Single-molecule Fluorescence Spectroscopy Research Articles

Single-Molecule Fluorescence Probes Interactions between Photoactive Protein-Silver Nanowire Conjugate and Monolayer Graphene.

In this work, we apply single-molecule fluorescence microscopy and spectroscopy to probe plasmon-enhanced fluorescence and Förster resonance energy transfer in a nanoscale assemblies. The structure where the interplay between these two processes was present consists of photoactive proteins conjugated with silver nanowires and deposited on a monolayer graphene. By comparing the results of continuous-wave and time-resolved fluorescence microscopy acquired for this structure with those obtained for the reference samples, where proteins were coupled with either a graphene monolayer or silver nanowires, we find clear indications of the interplay between plasmonic enhancement and the energy transfer to graphene. Namely, fluorescence intensities calculated for the structure, where proteins were coupled to graphene only, are less than for the structure playing the central role in this study, containing both silver nanowires and graphene. Conversely, decay times extracted for the latter are shorter compared to a protein-silver nanowire conjugate, pointing towards emergence of the energy transfer. Overall, the results show that monitoring the optical properties of single emitters in a precisely designed hybrid nanostructure provides an elegant way to probe even complex combination of interactions at the nanoscale.

DNA controls the dimerization of the human FoxP1 forkhead domain

Transcription factors (TFs) regulate gene expression by binding to specific DNA sequences and gating access to genes. Even when the binding of TFs and their cofactors to DNA is reversible, indicating a reversible control of gene expression, there is little knowledge about the molecular effect DNA has on TFs. Using single-molecule multiparameter fluorescence spectroscopy, molecular dynamics simulations, and biochemical assays, we find that the monomeric form of the forkhead (FKH) domain of the human FoxP1 behaves as a disordered protein and increases its folded population when it dimerizes. Notably, DNA binding promotes a disordered FKH dimer bound to DNA, negatively controlling the stability of the dimeric FoxP1:DNA complex. The DNA-mediated reversible regulation on FKH dimers suggests that FoxP1-dependent gene suppression is unstable, and it must require the presence of other dimerization domains or cofactors to revert the negative impact exerted by the DNA.

Closing the Gap between Experiment and Simulation─A Holistic Study on the Complexation of Small Interfering RNAs with Polyethylenimine.

Rational design is pivotal in the modern development of nucleic acid nanocarrier systems. With the rising prominence of polymeric materials as alternatives to lipid-based carriers, understanding their structure-function relationships becomes paramount. Here, we introduce a newly developed coarse-grained model of polyethylenimine (PEI) based on the Martini 3 force field. This model facilitates molecular dynamics simulations of true-sized PEI molecules, exemplified by molecules with molecular weights of 1.3, 5, 10, and 25 kDa, with degrees of branching between 50.0 and 61.5%. We employed this model to investigate the thermodynamics of small interfering RNA (siRNA) complexation with PEI. Our simulations underscore the pivotal role of electrostatic interactions in the complexation process. Thermodynamic analyses revealed a stronger binding affinity with increased protonation, notably in acidic (endosomal) pH, compared to neutral conditions. Furthermore, the molecular weight of PEI was found to be a critical determinant of binding dynamics: smaller PEI molecules closely enveloped the siRNA, whereas larger ones extended outward, facilitating the formation of complexes with multiple RNA molecules. Experimental validations, encompassing isothermal titration calorimetry and single-molecule fluorescence spectroscopy, aligned well with our computational predictions. Our findings not only validate the fidelity of our PEI model but also accentuate the importance of in silico data in the rational design of polymeric drug carriers. The synergy between computational predictions and experimental validations, as showcased here, signals a refined and precise approach to drug carrier design.

Dual-wavelength metalens enables Epi-fluorescence detection from single molecules

Single molecule fluorescence spectroscopy is at the heart of molecular biophysics research and the most sensitive biosensing assays. The growing demand for precision medicine and environmental monitoring requires the creation of miniaturized and portable sensing platforms. However, the need for highly sophisticated objective lenses has precluded the development of single molecule detection systems for truly portable devices. Here, we propose a dielectric metalens device of submicrometer thickness to excite and collect light from fluorescent molecules instead of an objective lens. The high numerical aperture, high focusing efficiency, and dual-wavelength operation of the metalens enable the implementation of fluorescence correlation spectroscopy with a single Alexa 647 molecule in the focal volume. Moreover, the metalens enables real-time monitoring of individual fluorescent nanoparticle transitions and identification of hydrodynamic diameters ranging from a few to hundreds of nanometers. This advancement in sensitivity extends the application of the metalens technology to ultracompact single-molecule sensors.

Future Paths in Cryogenic Single-Molecule Fluorescence Spectroscopy.

In the last three decades, cryogenic single-molecule fluorescence spectroscopy has provided average-free understanding of the photophysics and of fundamental interactions at molecular scales. Furthermore, they propose original pathways and applications in the treatment and storage of quantum information. The ultranarrow lifetime-limited zero-phonon line acts as an excellent sensor to local perturbations caused either by intrinsic dynamical degrees of freedom, or by external perturbations, such as those caused by electric fields, elastic and acoustic deformations, or light-induced dynamics. Single aromatic hydrocarbon molecules, being sensitive to nanoscale probing at nanometer scales, are potential miniaturized platforms for integrated quantum photonics. In this Perspective, we look back at some of the past advances in cryogenic optical microscopy and propose some perspectives for future development.

De novo fabrication of custom-sequence plasmids for the synthesis of long DNA constructs with extrahelical features

DNA constructs for single-molecule experiments often require specific sequences and/or extrahelical/noncanonical structures to study DNA-processing mechanisms. The precise introduction of such structures requires extensive control of the sequence of the initial DNA substrate. A commonly used substrate in the synthesis of DNA constructs is plasmid DNA. Nevertheless, the controlled introduction of specific sequences and extrahelical/noncanonical structures into plasmids often requires several rounds of cloning on pre-existing plasmids whose sequence one cannot fully control. Here, we describe a simple and efficient way to synthesize 10.1-kb plasmids de novo using synthetic gBlocks that provides full control of the sequence. Using these plasmids, we developed a 1.5-day protocol to assemble 10.1-kb linear DNA constructs with end and internal modifications. As a proof of principle, we synthesize two different DNA constructs with biotinylated ends and one or two internal 3′ single-stranded DNA flaps, characterize them using single-molecule force and fluorescence spectroscopy, and functionally validate them by showing that the eukaryotic replicative helicase Cdc45/Mcm2-7/GINS (CMG) binds the 3′ single-stranded DNA flap and translocates in the expected direction. We anticipate that our approach can be used to synthesize custom-sequence DNA constructs for a variety of force and fluorescence single-molecule spectroscopy experiments to interrogate DNA replication, DNA repair, and transcription.

Correlated Single-Molecule Magnetic Tweezers and Fluorescence Measurements of DNA-Enzyme Interactions.

Combining force spectroscopy and fluorescence microscopy provides a substantial improvement to the single-molecule toolbox by allowing simultaneous manipulation and orthogonal characterizations of the conformations, interactions, and activity of biomolecular complexes. Here, we describe a combined magnetic tweezers and total internal reflection fluorescence microscopy setup to carry out correlated single-molecule fluorescence spectroscopy and force/twisting experiments. We apply the setup to reveal the DNA interactions of the CRISPR-Cas surveillance complex Cascade. Single-molecule fluorescence of a labeled Cascade allows to follow the DNA association and dissociation of the protein. Simultaneously, the magnetic tweezers probe the DNA unwinding during R-loop formation by the bound Cascade complexes. Furthermore, the setup supports observation of 1D diffusion of protein complexes on stretched DNA molecules. This technique can be applied to study a vast range of protein-DNA interactions.

Time-resolved burst variance analysis

Quantifying biomolecular dynamics has become a major task of single-molecule fluorescence spectroscopy methods. In single-molecule Förster resonance energy transfer (smFRET), kinetic information is extracted from the stream of photons emitted by attached donor and acceptor fluorophores. Here, we describe a time-resolved version of burst variance analysis that can quantify kinetic rates at microsecond to millisecond timescales in smFRET experiments of diffusing molecules. Bursts are partitioned into segments with a fixed number of photons. The FRET variance is computed from these segments and compared with the variance expected from shot noise. By systematically varying the segment size, dynamics at different timescales can be captured. We provide a theoretical framework to extract kinetic rates from the decay of the FRET variance with increasing segment size. Compared to other methods such as filtered fluorescence correlation spectroscopy, recurrence analysis of single particles, and two-dimensional lifetime correlation spectroscopy, fewer photons are needed to obtain reliable timescale estimates, which reduces the required measurement time.

Photoisomerization of Heptamethine Cyanine Dyes Results in Red-Emissive Species: Implications for Near-IR, Single-Molecule, and Super-Resolution Fluorescence Spectroscopy and Imaging.

Photoisomerization kinetics of the near-infrared (NIR) fluorophore Sulfo-Cyanine7 (SCy7) was studied by a combination of fluorescence correlation spectroscopy (FCS) and transient state (TRAST) excitation modulation spectroscopy. A photoisomerized state with redshifted emission was identified, with kinetics consistent with a three-state photoisomerization model. Combining TRAST excitation modulation with spectrofluorimetry (spectral-TRAST) further confirmed an excitation-induced redshift in the emission spectrum of SCy7. We show how this red-emissive photoisomerized state contributes to the blinking kinetics in different emission bands of NIR cyanine dyes, and how it can influence single-molecule, super-resolution, as well as Förster resonance energy transfer (FRET) and multicolor readouts. Since this state can also be populated at moderate excitation intensities, it can also more broadly influence fluorescence readouts, also readouts not relying on high excitation conditions. However, this additional red-emissive state and its photodynamics, as identified and characterized in this work, can also be used as a strategy to push the emission of NIR cyanine dyes further into the NIR and to enhance photosensitization of nanoparticles with absorption spectra further into the NIR. Finally, we show that the photoisomerization kinetics of SCy7 and the formation of its redshifted photoisomer depend strongly on local environmental conditions, such as viscosity, polarity, and steric constraints, which suggests the use of SCy7 and other NIR cyanine dyes as environmental sensors. Such environmental information can be monitored by TRAST, in the NIR, with low autofluorescence and scattering conditions and on a broad range of samples and experimental conditions.

Photo-Isomerization of Heptamethine Cyanine Dyes Results in Red Emissive Species – Implications for Near-IR, Single-Molecule and Super-Resolution Fluorescence Spectroscopy and Imaging

Photo-Isomerization of Heptamethine Cyanine Dyes Results in Red Emissive Species – Implications for Near-IR, Single-Molecule and Super-Resolution Fluorescence Spectroscopy and Imaging

Interaction Dynamics of Intrinsically Disordered Proteins from Single-Molecule Spectroscopy.

Many proteins contain large structurally disordered regions or are entirely disordered under physiological conditions. The functions of these intrinsically disordered proteins (IDPs) often involve interactions with other biomolecules. An important emerging effort has thus been to identify the molecular mechanisms of IDP interactions and how they differ from the textbook notions of biomolecular binding for folded proteins. In this review, we summarize how the versatile tool kit of single-molecule fluorescence spectroscopy can aid the investigation of these conformationally heterogeneous and highly dynamic molecular systems. We discuss the experimental observables that can be employed and how they enable IDP complexes to be probed on timescales from nanoseconds to hours. Key insights include the diverse structural and dynamic properties of bound IDPs and the kinetic mechanisms facilitated by disorder, such as fly-casting; disorder-mediated encounter complexes; and competitive substitution via ternary complexes, which enables rapid dissociation even for high-affinity complexes. We also discuss emerging links to aggregation, liquid-liquid phase separation, and cellular processes, as well as current technical advances to further expand the scope of single-molecule spectroscopy.

Step-by-step binding and unwinding of RNA by a Ded1p trimer revealed by combined high-resolution tweezers and fluorescence measurements.

The DEAD-box family of RNA helicases is the largest of all known RNA helicase families, where they remodel RNA by binding and unwinding duplex RNA via an ATP-dependent mechanism. Lacking translocation capability, these helicases are known to unwind only a limited amount, <10–20 bp, locally nearby the site of binding. However, some DEAD-box RNA helicases have recently been shown to unwind efficiently upon oligomerization. The DEAD-box RNA helicase Ded1p has been observed to efficiently unwind RNA duplexes possessing a single-stranded RNA (ssRNA) tail as a trimer. The detailed mechanism of Ded1p trimer assembly and unwinding of RNA remains unknown. We have used combined high-resolution tweezers and single-molecule fluorescence spectroscopy in order to directly observe the detailed step-by-step binding and dissociation of Ded1p protomers and subsequent unwinding and rezipping of duplex RNA. The overall trimer activity previously observed in ensemble experiments was recapitulated. A complete general model of unwinding is developed whereby unwinding is initiated by binding of a single Ded1p to the duplex adjacent ssRNA tail. Subsequently, in sequence two additional Ded1p bind and unwind a 5-7 bp portion of the duplex, each resulting in the full 16 bp duplex unwinding. The reaction is highly dynamic and stochastic, with unwinding combining with frequent rezipping reversals. Rezipping reversals are suppressed when ATP hydrolysis is suppressed via use of an ATP analog. While unwinding and rezipping are easily captured by high-resolution tweezers, Ded1p binding and dissociation on ssRNA tail was measured via the protein-induced-fluorescence-enhancement (PIFE) signal from the fluorophore-labeled tail. The combined methods allowed us to fully observe the coordinated binding and unwinding of the RNA substrate by individual protomers of the Ded1p trimer.

Elucidating the dynamics of individual IDPs in a biomolecular condensate using single-molecule FRET and molecular simulation.

A wide range of biomolecules in solution can phase-separate and form membraneless organelles in the cell. These assemblies often have liquid-like properties, and the corresponding dynamics and exchange of molecules with the environment are important for biological function. The dynamics and materials properties of these systems are commonly assessed based on translational diffusion or rheological properties, typically covering timescales of milliseconds and longer. However, information on the structure and dynamics at the molecular level is lacking. We have studied coacervates of two intrinsically disordered highly oppositely charged nuclear proteins as a prototypical example of heterotypic liquid-liquid phase separation of components abundant in the cell nucleus. Using single-molecule fluorescence spectroscopy, we show that the proteins in the condensates remain disordered, and that their chain dynamics occur on timescale surprisingly close to the ∼100-nanosecond dynamics of the 1:1 complex in dilute solution, even though the condensate is almost three orders of magnitude more viscous than the dilute solution and translational diffusion of the same proteins is ∼50 times slower in the dense than in the dilute phase. The experimental results are in good agreement with large-scale all-atom explicit-solvent molecular dynamics simulations consisting of ∼200 proteins and ∼4 million atoms in total, which can thus provide detailed insights into the protein interactions and dynamics within the condensate. The simulations indicate that the rapid protein chain dynamics in the dense phase are linked to the similarity of the local environment experienced by the proteins in the dilute and dense phases and the short lifetime of intermolecular contacts in the dense phase.

DNA-induces intra- and inter-molecular changes in dimerization of the human FOXP1.

Transcription factors (TFs) are proteins that regulate gene expression by binding to specific DNA sequences and gating access to genes. This paradigm implies a one-directional gene regulation of DNA remodeling via TFs and their cofactors. Here, we present an example in which the DNA modulates the kinetics of dimerization of the forkhead (FKH) domain of the FoxP1 human TF, favoring its monomeric form over its domain-swapped dimer conformation. Hence, challenging the one-dimensional remodeling paradigm. To inquire about the structural dynamics of the FKH domain in its dimeric form and the presence of DNA, we use molecular dynamics simulations, single-molecule multiparameter fluorescence spectroscopy, and biochemical assays. We found that the monomeric form of the FoxP1 FKH domain primarily displays a disordered-like behavior, which is allosterically reverted by dimerization. Interestingly, DNA favors its interaction with the FKH in its monomeric form over its domain-swapped dimer at the cost of decreasing the FKH domain structural stability. Our findings imply that gene expression is tightly regulated by a negative feedback control over suppressor proteins such as FoxP1 by introducing a bidirectional control over gene expression. Moreover, it suggests that FoxP1 gene suppression action in its domain-swapped conformation, which brings together distant DNA segments and locks transcription access, requires the presence of other dimerization domains, cofactors, or the nuclear macromolecular composition to revert the negative feedback exerted by the DNA.

Uncovering the biophysical mechanisms of riboregulatory interactions involving the hepatitis C virus 3'X RNA using single-molecule FRET.

The last 98 nucleotides of the hepatitis C virus genome form what is called the 3ʹX RNA, which is a structured and highly conserved non-coding RNA element that has been implicated in the regulation of several different RNA-dependent viral processes like replication, translation, and particle assembly. Current research suggests that this riboregulatory behavior arises from the ability of 3ʹX to form multiple mutually exclusive interactions with highly conserved RNA elements across the HCV genome. Here, we use time-resolved single-molecule fluorescence spectroscopy in conjunction with analytical size exclusion HPLC to characterize the structural, energetic, and kinetic principles governing these RNA-RNA interactions. In the absence of any interaction partners, we see that 3ʹX adopts two distinct conformations. Furthermore, these two conformations have different preferences for various inter- and intragenomic RNA-RNA interactions, with the interactions themselves depending on various external factors (e.g., [Mg2+]). These findings have made it possible for us to predict which of these mutually exclusive interactions will be formed under specific conditions and ultimately how the formation and disruption of them regulates various RNA-dependent viral processes.

Combining simulations and single-molecule fluorescence spectroscopy to understand SARS-CoV-2 nucleocapsid protein RNA interaction.

SARS-CoV-2 causes the respiratory disease COVID-19 that has resulted in a worldwide pandemic. An outstanding question revolves around how SARS-CoV-2 and related coronaviruses package their relatively long 30 kb genomes into a relatively small (<100 nm) virion. Genome packaging is driven by the nucleocapsid protein (N), a five-domain protein that consists of an intrinsically disordered N-terminal domain (NTD), folded RNA binding domain (RBD), disordered linker domain, folded dimerization domain, and a C-terminal disordered domain. This domain architecture is conserved in coronaviruses, hinting at the importance of interspersed disordered and folded domains in mediating N-protein biological function.

Ready, steady, go: Rapid ribosomal scanning to reach start codons

Wang etal. (2022)1 employ real-time single-molecule fluorescence spectroscopy to monitor eukaryotic translation initiation events, revealing that, while mRNA engagement by ribosomal 43S subunits is slow, the subsequent mRNA scanning process is rapid- ∼10 times faster than translation.

The biophysics of disordered proteins from the point of view of single-molecule fluorescence spectroscopy.

Intrinsically disordered proteins (IDPs) and regions (IDRs) have emerged as key players across many biological functions and diseases. Differently from structured proteins, disordered proteins lack stable structure and are particularly sensitive to changes in the surrounding environment. Investigation of disordered ensembles requires new approaches and concepts for quantifying conformations, dynamics, and interactions. Here, we provide a short description of the fundamental biophysical properties of disordered proteins as understood through the lens of single-molecule fluorescence observations. Single-molecule Förster resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) provides an extensive and versatile toolbox for quantifying the characteristics of conformational distributions and the dynamics of disordered proteins across many different solution conditions, both in vitro and in living cells.

Rapid 40S scanning and its regulation by mRNA structure during eukaryotic translation initiation

How the eukaryotic 43S preinitiation complex scans along the 5' untranslated region (5' UTR) of a capped mRNA to locate the correct start codon remains elusive. Here, we directly track yeast 43S-mRNA binding, scanning, and 60S subunit joining by real-time single-molecule fluorescence spectroscopy. 43S engagement with mRNA occurs through a slow, ATP-dependent process driven by multiple initiation factors including the helicase eIF4A. Once engaged, 43S scanning occurs rapidly and directionally at ∼100 nucleotides per second, independent of multiple cycles of ATP hydrolysis by RNA helicases post ribosomal loading. Scanning ribosomes can proceed through RNA secondary structures, but 5' UTR hairpin sequences near start codons drive scanning ribosomes at start codons backward in the 5' direction, requiring rescanning to arrive once more at a start codon. Direct observation of scanning ribosomes provides a mechanistic framework for translational regulation by 5' UTR structures and upstream near-cognate start codons.

Spatiotemporally controlled generation of NTPs for single-molecule studies.

Many essential processes in the cell depend on proteins that use nucleoside triphosphates (NTPs). Methods that directly monitor the often-complex dynamics of these proteins at the single-molecule level have helped to uncover their mechanisms of action. However, the measurement throughput is typically limited for NTP-utilizing reactions, and the quantitative dissection of complex dynamics over multiple sequential turnovers remains challenging. Here we present a method for controlling NTP-driven reactions in single-molecule experiments via the local generation of NTPs (LAGOON) that markedly increases the measurement throughput and enables single-turnover observations. We demonstrate the effectiveness of LAGOON in single-molecule fluorescence and force spectroscopy assays by monitoring DNA unwinding, nucleosome sliding and RNA polymerase elongation. LAGOON can be readily integrated with many single-molecule techniques, and we anticipate that it will facilitate studies of a wide range of crucial NTP-driven processes.