High-resolution fleezers reveal duplex opening and stepwise assembly by an oligomer of the DEAD-box helicase Ded1p.

DEAD-box RNA helicases are ubiquitous in all domains of life where they bind and remodel RNA and RNA-protein complexes. DEAD-box helicases unwind RNA duplexes by local opening of helical regions without directional movement through the duplexes and some of these enzymes, including Ded1p from Saccharomyces cerevisiae, oligomerize to effectively unwind RNA duplexes. Whether and how DEAD-box helicases coordinate oligomerization and unwinding is not known and it is unclear how many base pairs are actively opened. Using high-resolution optical tweezers and fluorescence, we reveal a highly dynamic and stochastic process of multiple Ded1p protomers assembling on and unwinding an RNA duplex. One Ded1p protomer binds to a duplex-adjacent ssRNA tail and promotes binding and subsequent unwinding of the duplex by additional Ded1p protomers in 4-6 bp steps. The data also reveal rapid duplex unwinding and rezipping linked with binding and dissociation of individual protomers and coordinated with the ATP hydrolysis cycle.

Few-body hydrodynamic interactions probed by optical trap pulling experiment.

We study the hydrodynamic coupling of neighboring micro-beads placed in a multiple optical trap setup allowing us to precisely control the degree of coupling and directly measure time-dependent trajectories of entrained beads. We performed measurements on configurations with increasing complexity starting with a pair of entrained beads moving in one dimension, then in two dimensions, and finally a triplet of beads moving in two dimensions. The average experimental trajectories of a probe bead compare well with the theoretical computation, illustrating the role of viscous coupling and setting timescales for probe bead relaxation. The findings also provide direct experimental corroborations of hydrodynamic coupling at large, micrometer spatial scales and long, millisecond timescales, of relevance to, e.g., microfluidic device design and hydrodynamic-assisted colloidal assembly, improving the capability of optical tweezers, and understanding the coupling between micrometer-scale objects within a living cell.

High-Resolution Optical Tweezers Combined with Multicolor Single-Molecule Microscopy.

We present an instrument that combines high-resolution optical tweezers and multicolor confocal fluorescence spectroscopy. Biological macromolecules exhibit complex conformation and stoichiometry changes in coordination with their motion and activity. To further our understanding of the complex machinery of life, we need methods that can simultaneously probe more than one degree of freedom of single molecules and complexes. Fluorescence optical tweezers, or "fleezers," combine the capabilities of optical tweezers and single-molecule fluorescence microscopy into a single instrument. Here we present the latest generation of a high-resolution fleezers instrument integrated with multicolor fluorescence spectroscopy. The tweezers portion of the instrument can manipulate biological macromolecules with pN scale forces while measuring subnanometer distances. Simultaneous with tweezers measurements, the multicolor fluorescence capability allows the direct observation of multiple molecules or multiple degrees of freedom which allows, for example, the observation of multiple proteins simultaneously within a complex. The instrument incorporates three fluorescence excitation lasers, all sourced from a single-mode optical fiber allowing a reliable alignment scheme, that allows, for example, three independent fluorescent probes or fluorescence resonance energy transfer (FRET) measurements and also increases flexibility in the choice of fluorescent probes. To avoid photobleaching and improve tweezers stability, the instrument implements a timesharing (using a single trap laser to produce a pair of traps via rapid switching between two locations) and interlacing (turning the trapping beam off when the fluorescence excitation beams are on and vice versa) scheme using acousto-optic modulators (AOM) to rapidly and precisely modulate lasers. Our latest "random phase" trap AOM control method obliterates previous residual trap positioning and bead position measurement errors. Here we present the general design principles and detailed construction and testing protocols for the instrument.

Observation of processive telomerase catalysis using high-resolution optical tweezers.

Telomere maintenance by telomerase is essential for continuous proliferation of human cells and is vital for the survival of stem cells and 90% of cancer cells. To compensate for telomeric DNA lost during DNA replication, telomerase processively adds GGTTAG repeats to chromosome ends by copying the template region within its RNA subunit. Between repeat additions, the RNA template must be recycled. How telomerase remains associated with substrate DNA during this critical translocation step remains unknown. Using a single-molecule telomerase activity assay utilizing high-resolution optical tweezers, we demonstrate that stable substrate DNA binding at an anchor site within telomerase facilitates the processive synthesis of telomeric repeats. The product DNA synthesized by telomerase can be recaptured by the anchor site or fold into G-quadruplex structures. Our results provide detailed mechanistic insights into telomerase catalysis, a process of critical importance in aging and cancer.

Combined High-Resolution Optical Tweezers and Multicolor Single-Molecule Fluorescence with an Automated Single-Molecule Assembly Line.

We present an instrument that combines high-resolution optical tweezers and multicolor confocal fluorescence spectroscopy along with automated single-molecule assembly. The multicolor allows the simultaneous observation of multiple molecules or multiple degrees of freedom, which allows, for example, the observation of multiple proteins simultaneously within a complex. The instrument incorporates three fluorescence excitation lasers, with a reliable alignment scheme, which will allow three independent fluorescent probe or FRET measurements and also increases flexibility in the choice of fluorescent molecules. We demonstrate the ability to simultaneously measure angstrom-scale changes in tether extension and fluorescence signals. Simultaneous tweezers and fluorescence measurement are particularly challenging because of fluorophore photobleaching, even more so if multiple fluorophores are to be measured. Therefore, (1) fluorescence excitation and detection is interlaced with time-shared dual optical traps. (2) We investigated the photostability of common fluorophores. The mean number of photons emitted before bleaching was unaffected by the trap laser and decreased only slightly with increasing excitation laser intensity. Surprisingly, we found that Cy5 outperforms other commonly used fluorophores by more than fivefold. (3) We devised computer-controlled automation, which conserves fluorophore lifetime by quickly detecting fluorophore-labeled molecule binding, turning off lasers, and moving to add the next fluorophore-labeled component. The single-molecule assembly line enables the precise assembly of multimolecule complexes while preserving fluorophores.

Combined Force Ramp and Equilibrium High-Resolution Investigations Reveal Multipath Heterogeneous Unfolding of Protein G.

Over the past two decades, one of the standard models of protein folding has been the "two-state" model, in which a protein only resides in the folded or fully unfolded states with a single pathway between them. Recent advances in spatial and temporal resolution of biophysical measurements have revealed "beyond-two-state" complexity in protein folding, even for small, single-domain proteins. In this work, we used high-resolution optical tweezers to investigate the folding/unfolding kinetics of the B1 domain of immunoglobulin-binding protein G (GB1), a well-studied model system. Experiments were performed for GB1 both in and out of equilibrium using force spectroscopy. When the force was gradually ramped, simple single-peak folding force distributions were observed, while multiple rupture peaks were seen in the unfolding force distributions, consistent with multiple force-dependent parallel unfolding pathways. Force-dependent folding and unfolding rate constants were directly determined by both force-jump and fixed-trap measurements. Monte Carlo modeling using these rate constants was in good agreement with the force ramp data. The unfolding rate constants exhibited two different behaviors at low vs high force. At high force, the unfolding rate constant increased with increasing force, as previously reported by high force, high pulling speed force ramp measurements. However, at low force, the situation reversed and the unfolding rate constant decreased with increasing force. Taken together, these data indicate that this small protein has multiple distinct pathways to the native state on the free energy landscape.

A force sensor that converts fluorescence signal into force measurement utilizing short looped DNA.

A force sensor concept is presented where fluorescence signal is converted into force information via single-molecule Förster resonance energy transfer (smFRET). The basic design of the sensor is a ~100 base pair (bp) long double stranded DNA (dsDNA) that is restricted to a looped conformation by a nucleic acid secondary structure (NAS) that bridges its ends. The looped dsDNA generates a tension across the NAS and unfolds it when the tension is high enough. The FRET efficiency between donor and acceptor (D&A) fluorophores placed across the NAS reports on its folding state. Three dsDNA constructs with different lengths were bridged by a DNA hairpin and KCl was titrated to change the applied force. After these proof-of-principle measurements, one of the dsDNA constructs was used to maintain the G-quadruplex (GQ) construct formed by thrombin binding aptamer (TBA) under tension while it interacted with a destabilizing protein and stabilizing small molecule. The force required to unfold TBA-GQ was independently investigated with high-resolution optical tweezers (OT) measurements that established the relevant force to be a few pN, which is consistent with the force generated by the looped dsDNA. The proposed method is particularly promising as it enables studying NAS, protein, and small molecule interactions using a highly-parallel FRET-based assay while the NAS is kept under an approximately constant force.

Single molecule force spectroscopy at high data acquisition: A Bayesian nonparametric analysis.

Bayesian nonparametrics (BNPs) are poised to have a deep impact in the analysis of single molecule data as they provide posterior probabilities over entire models consistent with the supplied data, not just model parameters of one preferred model. Thus they provide an elegant and rigorous solution to the difficult problem encountered when selecting an appropriate candidate model. Nevertheless, BNPs' flexibility to learn models and their associated parameters from experimental data is a double-edged sword. Most importantly, BNPs are prone to increasing the complexity of the estimated models due to artifactual features present in time traces. Thus, because of experimental challenges unique to single molecule methods, naive application of available BNP tools is not possible. Here we consider traces with time correlations and, as a specific example, we deal with force spectroscopy traces collected at high acquisition rates. While high acquisition rates are required in order to capture dwells in short-lived molecular states, in this setup, a slow response of the optical trap instrumentation (i.e., trapped beads, ambient fluid, and tethering handles) distorts the molecular signals introducing time correlations into the data that may be misinterpreted as true states by naive BNPs. Our adaptation of BNP tools explicitly takes into consideration these response dynamics, in addition to drift and noise, and makes unsupervised time series analysis of correlated single molecule force spectroscopy measurements possible, even at acquisition rates similar to or below the trap's response times.

Randomizing phase to remove acousto-optic device wiggle errors for high-resolution optical tweezers.

Acousto-optic (AO) devices have been used extensively in optical tweezers because of their flexibility and speed; however, these devices have trap positioning inaccuracies that limit their usefulness, especially for high-resolution applications. We show that these inaccuracies are due to interference patterns within the AO device sound fields. We have devised a method that removes these inaccuracies by reducing the coherence of the sound fields by directly controlling and randomizing the phase of the radio frequency voltage input signal. We demonstrate that the trapping inaccuracies are eliminated, for both constant trap position and force-ramp measurements, and that no additional noise is added. We show that this random phase method is applicable to both acousto-optic modulator and deflector type devices and can be easily integrated via software upgrade into existing instruments.

Ultrashort Nucleic Acid Duplexes Exhibit Long Wormlike Chain Behavior with Force-Dependent Edge Effects.

Despite their importance in biology and use in nanotechnology, the elastic behavior of nucleic acids on "ultrashort" (<15 nt) length scales remains poorly understood. Here, we use optical tweezers combined with fluorescence imaging to observe directly the hybridization of oligonucleotides (7-12 nt) to a complementary strand under tension and to measure the difference in end-to-end extension between the single-stranded and duplex states. Data are consistent with long-polymer models at low forces (<8 pN) but smaller than predicted at higher forces (>8 pN), the result of the sequence-dependent duplex edge effects.

High-Resolution "Fleezers": Dual-Trap Optical Tweezers Combined with Single-Molecule Fluorescence Detection.

Recent advances in optical tweezers have greatly expanded their measurement capabilities. A new generation of hybrid instrument that combines nanomechanical manipulation with fluorescence detection-fluorescence optical tweezers, or "fleezers"-is providing a powerful approach to study complex macromolecular dynamics. Here, we describe a combined high-resolution optical trap/confocal fluorescence microscope that can simultaneously detect sub-nanometer displacements, sub-piconewton forces, and single-molecule fluorescence signals. The primary technical challenge to these hybrid instruments is how to combine both measurement modalities without sacrificing the sensitivity of either one. We present general design principles to overcome this challenge and provide detailed, step-by-step instructions to implement them in the construction and alignment of the instrument. Lastly, we present a set of protocols to perform a simple, proof-of-principle experiment that highlights the instrument capabilities.

Elasticity of the transition state for oligonucleotide hybridization.

Despite its fundamental importance in cellular processes and abundant use in biotechnology, we lack a detailed understanding of the kinetics of nucleic acid hybridization. In particular, the identity of the transition state, which determines the kinetics of the two-state reaction, remains poorly characterized. Here, we used optical tweezers with single-molecule fluorescence to observe directly the binding and unbinding of short oligonucleotides (7-12 nt) to a complementary strand held under constant force. Binding and unbinding rate constants measured across a wide range of forces (1.5-20 pN) deviate from the exponential force dependence expected from Bell's equation. Using a generalized force dependence model, we determined the elastic behavior of the transition state, which we find to be similar to that of the pure single-stranded state. Our results indicate that the transition state for hybridization is visited before the strands form any significant amount of native base pairs. Such a transition state supports a model in which the rate-limiting step of the hybridization reaction is the alignment of the two strands prior to base pairing.

The RNA helicase Mtr4p is a duplex-sensing translocase.

The conserved Saccharomyces cerevisiae Ski2-like RNA helicase Mtr4p plays essential roles in eukaryotic nuclear RNA processing. RNA helicase activity of Mtr4p is critical for biological functions of the enzyme, but the molecular basis for RNA unwinding is not understood. Here, single-molecule high-resolution optical trapping measurements reveal that Mtr4p unwinds RNA duplexes by 3'-to-5' translocation on the loading strand, that strand separation occurs in discrete steps of 6 base pairs and that a single Mtr4p molecule performs consecutive unwinding steps. We further show that RNA unwinding by Mtr4p requires interaction with upstream RNA duplex. Inclusion of Mtr4p within the TRAMP complex increases the rate constant for unwinding initiation but does not change the characteristics of Mtr4p's helicase mechanism. Our data indicate that Mtr4p utilizes a previously unknown unwinding mode that combines aspects of canonical translocating helicases and non-canonical duplex-sensing helicases, thereby restricting directional translocation to duplex regions.

Protein structure. Direct observation of structure-function relationship in a nucleic acid-processing enzyme.

The relationship between protein three-dimensional structure and function is essential for mechanism determination. Unfortunately, most techniques do not provide a direct measurement of this relationship. Structural data are typically limited to static pictures, and function must be inferred. Conversely, functional assays usually provide little information on structural conformation. We developed a single-molecule technique combining optical tweezers and fluorescence microscopy that allows for both measurements simultaneously. Here we present measurements of UvrD, a DNA repair helicase, that directly and unambiguously reveal the connection between its structure and function. Our data reveal that UvrD exhibits two distinct types of unwinding activity regulated by its stoichiometry. Furthermore, two UvrD conformational states, termed "closed" and "open," correlate with movement toward or away from the DNA fork.

Ultrahigh-resolution optical trap with single-fluorophore sensitivity.

We present a single-molecule instrument that combines a time-shared ultrahigh-resolution dual optical trap interlaced with a confocal fluorescence microscope. In a demonstration experiment, we observed individual single fluorophore-labeled DNA oligonucleotides to bind and unbind complementary DNA suspended between two trapped beads. Simultaneous with the single-fluorophore detection, we clearly observed coincident angstrom-scale changes in tether extension. Fluorescence readout allowed us to determine the duplex melting rate as a function of force. The new instrument will enable the simultaneous measurement of angstrom-scale mechanical motion of individual DNA-binding proteins (for example, single-base-pair stepping of DNA translocases) along with the detection of properties of fluorescently labeled protein (for example, internal configuration).

Determination of photoswitching dynamics through chiral mapping of single molecules using a scanning tunneling microscope.

Single-molecule-resolved scanning tunneling microscopy of tetra-tert-butyl azobenzene (TTB-AB) molecules adsorbed onto Au(111) reveals chirality selection rules in their photoswitching behavior. This observation is enabled by the fact that trans-TTB-AB molecules self-assemble into homochiral domains. Cis-TTB-AB molecules produced via photoisomerization are found in two distinct conformations with final state chirality determined by the initial trans isomer chirality. Based on these observations and ab initio calculations, we propose a new inversion-based dynamical photoswitching mechanism for azobenzene molecules at a surface.

Surface anchoring and dynamics of thiolated azobenzene molecules on Au(111).

We have investigated the temperature-dependent behavior of thiolated azobenzene molecules on Au(111) using scanning tunneling microscopy. The addition of a thiol functional group to azobenzene molecules leads to increased surface anchoring of single azobenzene molecules to gold. Thiolated azobenzene shows diverse surface morphology and does not form well-ordered structures at low coverage. At elevated temperatures, anchored molecules are observed to spin in place via hindered rotation. By measuring the number of rotating molecules as a function of temperature and using a simple model, we are able to estimate the energy barrier and attempt frequency for thermally induced hindered rotation to be 102+/-3 meV and 110+/-2 GHz, respectively.

Self-patterned molecular photoswitching in nanoscale surface assemblies.

Photomechanical switching (photoisomerization) of molecules at a surface is found to strongly depend on molecule-molecule interactions and molecule-surface orientation. Scanning tunneling microscopy was used to image photoswitching behavior in the single-molecule limit of tetra-tert-butyl-azobenzene molecules adsorbed onto Au(111) at 30 K. Photoswitching behavior varied strongly with surface molecular island structure, and self-patterned stripes of switching and nonswitching regions were observed having approximately 10 nm pitch. These findings can be summarized into photoswitching selection rules that highlight the important role played by a molecule's nanoscale environment in determining its switching properties.

Reversible photomechanical switching of individual engineered molecules at a metallic surface.

We have observed reversible light-induced mechanical switching for individual organic molecules bound to a metal surface. Scanning tunneling microscopy (STM) was used to image the features of individual azobenzene molecules on Au(111) before and after reversibly cycling their mechanical structure between trans and cis states using light. Azobenzene molecules were engineered to increase their surface photomechanical activity by attaching varying numbers of tert-butyl (TB) ligands ("legs") to the azobenzene phenyl rings. STM images show that increasing the number of TB legs "lifts" the azobenzene molecules from the substrate, thereby increasing molecular photomechanical activity by decreasing molecule-surface coupling.