Omar A. Saleh

Direct detection of antibody–antigen binding using an on-chip artificial pore

We demonstrate a rapid and highly sensitive all-electronic technique based on the resistive pulse method of particle sizing with a pore to detect the binding of unlabeled antibodies to the surface of latex colloids. Here, we use an on-chip pore to sense colloids derivatized with streptavidin and measure accurately their diameter increase on specific binding to several different types of antibodies. We show the sensitivity of this technique to the concentration of free antibody and that it can be used to perform immunoassays in both inhibition and sandwich configurations. Overall, our technique does not require labeling of the reactants and is performed rapidly by using very little solution, and the pore itself is fabricated quickly and inexpensively by using soft lithography. Finally, because this method relies only on the volume of bound ligand, it can be generally applied to detecting a wide range of ligand–receptor binding reactions.

KOPS: DNA motifs that control E. coli chromosome segregation by orienting the FtsK translocase

Bacterial chromosomes are organized in replichores of opposite sequence polarity. This conserved feature suggests a role in chromosome dynamics. Indeed, sequence polarity controls resolution of chromosome dimers in Escherichia coli. Chromosome dimers form by homologous recombination between sister chromosomes. They are resolved by the combined action of two tyrosine recombinases, XerC and XerD, acting at a specific chromosomal site, dif, and a DNA translocase, FtsK, which is anchored at the division septum and sorts chromosomal DNA to daughter cells. Evidences suggest that DNA motifs oriented from the replication origin towards dif provide FtsK with the necessary information to faithfully distribute chromosomal DNA to either side of the septum, thereby bringing the dif sites together at the end of this process. However, the nature of the DNA motifs acting as FtsK orienting polar sequences (KOPS) was unknown. Using genetics, bioinformatics and biochemistry, we have identified a family of DNA motifs in the E. coli chromosome with KOPS activity.

Quantitative sensing of nanoscale colloids using a microchip Coulter counter

We have fabricated a microchip Coulter counter on a quartz substrate, and have used it to detect individual nanoscale colloidal particles with a sensitivity proportional to each particles size. We demonstrate the ability of this device to sense colloids as small as 87 nm diameter, and to distinguish between colloids whose diameters differ by less than 10%. Further reductions in the pore size, easily done with current nanofabrication techniques, make our device applicable to measuring biological macromolecules, such as DNA and proteins.

Fast, DNA-sequence independent translocation by FtsK in a single-molecule experiment

Escherichia coli FtsK is an essential cell division protein, which is thought to pump chromosomal DNA through the closing septum in an oriented manner by following DNA sequence polarity. Here, we perform single‐molecule measurements of translocation by FtsK50C, a derivative that functions as a DNA translocase in vitro. FtsK50C translocation follows Michaelis–Menten kinetics, with a maximum speed of ∼6.7 kbp/s. We present results on the effect of applied force on the speed, distance translocated, and the mean times during and between protein activity. Surprisingly, we observe that FtsK50C can spontaneously reverse its translocation direction on a fragment of E. coli chromosomal DNA, indicating that DNA sequence is not the sole determinant of translocation direction. We conclude that in vivo polarization of FtsK translocation could require the presence of cofactors; alternatively, we propose a model in which tension in the DNA directs FtsK translocation.

Multiplexed single-molecule measurements with magnetic tweezers

We present a method for performing multiple single-molecule manipulation experiments in parallel with magnetic tweezers. We use a microscope with a low magnification, and thus a wide field of view, to visualize multiple DNA-tethered paramagnetic beads and apply an optimized image analysis routine to track the three-dimensional position of each bead simultaneously in real time. Force is applied to each bead using an externally applied magnetic field. Since variations in the field parameters are negligible across the field of view, nearly identical manipulation of all visible beads is possible. However, we find that the error in the position measurement is inversely proportional to the microscopes magnification. To mitigate the increased error caused by demagnification, we have developed a strategy based on tracking multiple fixed beads. Our system is capable of simultaneously manipulating and tracking up to 34 DNA-tethered beads at 60 Hz with approximately 1.5 nm resolution and with approximately 10% variation in applied force.

Analysis of DNA supercoil induction by FtsK indicates translocation without groove-tracking.

FtsK is a bacterial protein that translocates DNA in order to transport chromosomes within the cell. During translocation, DNAs double-helical structure might cause a relative rotation between FtsK and the DNA. We used a single-molecule technique to quantify this rotation by observing the supercoils induced into the DNA during translocation of an FtsK complex. We find that FtsK induces ∼0.07 supercoils per DNA helical pitch traveled. This rate indicates that FtsK does not track along DNAs groove, but it is consistent with our previous estimate of FtsKs step size. We show that this rate of supercoil induction is markedly near to the ideal value that would minimize in vivo disturbance to the chromosomal supercoil density, suggesting an origin for the unusual rotational behavior of FtsK.

A high-resolution magnetic tweezer for single-molecule measurements

Magnetic tweezers (MT) are single-molecule manipulation instruments that utilize a magnetic field to apply force to a biomolecule-tethered magnetic bead while using optical bead tracking to measure the biomolecule’s extension. While relatively simple to set up, prior MT implementations have lacked the resolution necessary to observe sub-nanometer biomolecular configuration changes. Here, we demonstrate a reflection-interference technique for bead tracking, and show that it has much better resolution than traditional diffraction-based systems. We enhance the resolution by fabricating optical coatings on all reflecting surfaces that optimize the intensity and contrast of the interference image, and we implement feedback control of the focal position to remove drift. To test the system, we measure the length change of a DNA hairpin as it undergoes a folding/unfolding transition.

DNA mechanics as a tool to probe helicase and translocase activity

Helicases and translocases are proteins that use the energy derived from ATP hydrolysis to move along or pump nucleic acid substrates. Single molecule manipulation has proved to be a powerful tool to investigate the mechanochemistry of these motors. Here we first describe the basic mechanical properties of DNA unraveled by single molecule manipulation techniques. Then we demonstrate how the knowledge of these properties has been used to design single molecule assays to address the enzymatic mechanisms of different translocases. We report on four single molecule manipulation systems addressing the mechanism of different helicases using specifically designed DNA substrates: UvrD enzyme activity detection on a stretched nicked DNA molecule, HCV NS3 helicase unwinding of a RNA hairpin under tension, the observation of RecBCD helicase/nuclease forward and backward motion, and T7 gp4 helicase mediated opening of a synthetic DNA replication fork. We then discuss experiments on two dsDNA translocases: the RuvAB motor studied on its natural substrate, the Holliday junction, and the chromosome-segregation motor FtsK, showing its unusual coupling to DNA supercoiling.

Power spectrum and Allan variance methods for calibrating single-molecule video-tracking instruments.

Single-molecule manipulation instruments, such as optical traps and magnetic tweezers, frequently use video tracking to measure the position of a force-generating probe. The instruments are calibrated by comparing the measured probe motion to a model of Brownian motion in a harmonic potential well; the results of calibration are estimates of the probe drag, α, and spring constant, κ. Here, we present both time- and frequency-domain methods to accurately and precisely extract α and κ from the probe trajectory. In the frequency domain, we discuss methods to estimate the power spectral density (PSD) from data (including windowing and blocking), and we derive an analytical formula for the PSD which accounts both for aliasing and the filtering intrinsic to video tracking. In the time domain, we focus on the Allan variance (AV): we present a theoretical equation for the AV relevant to typical single-molecule setups and discuss the optimal manner for computing the AV from experimental data using octave-sampled overlapping bins. We show that, when using maximum-likelihood methods to fit to the data, both the PSD and AV approaches can extract α and κ in an unbiased and low-error manner, though the AV approach is simpler and more robust.

DnaB Helicase Activity Is Modulated by DNA Geometry and Force

The replicative helicase for Escherichia coli is DnaB, a hexameric, ring-shaped motor protein that encircles and translocates along ssDNA, unwinding dsDNA in advance of its motion. The microscopic mechanisms of DnaB are unknown; further, prior work has found that DnaBs activity is modified by other replication proteins, indicating some mechanistic flexibility. To investigate these issues, we quantified translocation and unwinding by single DnaB molecules in three tethered DNA geometries held under tension. Our data support the following conclusions: 1), Unwinding by DnaB is enhanced by force-induced destabilization of dsDNA. 2), The magnitude of this stimulation varies with the geometry of the tension applied to the DNA substrate, possibly due to interactions between the helicase and the occluded ssDNA strand. 3), DnaB unwinding and (to a lesser extent) translocation are interrupted by pauses, which are also dependent on force and DNA geometry. 4), DnaB moves slower when a large tension is applied to the helicase-bound strand, indicating that it must perform mechanical work to compact the strand against the applied force. Our results have implications for the molecular mechanisms of translocation and unwinding by DnaB and for the means of modulating DnaB activity.