Optical traps are widely used to study nucleic-acid-processing enzymes. To investigate these enzymes, we developed “force-activated” DNA substrates that contain a pair of nicks, allowing displacement of single-stranded DNA when pulled into DNA’s overstretching transition. We designed these substrates to include DNA hairpins and are using them to investigate the mechanism of E. coli RecQ helicase, an enzyme that unwinds double-stranded DNA. Force-activated DNA substrates also have the potential to provide an easy-to-use intrinsic force standard at ~15 pN, suitable for all three major force spectroscopy modalities (i.e., optical traps, magnetic tweezers, and AFM).
Optical-trapping-based assays can measure individual proteins bind to and move along DNA with sub-nm resolution, and have yielded insight into a broad array of protein-DNA interactions. Unfortunately, collecting large numbers of high-resolution traces remains an ongoing challenge. Studying helicase motion along DNA exemplifies this challenge. One major difficulty is that helicase binding often requires a single stranded (ss)-double stranded (ds) DNA junction flanked by ssDNA with a minimum size and orientation. Historically, creating such DNA substrates is inefficient. More problematic is that data throughput is low in standard surface-based assays since all substrates are unwound upon introduction of ATP. The net result is ~2–4 high-resolution traces on a good day. To improve throughput, we sought to turn-on or activate a substrate for a helicase one molecule at a time and thereby sequentially study many molecules on an individual microscope slide. As a first step towards this goal, we engineered a dsDNA that contains two site-specific nicks along the same strand of the dsDNA but no ssDNA. Upon overstretching the DNA (F = 65 pN), the strand between the two nicks was mechanically dissociated. We demonstrated this with two different substrates: one yielding an internal ssDNA region of 1100 nt and the other yielding a 20-bp long hairpin flanked by 30 nt of ssDNA. Unwinding a hairpin yields a 3-fold larger signal while the 30-nt ssDNA serves as the binding site for the helicase. We expect that these force-activated substrates to significantly accelerate high-resolution optical-trapping studies of DNA helicases.
Single-molecule studies of the mechanical properties of individual double-stranded DNA have excited interest across
many scientific disciplines because of DNA’s fundamental role in biology and DNA’s remarkable overstretching
transition at higher forces. Here, we discuss a recent result on the overstretching transition of DNA and on the dynamics
of dye molecules intercalating into DNA under tension. Overstretching DNA is mechanical transition whereby DNA’s
extension increases by 70% at 65 pN. Notwithstanding more than a decade of experimental and theoretical studies, there
remains significant debate on the nature of overstretched DNA. We developed a topologically closed but torsionally
unconstrained DNA assay that contains no nicks or free ends. DNA in this assay exhibited the canonical overstretching
transition at 65 pN but without hysteresis upon retraction. Controlled introduction of a nick led to hysteresis in the force
extension curve. Moreover, the degree of hysteresis increased with the number of nicks. In the second study, we isolated
the effects of binding and intercalation of a DNA staining dye, by combining single molecule force spectroscopy with
simple buffer exchange. We showed that force-enhanced intercalation can occur from a reservoir of bound dye that was
not bis-intercalated, yet remained out of equilibrium with free dye for long periods (<5 min for YOPRO and <2 hr for
YOYO). Our work highlights that binding/unbinding and intercalation/de-intercalation are distinct processes that can
occur on very different time scales. Taken together, these works highlight ongoing discoveries based on a twenty year
old technique, force spectroscopy of single DNA molecules.
Mechanical drift between an atomic force microscope (AFM) tip and sample is a longstanding problem that limits tipsample
stability, registration, and the signal-to-noise ratio during imaging. We demonstrate a robust solution to drift that
enables novel precision measurements, especially of biological macromolecules in physiologically relevant conditions.
Our strategy - inspired by precision optical trapping microscopy - is to actively stabilize both the tip and the sample
using locally generated optical signals. In particular, we scatter a laser off the apex of commercial AFM tips and use the
scattered light to locally measure and thereby actively control the tip's three-dimensional position above a sample
surface with atomic precision in ambient conditions. With this enhanced stability, we overcome the traditional need to
scan rapidly while imaging and achieve a 5-fold increase in the image signal-to-noise ratio. Finally, we demonstrate
atomic-scale (~ 100 pm) tip-sample stability and registration over tens of minutes with a series of AFM images. The
stabilization technique requires low laser power (<1 mW), imparts a minimal perturbation upon the cantilever, and is
independent of the tip-sample interaction. This work extends atomic-scale tip-sample control, previously restricted to
cryogenic temperatures and ultrahigh vacuum, to a wide range of perturbative operating environments.
We developed a freely available interactive simulation of optical traps and their biological applications
(phet.colorado.edu). The target audience is undergraduate majors as well as more advanced researchers. The simulation
has three panels: optical traps, manipulating DNA, and measuring molecular motors. Each panel has options that allow
students to interactively explore key physical ideas. For instance, viscosity can be turned off to see the critical aspect of
dissipation, or time can be slowed down to see the oscillating electric field and the induced charge separation. An
overview of the simulation and specific exercises suitable for an undergraduate class are discussed.
Historically, precise vertical control of an atomic force microscope (AFM) tip while it is disengaged from the surface has
been an unsolved problem. By separately scattering a pair of lasers off the tip and a fiducial mark in the sample, we
locally measured and thereby actively controlled tip and sample position in three dimensions, achieving atomic-scale
(0.1 nm) precision at ambient conditions. We also measured cantilever deflection (force) using the standard optical-lever-
arm geometry. Both detection techniques were used to determine the vertical location of the surface (z = 0) relative
to the AFM tip assembly. The difference in these vertical determinations was 0.0 ± 0.3 nm (mean ± S.D.; N = 86). This
agreement allowed us to establish an optically based reference frame to measure the vertical position of the tip relative to
the surface. This reference frame is insensitive to long-term mechanical drift of the AFM assembly and complementary
to the cantilever deflection sensing, which measures force. We expect this dual z-detection to be useful in a broad array
of applications that demand precise tip-sample control, including tip-based nanofabrication and single-molecule force
spectroscopy.
Many precision measurement techniques (e.g. scanning probe microscopy, optical tweezers) are limited by sample drift.
This is particularly true at room temperature in air or in liquid. Previously, we developed a general solution for sample
control in three dimensions (3D) by first measuring the position of the sample and then using this position in a feedback
loop to move a piezo-electric stage accordingly (Carter et al., Optics Express, 2007). In that work, feedback was
performed using a software-based data acquisition program with limited bandwidth (≤ 100 Hz). By implementing
feedback through a field programmable gate array (FPGA), we achieved real-time, deterministic control and increased
the feedback rate to 500 Hz - half the resonance frequency of the piezo-electric stage in the feedback loop. This better
control led to a three-fold improvement in lateral stability to 10 pm (Δf = 0.01-10 Hz). Furthermore, we exploited the
rapid signal processing of FPGA to achieve fast stepping rates coupled with highly accurate and orthogonal scanning.
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