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A fast and versatile technique has been developed for detecting small quantities of specific microorganisms or molecules with high specificity. The target analytes are bound to a substrate and placed in the measurement cell of a microscope based on a high-transition temperature Superconducting Quantum Interference Device (SQUID). A solution containing nanometer-size magnetite particles, coated with antibodies specific to the target, is added. The particles, which bind to the target via the antibody- antigen interaction, are superparamagnetic with a Neel relaxation time of ~1s. A pulsed magnetic field aligns the dipole moments, and the SQUID measures the magnetic relaxation signal when the field is turned off. Unbound magnetic particles relax rapidly (~15microsecond(s) ) by Brownian rotation and are not detected. On the other hand, particles bound to targets cannot rotate and instead relax slowly by the Neel mechanism. As a result, only bound particles contribute to the signal, allowing for quantification of the number of targets present without the need for a wash step. The current system can detect as few as 2000 magnetic particles. This technique could be used to detect a wide range of bacteria, viruses, and molecules, with potential applications in the food industry, clinical settings, or research laboratories.

A technique is described for specific, sensitive, quantitative, and rapid detection of biological targets by using superparamagnetic nanoparticles and a “microscope” based on a high-transition temperature dc superconducting quantum interference device (SQUID). In this technique, a mylar film to which the targets have been bound is placed on the microscope. The film, at room temperature and atmospheric pressure, is typically 40 μm from the SQUID, which is at 77 K in a vacuum. A suspension of magnetic nanoparticles carrying antibodies directed against the target is added to the mixture in the well, and 1-s pulses of magnetic field are applied parallel to the SQUID. In the presence of this aligning field the nanoparticles develop a net magnetization, which relaxes when the field is turned off. Unbound nanoparticles relax rapidly by Brownian rotation and contribute no measurable signal. Nanoparticles that are bound to the target on the film are immobilized and undergo Néel relaxation, producing a slowly decaying magnetic flux, which is detected by the SQUID. The ability to distinguish between bound and unbound labels allows one to run homogeneous assays, which do not require separation and removal of unbound magnetic particles. The technique has been demonstrated with a model system of liposomes carrying the FLAG epitope. The SQUID microscope requires no more than (5 ± 2) × 10 4 magnetic nanoparticles to register a reproducible signal.

We present single-molecule in vitro measurements of the DNA unwinding activity of the Superfamily 1 UvrD helicase that reveal directly how helicase stoichiometry and conformation correspond to motor activity. Although a single UvrD molecule can translocate along ssDNA, it has been postulated that only a dimer is capable of unwinding duplex DNA. UvrD is known to exhibit two different conformations: the ‘closed' and ‘open' orientation of the 2B regulatory subdomain. The function of these conformations has remained elusive. using a new instrument combining high-resolution optical tweezers with simultaneous single-molecule fluorescence microscopy (Comstock, Ha, and Chemla, Nature Methods 2011), we obtain an unprecedented detailed view of helicase motor dynamics by recording single base pair-scale DNA unwinding activity (optical tweezers) simultaneously with helicase stoichiometry and conformation (fluorescence). We observe two classes of DNA unwinding by UvrD: long distance continuous unwinding (∼100 bp) and repetitive, non-processive (∼12 bp) unwinding. We determine directly the stoichiometry of UvrD molecules participating in unwinding by quantifying the fluorescence intensity using singly-labeled UvrD molecules. We observe that pairs of UvrD molecules are required for long distance unwinding but individual molecules exhibit limited non-processive unwinding activity. Furthermore, we observe directly the conformation of the 2B subdomain of individual UvrD molecules during unwinding by measuring the fluorescence of FRET-labeled proteins. We observe that UvrD is in the ‘closed' conformation during DNA unwinding but surprisingly switches to the ‘open' conformation when UvrD translocates on the opposing ssDNA strand away from the junction with the DNA junction re-annealing behind it. These measurements may explain UvrD helicase's remarkable ability to switch translocation strands. We hypothesize that the UvrD 2B subdomain acts as a switch that controls DNA unwinding vs. re-annealing.

Although it is known that single-stranded DNA binding proteins can stimulate helicase activity, the mechanism by which this occurs may be more complex than sequestering ssDNA products of duplex separation. Here, we present a single-molecule helicase assay with base-pair sensitivity, which utilizes high-resolution optical tweezers combined with microfluidics and fluorescence microscopy to decipher how FacXPD helicase is modulated by FacRPA2. FacXPD is the archaeal homolog of yeast Rad3 and human xeroderma pigmentosum group D protein (XPD) helicase from the organism Ferroplasma acidarmanus. This enzyme serves as a model for understanding the molecular mechanism of human Superfamily 2B helicase XPD involved in transcription initiation and nucleotide excision repair and related helicases FANCJ, RTEL and CHLR1 involved in maintenance of the genomic integrity. First, we demonstrated that monomeric XPD unwinds duplex DNA in single base-pair steps, yet is non-processive, unwinding for short distances (∼12 bp) and displaying a strong dependence on DNA sequence. Second, we investigated how RPA2 by itself interacts with DNA. We showed that RPA2 can unwind duplex DNA in steps of ∼5-8 bp in the presence of an assisting force of 12 pN. Finally, we examined the effect of RPA2 on XPD activity. Using our microfluidic platform, we performed experiments in which XPD and RPA2 were sequentially assembled on a DNA substrate in a controlled order. RPA2 increased XPD processivity. Duplex unwinding occurred in 1-bp steps, indicating that RPA2 is not directly involved in strand separation. Importantly, our data suggest that only 1-3 RPA2 molecules are sufficient to turn XPD into a processive helicase. We propose two scenarios. Either RPA2 forms a complex with XPD, or it alters its interaction with DNA upon binding, activating it for processive unwinding. We discuss the biological implications of our findings.

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.

Nearly all aspects of nucleic acid metabolism involve motor proteins. This diverse group of enzymes, which includes DNA and RNA polymerases, the ribosome, helicases, and other translocases, converts chemical energy in the form of bond hydrolysis into concerted motion along nucleic acid filaments. The direct observation of this motion at its fundamental distance scale of one base pair has required the development of new ultrasensitive techniques. Recent advances in optical traps have now made these length scales, once the exclusive realm of crystallographic techniques, accessible. Several new studies using optical traps have revealed for the first time how motor proteins translocate along their substrates in a stepwise fashion. Though these techniques have only begun to be applied to biological problems, the unprecedented access into nucleic acid motor protein movement has already provided important insights into their mechanism. In this perspective, we review these advances and offer our view on the future of this exciting development.

INTRODUCTIONOptical 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.

INTRODUCTIONOptical 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.

INTRODUCTIONOptical 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.

INTRODUCTIONOptical 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 Å. Here we briefly review the basic concepts and components of optical traps and the single-molecule experiments in which they are used.

INTRODUCTIONOptical 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.

DNA breaks can be repaired by homologous recombination, a process that maintains genetic stability in an organism. A protein that is essential for this mechanism in E. coli is RecA. During repair, RecA must bind and form nucleoprotein filaments on single-stranded DNA (ssDNA) in direct competition with single-stranded DNA binding protein (SSB). Despite extensive studies, the mechanism behind this competitive process remains unclear.Here, we use high-resolution optical tweezers with simultaneous fluorescence microscopy to observe directly the mechanical properties of ssDNA-SSB, ssDNA-RecA, and ssDNA-SSB-RecA complexes under a range of tensions. This single-molecule assay allows us to probe and visualize simultaneously the interactions of RecA and SSB with ssDNA in real time and with nanometer resolution. Using a 70-nucleotide, poly-dT ssDNA construct designed to accommodate a single SSB, we investigated different scenarios of protein-DNA complex formation under a range of tensions between 0-10 pN.Individual SSBs on their own bind and condense ssDNA in discrete steps, the size of which depend on tension. Under low tensions (1-3 pN), an SSB wraps 50-70 nt of ssDNA in a single step. At higher tensions (>4 pN), SSB exhibit transient, partially wrapped states on ssDNA. In the absence of SSB, RecA filaments nucleate rapidly on ssDNA regardless of tension. In contrast, when RecA is added to ssDNA coated with an SSB, filament formation is inhibited at low tensions. At higher tensions, where the SSB can partially unwrap from ssDNA, we observe RecA filaments form after an extended lag time. Our results suggest a mechanism in which SSBs inhibit RecA by sequestering ssDNA. Tension-induced unwrapping of the SSB makes ssDNA available for RecA to bind and nucleate a filament.

A significant subset of protein-DNA interactions occur between proteins that identify a specific target sequence amongst large sections of nonspecific DNA. The currently accepted model for this process states that sequence-specific DNA-binding proteins (SSDBPs) find their DNA targets by a ubiquitous process in which they bind loosely to DNA to scan DNA in 1-D, adopting a tightly bound state to the DNA only at the target sequence1. However, little consideration has been made for the vast array of properties exhibited by SSDBPs which could influence protein search behavior prior to finding the DNA target site. SSDBPs differ considerably in size, DNA target sequence length, function, and (in)dependence on cofactors. These properties affect protein activity at the target sites and may similarly affect protein search mechanisms.We propose that SSDBP search mechanisms may not be ubiquitously defined for all SSDBPs. We study protelomerase TelK, a cofactor-independent single-turnover protein responsible for the formation of DNA hairpins in prokaryotic DNA, and its mechanism of DNA target site identification2. High resolution optical trap studies show an unpredicted observation of nonspecific DNA condensation by TelK. TIRFM studies show that TelK 1D scanning of nonspecific DNA occurs only at low concentrations where TelK is a monomer. 1D scanning ceases at higher TelK concentrations, as TelK forms aggregates of multiple TelK units. This suggests that TelK searches for its target site while loosely bound to DNA as a monomer and aggregates once it encounters another TelK unit on DNA, before reaching the target site. These results suggest a mechanism contrary to the currently accepted model for SSDBP targeting mechanisms, indicating that these processes may vary from protein to protein.1. Halford et al. Nucl. Ac. Res 32, 3040 (2004).2. Aihara et al. Mol. Cell 27, 901 (2007).

We report our results on calibrating an optical trap in vivo while simultaneously trapping and tracking organelles with it. We have built an optical trap with microsecond time resolution and nm spatial resolution with a trapping laser that is positioned with an Acousto-Optic Deflector (AOD). This AOD is of fundamental importance to the in vivo stiffness calibration technique, as this technique requires high speed oscillation of the trap position (on the order of 10's of kHz), in order to compare an active spectrum (the system's response to laser driving) with a passive spectrum (the system's response to Brownian motion). In addition, we have an AOD in our detection laser's path, allowing us to calibrate our position detection Quadrant Photo Diode's (QPD) volts to nanometers conversion. Our system allows calibration of every necessary trap parameter in the cell on each individual organelle we trap. We currently trap lipid droplets in Cos-7 cells, and are planning on measuring motor stall forces, motor number, and looking at switching between kinesin based transport and dynein based transport.

We present a single-cell motility assay1, which allows the quantification of bacterial swimming in a well-controlled environment, for durations of up to an hour and with a temporal resolution greater than the flagellar rotation rates of ∼100 Hz. The assay is based on an instrument combining optical tweezers, light and fluorescence microscopy, and a microfluidic chamber. Using this device we characterized the long-term statistics of the run-tumble time series in individual Escherichia coli cells. We also quantified higher-order features of bacterial swimming, such as changes in velocity and reversals of swimming direction.[1] Min, T.L., Mears, P.J., Chubiz, L.M., Rao, C.V., Golding, I. & Chemla, Y.R. (2009) High-resolution, long-term characterization of bacterial motility using optical tweezers. Nature Methods (in press)View Large Image | View Hi-Res Image | Download PowerPoint Slide

Leukocytes are responsible for fighting infections in the body. When injuries occur, selectin molecules are expressed on the surface of nearby blood vessel walls. These selectin molecules transiently adhere to leukocytes flowing in the bloodstream and capture them, leading to leukocyte rolling towards the injury site. Rolling adhesion is critical for leukocytes to locate injury sites and to activate various signaling pathways for subsequent transmigration and chemotaxis.Individual adhesion components involved in rolling adhesion, such as adhesion molecules and membrane tethers have been characterized. However, models incorporating these properties are still unable to describe fully the rolling behavior. Current models assume uniformly distributed adhesion properties on cell surfaces due to the lack of measurements quantifying this distribution. Here, we determined experimentally the spatial distribution of adhesive properties on leukocyte surfaces. We used dark-field imaging and particle tracking techniques to extract not only the translational but also the rotational motion of a single rolling cell. The additional rotational information allows us to map precisely the whole cell motion and adhesion properties to a particular cell orientation. We find that the adhesion properties of the leukocyte surface are far from homogenous, with large, localized patches on the cell surface exhibiting strong or weak adhesive properties. This finding provides new insight into leukocyte adhesion properties, such as the asymmetric distribution of receptor and microvilli on rolling cells, and could lead to better modeling of rolling adhesion.

E. coli SSBs (single-stranded DNA binding proteins) are essential accessory proteins in replication, recombination and during DNA repair. They not only protect ssDNA from chemical and nucleolytic attack, but also associate with many genome maintenance proteins. E. coli SSBs are tetrameric and have at least two binding modes: SSB35 and SSB65, which bind 35 and 65 nucleotides, respectively. Proteins in the SSB35 mode bind cooperatively, interacting with each other and creating long nucleoprotein filaments. In this study, we present mechanical studies of SSB bound to ssDNA using dual trap, high-resolution optical tweezers. This assay allows us to probe the interaction of SSBs to ssDNA constructs of various lengths, in real time, with nanometer resolution. By directly detecting wrapping of ssDNA by a single protein, we are able to characterize the thermodynamics and kinetics of nucleoprotein complex formation. Mechanical pulling of our constructs in the presence of SSBs reveals that the protein condenses ssDNA in the force range 0-8pN and that tension can be used to modulate the binding mode of SSBs. Binding kinetics further indicate that SSBs bind to ssDNA in two successive processes consisting of a loose binding step in which the protein associates weakly with its substrate, followed by a wrapping step in which ssDNA is condensed.

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)