High-resolution Optical Tweezers Research Articles

Single Molecule Studies on the RNA Polymerase QDE-1 by Optical Tweezers

Cellular regulatory mechanisms which rely on small dsRNA molecules (RNA silencing) are major (new) discoveries in biology.In many eukaryotic organisms silencing is achieved post-transcriptionally through pathways where dsRNA is synthesized by RNA-dependent RNA polymerases (RdRPs) and processed into 21-25 nucleotide long RNA molecules.Here we study QDE-1, an RdRP involved in the quelling (RNA silencing) pathway of Neurospora crassa. This filamentous fungus displays noticeable genomic stability, which has been attributed to quelling and other silencing mechanisms. Recently it was shown that QDE-1 is more active as a DNA-dependent RNA polymerase (DdRP) than as an RdRp and that it is also involved in DNA damage response (1,2). Recombinant QDE-1 displays five different enzymatic activities in vitro (3). The structural and biochemical data on this enzyme is extensive (1-5).We are interested on the basic biophysical parameters of QDE-1 action. The approaches taken rely on single molecule assays using high resolution optical tweezers, combined with fluorescence imaging. An optically levitated ‘dumbbell’ assay is used: the nucleic acid (NA) construct features one or two biotinylated ends that tether two different microspheres. The protein of interest is attached to the microsphere or directly to the NA tether. Since in our double-trap instrument one trap is stable whereas the other mobile, we can manipulate the tethers, detect changes in tether length and stiffness, and apply different forces and simultaneously observe the mobility of the fluorescently labelled template or protein.1. Lee, H-C et al. (2009). Nature 459(7244), 274-277.2. Lee, H-C et al. (2010). PLoS Biol. 8 (10), e1000469.3. Aalto et al. (2010). The Journal of Biological Chemistry, 285, 29367-29374.4. Salgado et al. (2006). PLoS. Biol. 4 (12), e434.5. Makeyev E.V. & Bamford D.H. (2002). Mol Cell 10(6), 1417-1427.

The RSC Chromatin Remodeling ATPase can Translocate DNA with High Force and Small Step Size

ATP-dependent chromatin remodeling complexes (remodelers) use the energy of ATP hydrolysis to reposition and reconfigure nucleosomes. Despite their diverse functions, all remodelers share highly conserved catalytic ATPase domains, many of which are shown to translocate DNA. Understanding remodeling requires biophysical knowledge of the DNA translocation process: how the ATPase moves DNA and generates force, and how translocation and force generation are coupled on nucleosomes. Here we characterize the real-time activity of a minimal translocase ‘motor’ complex isolated from a prototypical remodeler (RSC) on bare DNA, using high-resolution optical tweezers and a ‘tethered’ translocase system. We observe on dsDNA a processivity of ∼35 bp, a speed of ∼25 bp/sec, and a step size of 1.9 (± 0.3, s.d.) bp. Surprisingly, the motor is capable of moving against high force, up to 30 pN, making it one of the most force-resistant motors known. We also provide evidence for DNA ‘buckling’ at initiation. These observations extend and clarify measurements of nucleosome-dependent translocation by the complete RSC or SWI/SNF complex, and reveal the ATPase as a powerful and versatile DNA translocating motor capable of disrupting DNA-histone interactions by mechanical force using a small step size.

Unzip Single Protein Zippers using Optical Tweezers

Basic leucine zippers (bZIPs) are dimeric alpha-helical domains found in many transcription factors. They comprise coiled coil regions (zippers) that mediate specific protein dimerization, and basic regions that undergo disorder-to-order transitions to grip their cognate DNA binding sites. It remains unclear how folding of the two regions and binding of DNA are coupled. We investigated the detailed folding kinetics of a model bZIP domain derived from GCN4 transcriptional factor, using high-resolution optical tweezers. When pulled from the same amino- or carboxyl-terminals, the coiled coil alone folds and unfolds rapidly in a two-state manner, with little energy barrier. The folding kinetics of the full GCN4 bZIP domain is similar to the coiled coil alone. However, the presence of its binding site (AP1) induces a third new state corresponding to the bZIP-DNA complex. The probability of this new state critically depends upon the AP1 concentration, which reaches a maximum at an intermediate AP1 concentration. Results from our single-molecule manipulation experiments reveal a complex folding energy landscape even for a small protein domain that can be further tuned by their binding ligands.

Displaced Strand Regulation of Facxpd Helicase Activity

FacXPD is the archaeal homolog of yeast Rad3 and human xeroderma pigmentosum group D protein (XPD) helicase from Ferroplasma acidarmanus. This enzyme serves as a model for understanding the molecular mechanism of human Superfamily 2 helicase XPD involved in both transcription initiation and nucleotide excision repair, and for the related 5’-3’ helicases FancJ, Rtel and ChlR1 important for maintaining genomic integrity and DNA repair. We developed a single-molecule, high-resolution optical tweezers assay to decipher the mechanism by which a single XPD helicase unwinds dsDNA while translocating in the 5’-3’ direction. This assay monitors the unwinding of an 89-bp DNA hairpin substrate with single base pair resolution. Our substrate design allows us to control the length of a poly-dT ssDNA “translocation” strand (the strand to which XPD binds and along which it translocates), and a “displaced” strand (the strand displaced upon unwinding the duplex), located at the 5’ and 3’ tails of the hairpin, respectively. We found that the displaced strand interacts with XPD and that this interaction controls the helicase activity. When the 3’ tail of the substrate hairpin is substituted for dsDNA, a single XPD molecule displays repetitive “non-processive” bursts of substrate unwinding in which only ∼10-bp of the hairpin is unwound at a time. However, in the presence of a ssDNA displaced strand (of length ranging from 3 to 10 nt), we observe two types of activity: the same 10-bp non-processive mode as above, and also a “processive” mode in which the entire 89-bp hairpin is unwound. These data suggest two different binding modes for XPD resulting in non-processive or processive unwinding which are regulated by its interaction with the displaced strand. We propose a model for how the domains of XPD bind to its DNA substrate in these two modes.

Lambda Exonuclease Activity Measured Using Optical Tweezers with Active Force Clamp Control

Exonucleases are part of many genetic recombination and repair systems. We use high-resolution optical tweezers to study bacteriophage lambda exonuclease. We trap a dumbbell construct (bead-DNA-bead) in an inverted microscope by dividing a CW laser beam into a stationary trap and a steerable trap. The inter-trap distance is actively controlled with 200 kHz update rate by acousto-optical deflectors (AOD) and field programmable gate array -card. In addition, we control the sample temperature by flowing heating fluid through aluminum jacket around the trapping objective. We follow the exonuclease activity through the length change of a 48kb dsDNA template to ssDNA-form. The dsDNA tether is attached from one strand to two streptavidin coated beads, leaving one 5’ phosphorylated end free for exonuclease to start catalytic removal of mononucleotides. At low forces the length of the ssDNA is significantly shorter than the dsDNA. The template is held in a dumbbell construct at constant force with active feedback control. This allows us to follow the enzymatic activity of the lambda exonuclease over ten micrometers while simultaneously applying a constant load between 0 and 50 pN. We apply different loads on the DNA template to measure how the template tension affects the activity of the lambda exonuclease. We also measure the activity under different ambient temperatures.

55 Association of mammographic density with selected nutrients in Norwegian women

XPD-like helicases constitute a prominent DNA helicase family critical for many aspects of genome maintenance. These enzymes share a unique structural feature, an auxiliary domain stabilized by an iron-sulphur (FeS) cluster, and a 5′–3′ polarity of DNA translocation and duplex unwinding. Biochemical analyses alongside two single-molecule approaches, total internal reflection fluorescence microscopy and high-resolution optical tweezers, have shown how the unique structural features of XPD helicase and its specific patterns of substrate interactions tune the helicase for its specific cellular function and shape its molecular mechanism. The FeS domain forms a duplex separation wedge and contributes to an extended DNA binding site. Interactions within this site position the helicase in an orientation to unwind the duplex, control the helicase rate, and verify the integrity of the translocating strand. Consistent with its cellular role, processivity of XPD is limited and is defined by an idiosyncratic stepping kinetics. DNA duplex separation occurs in single base pair steps punctuated by frequent backward steps and conformational rearrangements of the protein–DNA complex. As such, the helicase in isolation mainly stabilizes spontaneous base pair opening and exhibits a limited ability to unwind stable DNA duplexes. The presence of a cognate ssDNA binding protein converts XPD into a vigorous helicase by destabilizing the upstream dsDNA as well as by trapping the unwound strands. Remarkably, the two proteins can co-exist on the same DNA strand without competing for binding. The current model of the XPD unwinding mechanism will be discussed along with possible modifications to this mechanism by the helicase interacting partners and unique features of such bio-medically important XPD-like helicases as FANCJ (BACH1), RTEL1 and CHLR1 (DDX11).

DNA Polymerization in Optical Tweezers

Molecular motors associated with nucleic acids ensure replication, maintenance and expression of genetic information, which is crucial for life. Biochemical and biological studies have generated considerable knowledge about these molecular motors. However, these studies assess only sum reactions/effects due to ensemble averaging. Single molecule (SM) measurements overcome this shortcoming. Such measurements allow learning about individual motor functions, such as processivity, fidelity, step size, template dependence and generated forces. Transient intermediate states and rarely occurring events can also be observed.We are developing an assay based on bacteriophage phi29 polymerase to study DNA/RNA polymerases in action using high resolution optical tweezers (OT) combined with fluorescence imaging (OT-SMF). We use an optically levitated ‘dumbbell’ assay: the nucleic acid (NA) construct features biotinylated and digixigeninated ends that tether two different kinds of microspheres (coated with streptavidin and anti-digoxigenin); the protein of interest is attached directly to the NA tether. Since in our double OT instrument one trap is stable whereas the other mobile, we can manipulate the tethers, detect changes in tether length and stiffness, apply different forces and simultaneously observe the fluorescently stained template/protein. We present preliminary results on DNA replication by phi29 polymerase.

A Biological Friendly High-Resolution Optical Tweezers for Single Molecule Studies

Exciting progress has been made recently in biophysical techniques that allow optical manipulation and measurement for single molecules at subnanometer scale as they undergo conformational transitions in real time. However, a serious problem that could limit the future application of this technique is the photo damage to biological molecules brought by intense laser irradiation. The state-of-the-art high resolution optical tweezers instrument utilizes lasers that operate at 1064 nm, which coincides with the absorption of molecular oxygen in water.

Single Molecule Studies of the Recognition Sequence Finding Mechanism of Protelomerase Telk

Protelomerase TelK is an enzyme responsible for forming DNA hairpins in linear prokaryotic DNA. The mechanism by which this protein recognizes its target sequence and both quickly and accurately catalyzes DNA hairpin formation is poorly understood. To investigate the target recognition process, we used TIRF microscopy to visualize quantum dot-labeled TelK interacting with both nonspecific DNA and DNA containing the TelK target sequence. While many sequence-specific DNA-binding proteins (SSDBP) have been shown to scan DNA in 1D as their primary method for locating their recognition sequence1, we surprisingly find that TelK does not move laterally on either aforementioned DNA substrate and therefore does not search by 1D scanning. Measurements of a c-terminally truncated TelK mutant reveal the same behavior. Interestingly, this mutant forms DNA hairpins 50 times slower than wild type, and dissociates from nonspecific DNA at a comparably lower rate than full-length TelK. These results suggest that dissociation from nonspecific DNA is an essential step in the recognition sequence search. Complementary studies with high-resolution optical tweezers reveal that TelK binding to DNA is a highly tension dependent process and condenses the molecule by several nanometers, consistent with crystal structures of the protein-DNA complex2. Remarkably, this condensation is observed on nonspecific DNA as well, despite the fact that these DNA distortions are energetically expensive. These findings suggest that the TelK target sequence search may involve 3D hopping and intersegmental transfer in lieu of 1D scanning. This may represent a novel SSDBP recognition sequence search mechanism.1. Halford, S. et al. Nucl. Ac. Res. 32 (2004)2. Aihara, H. et al. Mol. Cell. 27, 901 (2007)

Active Force Clamp Control of Optical Tweezers

In a typical high-resolution optical tweezers (OT) experiment a molecular motor changes the contour length of a trapped dumbbell-construct. Unless the inter-trap distance is actively controlled the OT increases the load on the molecular motor as it steps along the template. To counter this phenomenon we implement a real-time controller for the OT to be used in constant force measurements.We trap a dumbbell construct (bead-DNA-bead) in an inverted microscope by dividing a CW laser beam into a stationary trap and a steerable trap. Separate low power detection lasers and position sensitive detectors in the back-focal plane measure the position of both beads. The position of the bead in the stationary trap is used for constant-force feedback control. The feedback algorithm runs a Proportional-Integral-Derivative-controller on a field programmable gate array, and acousto-optical deflectors update the steerable trap position at a rate of 200 kHz.We test the force clamp control with a 10kb dsDNA molecule and present a theory explaining the power spectrum of the force clamped bead's position. We study the effect of controller bandwidth by digitally filtering the signal used for feedback control, and test the response time of our real-time controlled optical tweezers with a RNA hairpin opening/closing reaction.

Towards Sub-Nanometer Resolution Optical Tweezers

Optical tweezers with sub-nanometer resolution can provide new insights into how nucleic-acid associated molecular motors work, since elementary steps of one base-pair or less can be resolved. For example, elementary 0.34 nm steps of RNA-Polymerase, as well as of a viral packaging motor, have been observed with high-resolution optical tweezers instruments. Techniques for achieving ultra-stable trapping/microscopy include housing the instrument in a Helium atmosphere, temperature control, and stabilizing laser-pointing and intensity.We present results on optimizing the spatial resolution of our optical tweezers instrument, including experiments with a Helium atmosphere, fiber-coupled actively stabilized detection lasers, and precise temperature control of the trapping objective.

High-Resolution Dual-Trap Optical Tweezers with Differential Detection: Instrument Design

Optical traps or "optical tweezers" have become an indispensable tool in understanding fundamental biological processes. In this article, we present the design of a high-resolution optical tweezers, which is designed to minimize sensitivity to environmental noise. When the tether is attached to a stage, resolution is typically limited by drift and fluctuations in the relative position of the stage and trap-forming objective, cued by environmental noise. In a dual-trap design, however, the biological system is decoupled entirely from the stage by utilizing two optical traps to hold both ends of the tether. By holding each end of the molecule in an identical fashion, such dual-trap optical tweezers are less sensitive to drift and fluctuations of mechanical components. In our instrument, we form two traps from two orthogonally polarized beams generated by a single laser. As a result, many optical components and much of the optical path traveled are common to both trapping beams, thus minimizing differential movements of one optical trap relative to the other. Additionally, this economical use of optics creates an instrument that is simple and compact, reducing the number of elements susceptible to environmental fluctuations.

High-Resolution Dual-Trap Optical Tweezers with Differential Detection: Managing Environmental Noise: Figure 1.

Optical traps or "optical tweezers" have become an indispensable tool in understanding fundamental biological processes. The ability to manipulate and probe individual molecules or molecular complexes has led to a new, more refined understanding of the mechanical properties of the fundamental building blocks of the cell, and of the mechanism by which molecular machines function. The minimization and management of noise are the keys to building a high-resolution optical tweezers. The high degree of stability needed to observe angstrom motions of a biological system places rather stringent requirements on the environment in which the instrument operates. Specifically, environmental noise is a concern when it leads to differential movements of the optical trap relative to the second attachment point of the tether. In this article, we discuss what entails a quiet environment--what sources of noise are relevant and how they affect the instrument--and provide guidelines for choosing an appropriate location for a high-resolution instrument.

High-Resolution Dual-Trap Optical Tweezers with Differential Detection: Minimizing the Influence of Measurement Noise

Optical traps or "optical tweezers" have become an indispensable tool in understanding fundamental biological processes. The environment, the instrument components, and the overall design all have important roles in determining the spatial resolution of an optical trap. However, even with careful attention to these details, the spatial resolution of the optical tweezers is still fundamentally limited by the forces that induce Brownian fluctuations of the trapped microspheres. In this article, we focus on the ways in which the design and implementation of an experiment--the choice of bead size, tether length, and even the method in which the motions of the beads are monitored--can have a large impact on the fundamental resolution of the measurement. By investigating how the Brownian fluctuations depend on experimental parameters and the detection method, we develop a set of guidelines for designing a high-resolution experiment, with a prescription for minimizing the effect of Brownian fluctuations, thus maximizing the resolution of the experiment.

High-Resolution Dual-Trap Optical Tweezers with Differential Detection: Data Collection and Instrument Calibration: Figure 1.

Optical traps or "optical tweezers" have become an indispensable tool in understanding fundamental biological processes. Dual-trap optical tweezers with differential detection have the advantage that the instrument is decoupled from sources of environmental noise, thereby minimizing interference of signal and maximizing stability and spatial resolution. In this article, we describe methods of data detection and calibration for our dual-trap design.

High-Resolution Dual-Trap Optical Tweezers with Differential Detection: Alignment of Instrument Components: Figure 1.

Optical traps or "optical tweezers" have become an indispensable tool in understanding fundamental biological processes. Using our design, a dual-trap optical tweezers with differential detection, we can detect length changes to a DNA molecule tethering the trapped beads of 1 bp. By forming two traps from the same laser and maximizing the common optical paths of the two trapping beams, we decouple the instrument from many sources of environmental and instrumental noise that typically limit spatial resolution. The performance of a high-resolution instrument--the formation of strong traps, the minimization of background signals from trap movements, or the mitigation of the axial coupling, for example--can be greatly improved through careful alignment. This procedure, which is described in this article, starts from the laser and advances through the instrument, component by component. Alignment is complicated by the fact that the trapping light is in the near infrared (NIR) spectrum. Standard infrared viewing cards are commonly used to locate the beam, but unfortunately, bleach quickly. As an alternative, we use an IR-viewing charge-coupled device (CCD) camera equipped with a C-mount telephoto lens and display its image on a monitor. By visualizing the scattered light on a pair of irises of identical height separated by >12 in., the beam direction can be set very accurately along a fixed axis.

High-Resolution Dual-Trap Optical Tweezers with Differential Detection: An Introduction: Figure 1.

Optical traps or "optical tweezers" have become an indispensable tool in understanding fundamental biological processes. The ability to manipulate and probe individual molecules or molecular complexes has led to a new, more refined understanding of the mechanical properties of the fundamental building blocks of the cell, and of the mechanism by which molecular machines function. The field has seen a steady stream of technological advances that have greatly refined the technique. One major effort has been in developing methods to resolve motions at the angstrom level--the fundamental length scale for many biological processes. This drive has only recently come to fruition with the advent of high-resolution optical trapping techniques that can now detect movements on the scale of a single base pair of DNA, 3.4 A. Here we briefly review the basic concepts and components of optical traps and the single-molecule experiments in which they are used.

Mechanisms of ATP-dependent Chromatin Remodeling Revealed by Single-molecule Manipulation Studies

Genome-wide chromatin structures, such as nucleosome positioning and various histone modifications, have recently been mapped relative to the underlying DNA sequences, revealing an exquisite and dynamic organization of chromatin. The chromatin structures are established and remodeled mainly by a large family of highly conserved and specialized ATP-dependent chromatin remodeling complexes (remodelers) in cooperation with histone-modifying enzymes. The core of these remodelers is a DNA translocase, a molecular motor capable of actively moving along DNA. It remains unclear how the energy of ATP hydrolysis is converted to the mechanical work for DNA translocation and in turn to nucleosome remodeling by remodelers, and how the remodeling process is regulated by different protein subunits of remodelers, nucleosome substrates, and histone modifications. Using high-resolution optical tweezers, we studied the nucleosome remodeling process by SWI/SNF and RSC, two prototypes of remodelers containing 11 and 15 subunits, respectively. We found that both remodelers can translocate along DNA at rates of ∼13 bp/s and generate forces up to ∼12 pN, producing DNA loops of a broad range of sizes (5-1200 bp) in a nucleosome-dependent manner. Interestingly, when attached by a strong DNA binding domain and anchored to a bare DNA molecule, the isolated ATPase subunit of RSC alone can efficiently translocate along DNA to produce DNA loops in a nucleosome-independent manner. Surprisingly, the tethered translocase can now move against forces as high as 26 pN, making the remodeler translocase one of the strongest molecular motors. Our single-molecule experiments revealed a powerful and versatile DNA translocase engine for remodelers, which may be crucial for their role of disrupting DNA-histone interactions in a regulatory fashion.

High-resolution Optical Trap Measurements Of A Ringed DNA Translocase

Ringed ATPases are ubiquitous in the cell, involved in such diverse tasks as cell division, chromosome segregation, DNA recombination, strand separation, and conjugation. Despite their importance, the mechanism of coordination between the subunits of ringed ATPases remains poorly understood. Viral DNA packaging, a process driven by a ringed, homomeric DNA translocase, provides a model system to study this coordination. The Bacillus subtilis bacteriophage phi29 packaging motor has been proposed to operate by sequential action of its subunits, each packaging DNA in stepwise increments of 2 base pairs (bp) per ATP hydrolyzed. Here, we present the first direct observations of individual packaging steps of this motor with recently developed high-resolution optical tweezers. Surprisingly, we find that packaging occurs in increments of 10 bp, each consisting of four 2.5-bp substeps. The dwell time statistics between these 10-bp “bursts” indicate that the motor loads multiple ATPs prior to translocation, and suggest a two-phase mechanochemical cycle in which ATP binding and translocation are temporally segregated. These studies reveal a much more complex coordination between motor ATPases than previously anticipated that contrasts sharply with most published models. In addition, the observation of a non-integer step size demands new models for motor-DNA interactions. Our findings are not only relevant to other viral packaging systems, but may also provide insight into the mechanisms of the large class of ringed molecular motors that share many structural similarities.

Intersubunit coordination in a homomeric ring ATPase.

Homomeric ring-ATPases perform many vital and varied tasks in the cell, ranging from chromosome segregation to protein degradation. Here we report the first direct observation of the inter-subunit coordination and the step size of such a ring-ATPase, the dsDNA packaging motor in the bacteriophage φ29. Using high-resolution optical tweezers, we find that packaging occurs in increments of 10 bp. Statistical analysis of the preceding dwell times reveals that multiple ATPs bind during each dwell, and application of high force reveals that these 10-bp increments are composed of four 2.5-bp steps. These results indicate that the hydrolysis cycles of the individual subunits are highly coordinated via a mechanism novel for ring-ATPases. In addition, a step size that is a non-integer number of base pairs demands new models for motor-DNA interactions.