Transition Path Times Measured by Single-Molecule Spectroscopy

The transition path is a tiny fraction of a molecular trajectory during which the free-energy barrier is crossed. It is a single-molecule property and contains all mechanistic information of folding processes of biomolecules such as proteins and nucleic acids. However, the transition path has been difficult to probe because it is short and rarely visited when transitions actually occur. Recent technical advances in single-molecule spectroscopy have made it possible to directly probe transition paths, which has opened up new theoretical and experimental approaches to investigating folding mechanisms. This article reviews recent single-molecule fluorescence and force spectroscopic measurements of transition path times and their connection to both theory and simulations.     

Fluorescence resonance energy transfer as a structural tool for nucleic acids

Fluorescence resonance energy transfer is a spectroscopic method that provides distance information on macromolecules in solution in the range 20-80 A. It is particularly suited to the analysis of the global structure of nucleic acids because the long-range distance information provides constraints when modelling these important structures. The application of fluorescence resonance energy transfer to nucleic acid structure has seen a resurgence of interest in the past decade, which continues to increase. An especially exciting development is the recent extension to single-molecule studies.     

Fast and robust two-dimensional inverse Laplace transformation of single-molecule fluorescence lifetime data

Fluorescence spectroscopy at the single-molecule scale has been indispensable for studying conformational dynamics and rare states of biological macromolecules. Single-molecule two-dimensional (2D) fluorescence lifetime correlation spectroscopy is an emerging technique that holds promise for the study of protein and nucleic acid dynamics, as the technique is 1) capable of resolving conformational dynamics using a single chromophore, 2) resolves forward and reverse transitions independently, and 3) has a dynamic window ranging from microseconds to seconds. However, the calculation of a 2D fluorescence relaxation spectrum requires an inverse Laplace transform (ILT), which is an ill-conditioned inversion that must be estimated numerically through a regularized minimization. Current methods for performing ILTs of fluorescence relaxation can be computationally inefficient, sensitive to noise corruption, and difficult to implement. Here, we adopt an approach developed for NMR spectroscopy (T1-T2 relaxometry) to perform one-dimensional (1D) and 2D-ILTs on single-molecule fluorescence spectroscopy data using singular-valued decomposition and Tikhonov regularization. This approach provides fast, robust, and easy to implement Laplace inversions of single-molecule fluorescence data. We compare this approach to the widely used maximal entropy method.     

Mechanisms of nucleic acid translocases: lessons from structural biology and single-molecule biophysics

Enzymes that translocate nucleic acids using ATP hydrolysis include DNA and RNA helicases, viral genome packaging motors and chromatin remodeling ATPases. Recent structural analysis, in conjunction with single-molecule studies, has revealed a wealth of new insights into how these enzymes use ATP-driven conformational changes to move on nucleic acids.     

Single-molecule perspectives on helicase mechanisms and functions

Helicases are a diverse group of molecular motors that utilize energy derived from the hydrolysis of nucleoside triphosphates (NTPs) to unwind and translocate along nucleic acids. These enzymes play critical roles in nearly all aspects of nucleic acid metabolism, and consequently, a detailed understanding of helicase mechanisms at the molecular level is essential. Over the past few decades, single-molecule techniques, such as optical tweezers, magnetic tweezers, laminar flow, fluorescence resonance energy transfer (FRET), and DNA curtains, have proved to be powerful tools to investigate the functional properties of both DNA and RNA helicases. These approaches allow researchers to manipulate single helicase molecules, perturb their free energy landscape to probe the chemo-mechanical activities of these motors, and to detect the conformational changes of helicases during unwinding. Furthermore, these techniques also provide the capability to distinguish helicase heterogeneity and monitor helicase motion at nanometer spatial and millisecond temporal resolutions, ultimately providing new insights into the mechanisms that could not be resolved by ensemble assays. This review outlines the single-molecule techniques that have been utilized for measurements of helicase activities and discusses helicase mechanisms with a focus on functional and mechanistic insights revealed through single-molecule investigations in the past five years.     

Enzyme-Free Detection of Mutations in Cancer DNA Using Synthetic Oligonucleotide Probes and Fluorescence Microscopy

Background

Rapid reliable diagnostics of DNA mutations are highly desirable in research and clinical assays. Current development in this field goes simultaneously in two directions: 1) high-throughput methods, and 2) portable assays. Non-enzymatic approaches are attractive for both types of methods since they would allow rapid and relatively inexpensive detection of nucleic acids. Modern fluorescence microscopy is having a huge impact on detection of biomolecules at previously unachievable resolution. However, no straightforward methods to detect DNA in a non-enzymatic way using fluorescence microscopy and nucleic acid analogues have been proposed so far.

Methods and Results

Here we report a novel enzyme-free approach to efficiently detect cancer mutations. This assay includes gene-specific target enrichment followed by annealing to oligonucleotides containing locked nucleic acids (LNAs) and finally, detection by fluorescence microscopy. The LNA containing probes display high binding affinity and specificity to DNA containing mutations, which allows for the detection of mutation abundance with an intercalating EvaGreen dye. We used a second probe, which increases the overall number of base pairs in order to produce a higher fluorescence signal by incorporating more dye molecules. Indeed we show here that using EvaGreen dye and LNA probes, genomic DNA containing BRAF V600E mutation could be detected by fluorescence microscopy at low femtomolar concentrations. Notably, this was at least 1000-fold above the potential detection limit.

Conclusion

Overall, the novel assay we describe could become a new approach to rapid, reliable and enzyme-free diagnostics of cancer or other associated DNA targets. Importantly, stoichiometry of wild type and mutant targets is conserved in our assay, which allows for an accurate estimation of mutant abundance when the detection limit requirement is met. Using fluorescence microscopy, this approach presents the opportunity to detect DNA at single-molecule resolution and directly in the biological sample of choice.     

Molecular scaffolds: when DNA becomes the hardware for single-molecule investigations

Over the past few decades, single-molecule manipulation has been widely applied to the real-time analysis of biomolecular interactions. It has enabled researchers to decipher structure-function relationships for polymers, enzymes, and larger-scale molecular machines, in particular by harnessing force to probe both chemical and mechanical stabilities. Nucleic acids have played a central role in this effort because, in addition to their biological significance, they exhibit unique polymeric properties which have recast them as key components participating in numerous experimental designs. In this review, we introduce recent developments highlighting this dual nature of nucleic acids in biophysics, as objects of study but also as tools allowing novel approaches. More specifically, we present molecular scaffolds as an emerging concept and describe their use in single-molecule force spectroscopy. Aspects related to folding and noncovalent interactions will be presented in parallel to research in enzymology, with a focus on the acquisition of thermodynamic and kinetic data.     

Revisiting the central dogma one molecule at a time

The faithful relay and timely expression of genetic information depend on specialized molecular machines, many of which function as nucleic acid translocases. The emergence over the last decade of single-molecule fluorescence detection and manipulation techniques with nm and ? resolution and their application to the study of nucleic acid translocases are painting an increasingly sharp picture of the inner workings of these machines, the dynamics and coordination of their moving parts, their thermodynamic efficiency, and the nature of their transient intermediates. Here we present an overview of the main results arrived at by the application of single-molecule methods to the study of the main machines of the central dogma.     

Single-molecule,hybridization-based strategies for short nucleic acids detection and recognition with nanopores

DNA nanotechnology has seen large developments over the last 30 years through the combination of detection and discovery of DNAs, and solid phase synthesis to increase the chemical functionalities on nucleic acids, leading to the emergence of novel and sophisticated in features, nucleic acids-based biopolymers. Arguably, nanopores developed for fast and direct detection of a large variety of molecules, are part of a revolutionary technological evolution which led to cheaper, smaller and considerably easier to use devices enabling DNA detection and sequencing at the single-molecule level. Through their versatility, the nanopore-based tools proved useful biomedicine, nanoscale chemistry, biology and physics, as well as other disciplines spanning materials science to ecology and anthropology. This mini-review discusses the progress of nanopore- and hybridization-based DNA detection, and explores a range of state-of-the-art applications afforded through the combination of certain synthetically-derived polymers mimicking nucleic acids and nanopores, for the single-molecule biophysics on short DNA structures.     

The Effect of Dye-Dye Interactions on the Spatial Resolution of Single-Molecule FRET Measurements in Nucleic Acids

We study the effect of dye-dye interactions in labeled double-stranded DNA molecules on the Förster resonance energy transfer (FRET) efficiency at the single-molecule level. An extensive analysis of internally labeled double-stranded DNA molecules in bulk and at the single-molecule level reveals that donor-acceptor absolute distances can be reliably extracted down to ∼3-nm separation, provided that dye-dye quenching is accounted for. At these short separations, we find significant long-lived fluorescence fluctuations among discrete levels originating from the simultaneous and synchronous quenching of both dyes. By comparing four different donor-acceptor dye pairs (TMR-ATTO647N, Cy3-ATTO647N, TMR-Cy5, and Cy3-Cy5), we find that this phenomenon depends on the nature of the dye pair used, with the cyanine pair Cy3-Cy5 showing the least amount of fluctuations. The significance of these results is twofold: First, they illustrate that when dye-dye quenching is accounted for, single-molecule FRET can be used to accurately measure inter-dye distances, even at short separations. Second, these results are useful when deciding which dye pairs to use for nucleic acids analyses using FRET.     

Comparative analysis of RNA/protein dynamics for the arginine-rich-binding motif and zinc-finger-binding motif proteins encoded by HIV-1

We report a comparative study in which a single-molecule fluorescence resonance energy transfer approach was used to examine how the binding of two families of HIV-1 viral proteins to viral RNA hairpins locally changes the RNA secondary structures. The single-molecule fluorescence resonance energy transfer results indicate that the zinc finger protein (nucleocapsid) locally melts the TAR RNA and RRE-IIB RNA hairpins, whereas arginine-rich motif proteins (Tat and Rev) may strengthen the hairpin structures through specific binding interactions. Competition experiments show that Tat and Rev can effectively inhibit the nucleocapsid-chaperoned annealing of complementary DNA oligonucleotides to the TAR and RRE-IIB RNA hairpins, respectively. The competition binding data presented here suggest that the specific nucleic acid binding interactions of Tat and Rev can effectively compete with the general nucleic acid binding/chaperone functions of the nucleocapsid protein, and thus may in principle help regulate critical events during the HIV life cycle.     

Spectroscopic properties of ethidium monoazide: a fluorescent photoaffinity label for nucleic acids.

The non-covalent binding of ethidium monoazide to nucleic acids is entirely analogous to that of ethidium (binding constant approximately 2-3 X 10(5) M). The ethidium monoazide can be photochemically covalently linked to nucleic acids in high yield, up to 75%, by long wavelength light. The fluorescence of ethidium monoazide and ethidium crosslinked to nucleic acids show the same environmental sensitivity as does the fluorescence of ethidium. These properties of ethidium monoazide indicate its use as a fluorescent photoaffinity label for nucleic acids. Ethidium diazide can be photochemically linked to nucleic acids but appears to have properties substantially different from those of ethidium.     

Identification of an antitumor immune response of polyhistidine through a toll-like receptor 4-dependent manner

Polyhistidine is widely used for the delivery of nucleic acids and antibodies into the cell cytoplasm. However, little attention has been concerned on the effect of polyhistidine on the immune system. In this work, we identify a novel function of polyhistidine as an activator of the immune system. Single-molecule fluorescence imaging and single-molecule force measurements show that polyhistidine binds specifically to the toll-like receptor 4 (TLR4), inducing receptor dimerization and activation. Moreover, in a B16 melanoma model we demonstrate that polyhistidine treatment inhibits tumor growth in TLR4^+/+ but not TLR4^−/− mice. These results suggest the potential use of polyhistidine for cancer immunotherapy.     

PNA and LNA throw light on DNA

In some aspects, homogeneous (all-in-solution) nucleic acid hybridization assays are superior to the traditionally used heterogeneous (solution-to-surface) alternatives. Profluorescent probes, which reveal fluorescence enhancement or fluorescence polarization upon their binding to DNA and RNA targets, are a paradigm for the real-time sequence-specific homogeneous detection of nucleic acids. A variety of such DNA or RNA-derived probes of different constructs has already been developed with numerous applications. However, the recent additions to the field - locked nucleic acids (LNAs) and peptide nucleic acids (PNAs) - significantly increase the potential of profluorescent probes and provide a robust impulse for their new uses.     

Placing single-molecule T4 lysozyme enzymes on a bacterial cell surface: toward probing single-molecule enzymatic reaction in living cells

The T4 lysozyme enzymatic hydrolyzation reaction of bacterial cell walls is an important biological process, and single-molecule enzymatic reaction dynamics have been studied under physiological condition using purified Escherichia coli cell walls as substrates. Here, we report progress toward characterizing the T4 lysozyme enzymatic reaction on a living bacterial cell wall using a combined single-molecule placement and spectroscopy. Placing a dye-labeled single T4 lysozyme molecule on a targeted bacterial cell wall by using a hydrodynamic microinjection approach, we monitored single-molecule rotational motions during binding, attachment to, and dissociation from the cell wall by tracing single-molecule fluorescence intensity time trajectories and polarization. The single-molecule attachment duration of the T4 lysozyme to the cell wall during enzymatic reactions was typically shorter than the photobleaching time under physiological conditions. Applying single-molecule fluorescence polarization measurements to characterize the binding and motions of the T4 lysozyme molecules, we observed that the motions of wild-type and mutant T4 lysozyme proteins are essentially the same whether under an enzymatic reaction or not. The changing of the fluorescence polarization suggests that the motions of the T4 lysozyme are associated with orientational rotations. This observation also suggests that the T4 lysozyme binding-unbinding motions on cell walls involve a complex mechanism beyond a single-step first-order rate process.     

DNA computing using single-molecule hybridization detection

DNA computing aims at using nucleic acids for computing. Since micromolar DNA solutions can act as billions of parallel nanoprocessors, DNA computers can in theory solve optimization problems that require vast search spaces. However, the actual parallelism currently being achieved is at least a hundred million-fold lower than the number of DNA molecules used. This is due to the quantity of DNA molecules of one species that is required to produce a detectable output to the computations. In order to miniaturize the computation and considerably reduce the amount of DNA needed, we have combined DNA computing with single-molecule detection. Reliable hybridization detection was achieved at the level of single DNA molecules with fluorescence cross-correlation spectroscopy. To illustrate the use of this approach, we implemented a DNA-based computation and solved a 4-variable 4-clause instance of the computationally hard Satisfiability (SAT) problem.     

In Planta Single-Molecule Pull-Down Reveals Tetrameric Stoichiometry of HD-ZIPIII:LITTLE ZIPPER Complexes

Deciphering complex biological processes markedly benefits from approaches that directly assess the underlying biomolecular interactions. Most commonly used approaches to monitor protein-protein interactions typically provide nonquantitative readouts that lack statistical power and do not yield information on the heterogeneity or stoichiometry of protein complexes. Single-molecule pull-down (SiMPull) uses single-molecule fluorescence detection to mitigate these disadvantages and can quantitatively interrogate interactions between proteins and other compounds, such as nucleic acids, small molecule ligands, and lipids. Here, we establish SiMPull in plants using the HOMEODOMAIN LEUCINE ZIPPER III (HD-ZIPIII) and LITTLE ZIPPER (ZPR) interaction as proof-of-principle. Colocalization analysis of fluorophore-tagged HD-ZIPIII and ZPR proteins provides strong statistical evidence of complex formation. In addition, we use SiMPull to directly quantify YFP and mCherry maturation probabilities, showing these differ substantially from values obtained in mammalian systems. Leveraging these probabilities, in conjunction with fluorophore photobleaching assays on over 2000 individual complexes, we determined HD-ZIPIII:ZPR stoichiometry. Intriguingly, these complexes appear as heterotetramers, comprising two HD-ZIPIII and two ZPR molecules, rather than heterodimers as described in the current model. This surprising result raises new questions about the regulation of these key developmental factors and is illustrative of the unique contribution SiMPull is poised to make to in planta protein interaction studies.