subunit of the dimer. Using this strategy, we evaluated smFRET efficiencies for labels positioned on the extracellular side of the protein, to monitor the status of the outer permeation pathway. When [H+] is increased to enrich the outward-facing state, the smFRET efficiencies for this pair decrease. In a triple-mutant CLC-ec1 that mimics the protonated state of the protein and is known to favor the outward-facing conformation, the lower smFRET efficiency is observed at both low and high [H+]. These results confirm that the smFRET assay is following the transition to the outward-facing state and demonstrate the feasibility of using smFRET to monitor the relatively small (~1 Å) motions involved in CLC transporter conformational change. Using the smFRET assay, we show that the conformation of the partner subunit does not influence the conformation of the subunit being monitored by smFRET, thus providing evidence for the independence of the two subunits in the transport process.SUMMARYCheng, Krishnamoorti et al. use single-molecule Förster energy resonance transfer measurements to monitor the conformation of a CLC transporter and to show that the conformational state is not influenced by the neighboring subunit.

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Accurate Determination of Human CPR Conformational Equilibrium by smFRET using Dual Orthogonal Non-Canonical Amino Acid Labeling

10.1101/407684 ◽

2018 ◽

Author(s):

Robert B. Quast ◽

Fataneh Fatemi ◽

Michel Kranendonk ◽

Emmanuel Margeat ◽

Gilles Truan

Keyword(s):

Single Molecule ◽

Conformational Equilibrium ◽

Fluorescent Dyes ◽

Photophysical Properties ◽

Accurate Determination ◽

Conformational Dynamics ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

General Strategy

ABSTRACTConjugation of fluorescent dyes to proteins - a prerequisite for the study of conformational dynamics by single molecule Förster resonance energy transfer (smFRET) - can lead to substantial changes of the dye’s photophysical properties, ultimately biasing the quantitative determination of inter-dye distances. In particular the popular cyanine dyes and their derivatives, which are by far the most used dyes in smFRET experiments, exhibit such behavior. To overcome this, a general strategy to site-specifically equip proteins with FRET pairs by chemo-selective reactions using two distinct non-canonical amino acids simultaneously incorporated through genetic code expansion in Escherichia coli was developed. Applied to human NADPH- cytochrome P450 reductase (CPR), the importance of homogenously labeled samples for accurate determination of FRET efficiencies was demonstrated. Furthermore, the effect of NADP+ on the ionic strength dependent modulation of the conformational equilibrium of CPR was unveiled. Given its generality and accuracy, the presented methodology establishes a new benchmark to decipher complex molecular dynamics on single molecules.

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Extreme mechanical diversity of human telomeric DNA revealed by fluorescence-force spectroscopy

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1815162116 ◽

2019 ◽

Vol 116 (17) ◽

pp. 8350-8359 ◽

Cited By ~ 19

Author(s):

Jaba Mitra ◽

Monika A. Makurath ◽

Thuy T. M. Ngo ◽

Alice Troitskaia ◽

Yann R. Chemla ◽

...

Keyword(s):

Optical Tweezers ◽

Single Molecule ◽

Conformational Changes ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Force Spectroscopy ◽

Telomeric Dna ◽

Telomeric Sequences ◽

Substitution Mutant

G-quadruplexes (GQs) can adopt diverse structures and are functionally implicated in transcription, replication, translation, and maintenance of telomere. Their conformational diversity under physiological levels of mechanical stress, however, is poorly understood. We used single-molecule fluorescence-force spectroscopy that combines fluorescence resonance energy transfer with optical tweezers to measure human telomeric sequences under tension. Abrupt GQ unfolding with K+in solution occurred at as many as four discrete levels of force. Added to an ultrastable state and a gradually unfolding state, there were six mechanically distinct structures. Extreme mechanical diversity was also observed with Na+, although GQs were mechanically weaker. Our ability to detect small conformational changes at low forces enabled the determination of refolding forces of about 2 pN. Refolding was rapid and stochastically redistributed molecules to mechanically distinct states. A single guanine-to-thymine substitution mutant required much higher ion concentrations to display GQ-like unfolding and refolded via intermediates, contrary to the wild type. Contradicting an earlier proposal, truncation to three hexanucleotide repeats resulted in a single-stranded DNA-like mechanical behavior under all conditions, indicating that at least four repeats are required to form mechanically stable structures.

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Quantitative single molecule FRET efficiencies using TIRF microscopy

Faraday Discussions ◽

10.1039/c5fd00100e ◽

2015 ◽

Vol 184 ◽

pp. 131-142 ◽

Cited By ~ 8

Author(s):

Lasse L. Hildebrandt ◽

Søren Preus ◽

Victoria Birkedal

Keyword(s):

Single Molecule ◽

Structural Information ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Molecular Complexes ◽

Distance Information ◽

Single Molecule Level ◽

Single Molecule Fret ◽

Wide Range ◽

Intermolecular Distances

Förster resonance energy transfer (FRET) microscopy at the single molecule level has the potential to yield information on intra and intermolecular distances within the 2–10 nm range of molecules or molecular complexes that undergo frequent conformation changes. A pre-requirement for obtaining accurate distance information is to determine quantitative instrument independent FRET efficiency values. Here, we applied and evaluated a procedure to determine quantitative FRET efficiencies directly from individual fluorescence time traces of surface immobilized DNA molecules without the need for external calibrants. To probe the robustness of the approach over a wide range of FRET efficiencies we used a set of doubly labelled double stranded DNA samples, where the acceptor position was varied systematically. Interestingly, we found that fluorescence contributions arising from direct acceptor excitation following donor excitation are intrinsically taken into account in these conditions as other correction factors can compensate for inaccurate values of these parameters. We give here guidelines, that can be used through tools within the iSMS software (http://www.isms.au.dk), for determining quantitative FRET and assess uncertainties linked with the procedure. Our results provide insights into the experimental parameters governing quantitative FRET determination, which is essential for obtaining accurate structural information from a wide range of biomolecules.

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Single molecule fluorescence resonance energy transfer scanning near-field optical microscopy: potentials and challenges

Faraday Discussions ◽

10.1039/c5fd00097a ◽

2015 ◽

Vol 184 ◽

pp. 51-69 ◽

Cited By ~ 6

Author(s):

S. K. Sekatskii ◽

K. Dukenbayev ◽

M. Mensi ◽

A. G. Mikhaylov ◽

E. Rostova ◽

...

Keyword(s):

Energy Transfer ◽

Fluorescence Resonance Energy Transfer ◽

Single Molecule ◽

Near Field ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Nitrogen Vacancy ◽

Single Molecule Fluorescence ◽

Single Molecule Fret ◽

Fluorescence Resonance

A few years ago, single molecule Fluorescence Resonance Energy Transfer Scanning Near-Field Optical Microscope (FRET SNOM) images were demonstrated using CdSe semiconductor nanocrystal–dye molecules as donor–acceptor pairs. Corresponding experiments reveal the necessity to exploit much more photostable fluorescent centers for such an imaging technique to become a practically used tool. Here we report the results of our experiments attempting to use nitrogen vacancy (NV) color centers in nanodiamond (ND) crystals, which are claimed to be extremely photostable, for FRET SNOM. All attempts were unsuccessful, and as a plausible explanation we propose the absence (instability) of NV centers lying close enough to the ND border. We also report improvements in SNOM construction that are necessary for single molecule FRET SNOM imaging. In particular, we present the first topographical images of single strand DNA molecules obtained with fiber-based SNOM. The prospects of using rare earth ions in crystals, which are known to be extremely photostable, for single molecule FRET SNOM at room temperature and quantum informatics at liquid helium temperatures, where FRET is a coherent process, are also discussed.

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Sub-millisecond conformational transitions of short single-stranded DNA lattices by photon correlation single-molecule FRET

10.1101/2021.04.09.439239 ◽

2021 ◽

Author(s):

Brett Israels ◽

Claire S. Albrecht ◽

Anson Dang ◽

Megan Barney ◽

Peter H. von Hippel ◽

...

Keyword(s):

Single Molecule ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Time Correlation ◽

Distribution Functions ◽

Ssdna Binding ◽

Single Molecule Fret ◽

Thermally Driven ◽

Ssb Proteins ◽

Minority Species

Thermally-driven conformational fluctuations (or 'breathing') of DNA plays important roles in the function and regulation of the 'macromolecular machinery of genome expression.' Fluctuations in double-stranded (ds) DNA are involved in the transient exposure of pathways to protein binding sites within the DNA framework, leading to the binding of functional and regulatory proteins to single-stranded (ss) DNA templates. These interactions often require that the ssDNA sequences, as well as the proteins involved, assume transient conformations critical for successful binding. Here we use microsecond-resolved single-molecule F&oumlrster Resonance Energy Transfer (smFRET) experiments to investigate the backbone fluctuations of short (ss) oligo- oligo(dT)n templates within DNA constructs that can also serve as models for ss-dsDNA junctions. Such junctions, as well as the attached ssDNA sequences, are involved in the binding of ssDNA binding (ssb) proteins that control and integrate the mechanisms of DNA replication complexes. We have used these data to determine multi-order time-correlation functions (TCFs) and probability distribution functions (PDFs) that characterize the kinetic and thermodynamic behavior of the system. We find that the oligo(dT)n tails of ss-dsDNA constructs inter-convert, on sub-millisecond time-scales, between three macrostates with distinctly different end-to-end distances. These are: (i) a 'compact' macrostate that represents the dominant species at equilibrium; (ii) a 'partially extended' macrostate that exists as a minority species; and (iii) a 'highly extended' macrostate that is present in trace amounts. We propose a model for ssDNA secondary structure that advances our understanding of how spontaneously formed nucleic acid conformations may facilitate the activities of ssDNA associating proteins.

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DeepFRET: Rapid and automated single molecule FRET data classification using deep learning

10.1101/2020.06.26.173260 ◽

2020 ◽

Cited By ~ 1

Author(s):

Johannes Thomsen ◽

Magnus B. Sletfjerding ◽

Stefano Stella ◽

Bijoya Paul ◽

Simon Bo Jensen ◽

...

Keyword(s):

Deep Learning ◽

Structural Biology ◽

Single Molecule ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Ground Truth ◽

Real Data ◽

Ground Truth Data ◽

Single Molecule Fret ◽

Human Operators

AbstractSingle molecule Förster Resonance energy transfer (smFRET) is a mature and adaptable method for studying the structure of biomolecules and integrating their dynamics into structural biology. The development of high throughput methodologies and the growth of commercial instrumentation have outpaced the development of rapid, standardized, and fully automated methodologies to objectively analyze the wealth of produced data. Here we present DeepFRET, an automated standalone solution based on deep learning, where the only crucial human intervention in transiting from raw microscope images to histogram of biomolecule behavior, is a user-adjustable quality threshold. Integrating all standard features of smFRET analysis, DeepFRET will consequently output common kinetic information metrics for biomolecules. We validated the utility of DeepFRET by performing quantitative analysis on simulated, ground truth, data and real smFRET data. The accuracy of classification by DeepFRET outperformed human operators and current commonly used hard threshold and reached >95% precision accuracy only requiring a fraction of the time (<1% as compared to human operators) on ground truth data. Its flawless and rapid operation on real data demonstrates its wide applicability. This level of classification was achieved without any preprocessing or parameter setting by human operators, demonstrating DeepFRET’s capacity to objectively quantify biomolecular dynamics. The provided a standalone executable based on open source code capitalises on the widespread adaptation of machine learning and may contribute to the effort of benchmarking smFRET for structural biology insights.

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Conformational dynamics of Cas9 governing DNA cleavage revealed by single molecule FRET

10.1101/167627 ◽

2017 ◽

Cited By ~ 2

Author(s):

Mengyi Yang ◽

Sijia Peng ◽

Ruirui Sun ◽

Jingdi Lin ◽

Nan Wang ◽

...

Keyword(s):

Single Molecule ◽

Dna Cleavage ◽

Conformational Dynamics ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Nuclease Activity ◽

Single Molecule Fluorescence ◽

Single Molecule Fret ◽

Allosteric Communication ◽

Target Binding

SummaryOff-target binding and cleavage by Cas9 pose as major challenges in its applications. How conformational dynamics of Cas9 governs its nuclease activity under on- and off-target conditions remains largely unknown. Here, using intra-molecular single molecule fluorescence resonance energy transfer measurements, we revealed that Cas9 in apo, sgRNA-bound, and dsDNA/sgRNA-bound forms all spontaneously transits between three major conformational states, mainly reflecting significant conformational mobility of the catalytic HNH domain. We furthermore uncovered a surprising long-range allosteric communication between the HNH domain and RNA/DNA heteroduplex at the PAM-distal end to ensure correct positioning of the catalytic site, which demonstrated a unique proofreading mechanism served as the last checkpoint before DNA cleavage. Several Cas9 residues were likely to mediate the allosteric communication and proofreading step. Modulating interactions between Cas9 and heteroduplex at the distal end by introducing mutations on these sites provides an alternative route to improve and optimize the CRISPR/Cas9 toolbox.

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ABEL-FRET: tether-free single-molecule FRET with hydrodynamic profiling

10.1101/786897 ◽

2019 ◽

Cited By ~ 2

Author(s):

Hugh Wilson ◽

Quan Wang

Keyword(s):

Single Molecule ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Biological Processes ◽

Resolution Limit ◽

Single Molecule Fret ◽

Experimental Protocols ◽

Fret Efficiency ◽

Nanoscale Metrology ◽

Observation Times

ABSTRACTSingle-molecule Förster resonance energy transfer (smFRET) has become a versatile and widespread method to probe nanoscale conformation and dynamics. However, current experimental protocols often resort to molecule immobilization for long observation times and rarely approach the resolution limit of FRET-based nanoscale metrology. Here we present ABEL-FRET, an immobilization-free platform for smFRET measurements with near shot-noise limited, Angstrom-level resolution in FRET efficiency. Furthermore, ABEL-FRET naturally integrates hydrodynamic profiling, which harnesses single-molecule diffusion coefficient to enhance FRET sensing of biological processes.

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