Using Single-Molecule FRET to Evaluate DNA Nanodevices at Work.

The observation of DNA nanodevices at a single molecule (i.e., device) level and in real time provides rich information that is typically masked in ensemble measurements. Single-molecule fluorescence resonance energy transfer (smFRET) offers a means to directly follow dynamic conformational or compositional changes that DNA nanodevices undergo while operating, thereby retrieving insights critical for refining them toward optimal function. To be successful, smFRET measurements require careful execution and meticulous data analysis for robust statistics. Here we outline the elemental steps for smFRET experiments on DNA nanodevices, starting from microscope slide preparation for single-molecule observation to data acquisition and analysis.

Single-Molecule FRET: A Tool to Characterize DNA Nanostructures.

DNA nanostructures often involve temporally evolving spatial features. Tracking these temporal behaviors in real time requires sophisticated experimental methods with sufficiently high spatial and temporal resolution. Among the several strategies developed for this purpose, single-molecule FRET (smFRET) offers avenues to observe the structural rearrangement or locomotion of DNA nanostructures in real time and quantitatively measure the kinetics as well at the single nanostructure level. In this mini review, we discuss a few applications of smFRET-based techniques to study DNA nanostructures. These examples exemplify how smFRET signals not only have played an important role in the characterization of the nanostructures but also often have helped to improve the design and overall performance of the nanostructures and the devices designed from those structures. Overall, this review consolidates the potential of smFRET in providing crucial quantitative information on structure-function relations in DNA nanostructures.

Regulating DNA Self-Assembly Dynamics with Controlled Nucleation.

Controlling the nucleation step of a self-assembly system is essential for engineering structural complexity and dynamic behaviors. Here, we design a "frame-filling" model system that comprises one type of self-complementary DNA tile and a hosting DNA origami frame to investigate the inherent dynamics of three general nucleation modes in nucleated self-assembly: unseeded, facet, and seeded nucleation. Guided by kinetic simulation, which suggested an optimal temperature range to differentiate the individual nucleation modes, and complemented by single-molecule observations, the transition of tiles from a metastable, monomeric state to a stable, polymerized state through the three nucleation pathways was monitored by Mg2+-triggered kinetic measurements. The temperature-dependent kinetics for all three nucleation modes were correlated by a "nucleation-growth" model, which quantified the tendency of nucleation using an empirical nucleation number. Moreover, taking advantage of the temperature dependence of nucleation, tile assembly can be regulated externally by the hosting frame. An ultraviolet (UV)-responsive trigger was integrated into the frame to simultaneously control "when" and "where" nucleation started. Our results reveal the dynamic mechanisms of the distinct nucleation modes in DNA tile-based self-assembly and provide a general strategy for controlling the self-assembly process.

Single bacterial resolvases first exploit, then constrain intrinsic dynamics of the Holliday junction to direct recombination.

Homologous recombination forms and resolves an entangled DNA Holliday Junction (HJ) crucial for achieving genetic reshuffling and genome repair. To maintain genomic integrity, specialized resolvase enzymes cleave the entangled DNA into two discrete DNA molecules. However, it is unclear how two similar stacking isomers are distinguished, and how a cognate sequence is found and recognized to achieve accurate recombination. We here use single-molecule fluorescence observation and cluster analysis to examine how prototypic bacterial resolvase RuvC singles out two of the four HJ strands and achieves sequence-specific cleavage. We find that RuvC first exploits, then constrains the dynamics of intrinsic HJ isomer exchange at a sampled branch position to direct cleavage toward the catalytically competent HJ conformation and sequence, thus controlling recombination output at minimal energetic cost. Our model of rapid DNA scanning followed by 'snap-locking' of a cognate sequence is strikingly consistent with the conformational proofreading of other DNA-modifying enzymes.

Author Correction: Ras hyperactivation versus overexpression: Lessons from Ras dynamics in Candida albicans.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

A bio-hybrid DNA rotor-stator nanoengine that moves along predefined tracks.

Biological motors are highly complex protein assemblies that generate linear or rotary motion, powered by chemical energy. Synthetic motors based on DNA nanostructures, bio-hybrid designs or synthetic organic chemistry have been assembled. However, unidirectionally rotating biomimetic wheel motors with rotor-stator units that consume chemical energy are elusive. Here, we report a bio-hybrid nanoengine consisting of a catalytic stator that unidirectionally rotates an interlocked DNA wheel, powered by NTP hydrolysis. The engine consists of an engineered T7 RNA polymerase (T7RNAP-ZIF) attached to a dsDNA nanoring that is catenated to a rigid rotating dsDNA wheel. The wheel motor produces long, repetitive RNA transcripts that remain attached to the engine and are used to guide its movement along predefined ssDNA tracks arranged on a DNA nanotube. The simplicity of the design renders this walking nanoengine adaptable to other biological nanoarchitectures, facilitating the construction of complex bio-hybrid structures that achieve NTP-driven locomotion.

Ras hyperactivation versus overexpression: Lessons from Ras dynamics in Candida albicans.

Ras signaling in response to environmental cues is critical for cellular morphogenesis in eukaryotes. This signaling is tightly regulated and its activation involves multiple players. Sometimes Ras signaling may be hyperactivated. In C. albicans, a human pathogenic fungus, we demonstrate that dynamics of hyperactivated Ras1 (Ras1G13V or Ras1 in Hsp90 deficient strains) can be reliably differentiated from that of normal Ras1 at (near) single molecule level using fluorescence correlation spectroscopy (FCS). Ras1 hyperactivation results in significantly slower dynamics due to actin polymerization. Activating actin polymerization by jasplakinolide can produce hyperactivated Ras1 dynamics. In a sterol-deficient hyperfilamentous GPI mutant of C. albicans too, Ras1 hyperactivation results from Hsp90 downregulation and causes actin polymerization. Hyperactivated Ras1 co-localizes with G-actin at the plasma membrane rather than with F-actin. Depolymerizing actin with cytochalasin D results in faster Ras1 dynamics in these and other strains that show Ras1 hyperactivation. Further, ergosterol does not influence Ras1 dynamics.

Effect of T·T Mismatch on DNA Dynamics Probed by Minor Groove Binders: Comparison of Dynamic Stokes Shifts of Hoechst and DAPI.

Recognition of DNA base mismatches and their subsequent repair by enzymes is vital for genomic stability. However, it is difficult to comprehend such a process in which enzymes sense and repair different types of mismatches with different ability. It has been suggested that the differential structural changes of mismatched bases act as cues to the repair enzymes, although the effect of such DNA structural changes on surrounding water and ion dynamics is inevitable due to strong electrostatic coupling among them. Thus, collective dynamics of DNA, water, and ions near the mismatch site is believed to be important for mismatch recognition and repair mechanism. Here we show that introduction of a T·T mismatch in the minor groove of DNA induces dispersed (collective) power-law solvation dynamics (of exponent ∼0.24), measured by monitoring the time-resolved fluorescence Stokes shifts (TRFSS) of two popular minor groove binders (Hoechst 33258 and DAPI) over five decades of time from 100 fs to 10 ns. The same ligands however sense different dynamics (power-law of exponent ∼0.15 or power-law multiplied with biexponential relaxation) in the minor groove of normal-DNA. The similar fluorescence anisotropy decays of ligands measured in normal- and T·T-DNA suggest that Stokes shift dynamics and their changes in T·T-DNA purely originate from the solvation process, and not from any internal rotational motion of probe-ligands. The dispersed power-law solvation dynamics seen in T·T-DNA indicate that the ligands do not sense any particular (exponential) relaxation specific to T·T wobbling and/or other conformational changes. This could be the reason why T·T mismatch is recognized by enzymes with lower efficiency compared to purine-pyrimidine and purine-purine mismatches.

Manipulating motions of targeted single cells in solution by an integrated double-ring magnetic tweezers imaging microscope.

Controlling and manipulating living cell motions in solution hold a high promise in developing new biotechnology and biological science. Here, we developed a magnetic tweezers device that employs a combination of two permanent magnets in up-down double-ring configuration axially fitting with a microscopic objective, allowing a picoNewton (pN) bidirectional force and motion control on the sample beyond a single upward pulling direction. The experimental force calibration and magnetic field simulation using finite element method magnetics demonstrate that the designed magnetic tweezers covers a linear-combined pN force with positive-negative polarization changes in a tenability of sub-pN scale, which can be utilized to further achieve motion manipulation by shifting the force balance. We demonstrate an application of the up-down double-ring magnetic tweezers for single cell manipulation, showing that the cells with internalized paramagnetic beads can be selectively picked up and guided in a controlled fine motion.

Probing conformational dynamics of an enzymatic active site by an in situ single fluorogenic probe under piconewton force manipulation.

Unraveling the conformational details of an enzyme during the essential steps of a catalytic reaction (i.e., enzyme-substrate interaction, enzyme-substrate active complex formation, nascent product formation, and product release) is challenging due to the transient nature of intermediate conformational states, conformational fluctuations, and the associated complex dynamics. Here we report our study on the conformational dynamics of horseradish peroxidase using single-molecule multiparameter photon time-stamping spectroscopy with mechanical force manipulation, a newly developed single-molecule fluorescence imaging magnetic tweezers nanoscopic approach. A nascent-formed fluorogenic product molecule serves as a probe, perfectly fitting in the enzymatic reaction active site for probing the enzymatic conformational dynamics. Interestingly, the product releasing dynamics shows the complex conformational behavior with multiple product releasing pathways. However, under magnetic force manipulation, the complex nature of the multiple product releasing pathways disappears and more simplistic conformations of the active site are populated.

Protein-fluctuation-induced water-pore formation in ion channel voltage-sensor translocation across a lipid bilayer membrane.

We have applied a combined fluorescence microscopy and single-ion-channel electric current recording approach, correlating with molecular dynamics (MD) simulations, to study the mechanism of voltage-sensor domain translocation across a lipid bilayer. We use the colicin Ia ion channel as a model system, and our experimental and simulation results show the following: (1) The open-close activity of an activated colicin Ia is not necessarily sensitive to the amplitude of the applied cross-membrane voltage when the cross-membrane voltage is around the resting potential of excitable membranes; and (2) there is a significant probability that the activation of colicin Ia occurs by forming a transient and fluctuating water pore of ∼15 Šdiameter in the lipid bilayer membrane. The location of the water-pore formation is nonrandom and highly specific, right at the insertion site of colicin Ia charged residues in the lipid bilayer membrane, and the formation is intrinsically associated with the polypeptide conformational fluctuations and solvation dynamics. Our results suggest an interesting mechanistic pathway for voltage-sensitive ion channel activation, and specifically for translocation of charged polypeptide chains across the lipid membrane under a transmembrane electric field: the charged polypeptide domain facilitates the formation of hydrophilic water pore in the membrane and diffuses through the hydrophilic pathway across the membrane; i.e., the charged polypeptide chain can cross a lipid membrane without entering into the hydrophobic core of the lipid membrane but entirely through the aqueous and hydrophilic environment to achieve a cross-membrane translocation. This mechanism sheds light on the intensive and fundamental debate on how a hydrophilic and charged peptide domain diffuses across the biologically inaccessible high-energy barrier of the hydrophobic core of a lipid bilayer: The peptide domain does not need to cross the hydrophobic core to move across a lipid bilayer.

Power-Law Solvation Dynamics in G-Quadruplex DNA: Role of Hydration Dynamics on Ligand Solvation inside DNA.

G-quadruplex DNA (GqDNA) structures act as promising anticancer targets for small-molecules (ligands). Solvation dynamics of a ligand (DAPI: 4',6-diamidino-2-phenylindole) inside antiparallel-GqDNA is studied through direct comparison of time-resolved experiments to molecular dynamics (MD) simulation. Dynamic Stokes shifts of DAPI in GqDNA prepared in H2O buffer and D2O are compared to find the effect of water on ligand solvation. Experimental dynamics (in H2O) is then directly compared with the dynamics computed from 65 ns simulation on the same DAPI-GqDNA complex. Ligand solvation follows power-law relaxation (summed with fast exponential relaxation) from ~100 fs to 10 ns. Simulation results show relaxation below ~5 ps is dominated by water motion, while both water and DNA contribute comparably to dictate long-time power-law dynamics. Ion contribution is, however, found to be negligible. Simulation results also suggest that anomalous solvation dynamics may have origin in subdiffusive motion of perturbed water near GqDNA.

Sequence-Dependent Solvation Dynamics of Minor-Groove Bound Ligand Inside Duplex-DNA.

Ligand binding to minor-grooves of DNA depends on DNA-base sequence near its binding-site. However, it is not known how base-sequences affect the local solvation of ligand inside minor-grooves of DNA. Here we present a comprehensive study on sequence-dependent solvation dynamics of ligand inside duplex-DNA by measuring the static and dynamic fluorescence Stokes shifts of a popular groove-binder, DAPI, inside DNA minor-grooves created by four different sequences; d(5'-CGCGAATTCGCG-3')2, d(5'-CGCGTTAACGCG-3')2, d(5'-CGCGCAATTGCGCG-3')2, and d(5'-CGCGCTTAAGCGCG-3')2, having different sequences near DAPI-binding site. Fluorescence up-conversion and time-correlated single photon counting techniques are employed to capture the dynamic Stokes shifts of DAPI over five decades in time from 100 fs to 10 ns. We show that the ligands sense different static and dynamic solvation inside minor-grooves created by different sequences: Only subtle change in the dynamics is seen in DNA containing -AATTG-, -TTAAG-, and -AATTC- sequences, which show power-law relaxation in initial time-decades, followed by biexponential decay in nanosecond time-scales. However, changing a single base (and the complementary base) near ligand-binding site from -TTAAG- to -TTAAC- drastically induces the dynamics to follow a single power-law relaxation over the entire five decades. The observed variation of dynamics possibly relate to the local DNA motions, coupled to the hydration dynamics near the ligand-binding site.

Understanding growth kinetics of nanorods in microemulsion: a combined fluorescence correlation spectroscopy, dynamic light scattering, and electron microscopy study.

Even though nanostructures of various shapes and sizes can be controlled by microemulsions, there is substantial difficulty in understanding their growth mechanism. The evolution of nanostructures from the time of mixing of reactants to their final stage is a heterogeneous process involving a variety of intermediates. To obtain a deeper insight into these kinetic steps, we studied the slow growth kinetics (extending over eight days) of iron oxalate nanorods inside the polar core of water-in-oil microemulsion droplets made of cetyltrimethylammonium bromide/1-butanol/isooctane. Fluorescence correlation spectroscopy (FCS), dynamic light scattering (DLS), and transmission electron microscopy (TEM) have been employed to monitor the nanostructure growth at (near) the single-droplet level and in an ensemble. Analyzing FCS data with suitable kinetic model we obtain transient dimer lifetime (28 µs) and the droplet fusion rates (and fusion tendency) on each day as the reaction proceeds. The droplet fusion rate is found to directly control the nanorod growth in microemulsion solution and attains its maximum value (3.55 × 10(4) s(-1)) on day 6, when long nanorods are found in TEM data, implying that more and more reactants are fed into the growing system at this stage. Combining FCS, DLS, and TEM results, we find three distinct periods in the entire growth process: a long nucleation-dominant nanoparticle growth period which forms nanoparticles of critical (average) size of ∼53 nm, followed by a short period where isotropic nanoparticles switch to anisotropic growth to form nanorods, and finally elongation of nanorods and growth (and shrinking) of nanoparticles.

Understanding ligand interaction with different structures of G-quadruplex DNA: evidence of kinetically controlled ligand binding and binding-mode assisted quadruplex structure alteration.

The study of ligand interaction with G-quadruplex DNA is an active research area, because many ligands are shown to bind G-quadruplex structures, showing anticancer effects. Here, we show, for the first time, how fluorescence correlation spectroscopy (FCS) can be used to study binding kinetics of ligands with G-quadruplex DNA at the single molecule level. As an example, we study interaction of a benzo-phenoxazine ligand (Cresyl Violet, CV) with antiparallel and (3 + 1) hybrid G-quadruplex structures formed by human telomeric sequence. By using simple modifications in FCS setup, we describe how one can extract the reaction kinetics from diffusion-coupled correlation curves. It is found that the ligand (CV) binds stronger, by an order of magnitude, to a (3 + 1) hybrid structure, compared to an antiparallel one. Ensemble-averaged time-resolved fluorescence experiments are also carried out to obtain the binding equilibrium constants (K) of ligand-quadruplex interactions in bulk solution for the first time, which are found to match very well with FCS results. Global analysis of FCS data provides association (k(+)) and dissociation (k(-)) rates of the ligand in the two structures. Results indicate that stronger ligand binding to the (3 + 1) hybrid structure is controlled by the dissociation rate, rather than the association rate of ligand in the quadruplexes. Circular dichroism (CD) and induced-CD spectra show that the ligand not only binds at different conformations in the quadruplexes, but also induces antiparallel structure to form a mixed-type hybrid structure in Na(+) solution. However, in K(+) solution, the ligand stabilizes the (3 + 1) hybrid structure. Molecular docking studies predict the possible differences in binding sites of the ligand inside two quadruplexes, which strongly support the experimental observations. Results suggest that different binding modes of the ligand to the quadruplex structures actually assist the alteration of structures differently.

Probe Position-Dependent Counterion Dynamics in DNA: Comparison of Time-Resolved Stokes Shift of Groove-Bound to Base-Stacked Probes in the Presence of Different Monovalent Counterions.

Time-resolved fluorescence Stokes shifts (TRFSS) of 4',6-diamidino-2-phenylindole (DAPI) inside the minor groove of DNA are measured in the presence of three different monovalent counterions: sodium (Na(+)), rubidium (Rb(+)), and tetrabutylammonium (TBA(+)). Fluorescence up-conversion and time-correlated single photon counting are combined to obtain the time-resolved emission spectra (TRES) of DAPI in DNA from 100 fs to 10 ns. Time-resolved Stokes shift data suggest that groove-bound DAPI can not sense the counterion dynamics because the ions are displaced by DAPI far from the probe-site. However, when these results are compared to the earlier base-stacked coumarin data, the same ions are found to affect the nanosecond dynamics significantly. This suggests that the ions come close to the probe-site, such that they can affect the dynamics when measured by base-stacked coumarin. These results support previous molecular dynamics (MD) simulation data of groove-bound and base-stacked probes inside DNA.

Fluorescence correlation spectroscopy: an efficient tool for measuring size, size-distribution and polydispersity of microemulsion droplets in solution.

Fluorescence correlation spectroscopy (FCS) is an ideal tool for measuring molecular diffusion and size under extremely dilute conditions. However, the power of FCS has not been utilized to its best to measure diffusion and size parameters of complex chemical systems. Here, we apply FCS to measure the size, and, most importantly, the size distribution and polydispersity of a supramolecular nanostructure (i.e., microemulsion droplets, MEDs) in dilute solution. It is shown how the refractive index mismatch of a solution can be corrected in FCS to obtain accurate size parameters of particles, bypassing the optical matching problem of light scattering techniques that are used often for particle-size measurements. We studied the MEDs of 13 different W(0) values from 2 to 50 prepared in a ternary mixture of water, sodium bis(2-ethylhexyl) sulfosuccinate (AOT), and isooctane, with sulforhodamine-B as a fluorescent marker. We find that, near the optical matching point of MEDs, the dynamic light scattering (DLS) measurements underestimate the droplet sizes while FCS estimates the accurate ones. A Gaussian distribution model (GDM) and a maximum-entropy-based FCS data fitting model (MEMFCS) are used to analyze the fluorescence correlation curves that unfold Gaussian-type size distributions of MEDs in solution. We find the droplet size varies linearly with W(0) up to ~20, but beyond this W(0) value, the size variation deviates from this linearity. To explain nonlinear variation of droplet size for W(0) values beyond ~20, we invoke a model (the coated-droplet model) that incorporates the size polydispersity of the droplets.

Probe position dependence of DNA dynamics: comparison of the time-resolved Stokes shift of groove-bound to base-stacked probes.

Time-resolved fluorescence Stokes shift dynamics of a fluorescent probe, 4',6-diamidino-2-phenylindole (DAPI), inside the minor groove of the DNA is measured over five decades in time spans from 100 fs to 10 ns. Two different techniques, fluorescence up-conversion and time correlated single photon counting, are combined to obtain the time-resolved emission spectra of DAPI in DNA over the entire five decades in time. Having the dynamics of groove-bound DAPI in DNA measured over such a broad time window, we are able to convincingly compare our data to earlier time-resolved fluorescence results of a base-stacked probe that replaces a DNA base pair. Results show that the dynamics measured with either the groove-bound or the base-stacked probe are similar in the time span of 100 fs to approximately 100 ps but differ substantially from approximately 100 ps to 10 ns. Our present data also help to reconcile the previously reported molecular dynamics simulation results and provide important clues that the groove-bound water molecules inside DNA are mainly responsible for the slow dynamics seen in native DNA.