Kinetic and structural mechanism for DNA unwinding by a non-hexameric helicase

UvrD, a model for non-hexameric Superfamily 1 helicases, utilizes ATP hydrolysis to translocate stepwise along single-stranded DNA and unwind the duplex. Previous estimates of its step size have been indirect, and a consensus on its stepping mechanism is lacking. To dissect the mechanism underlying DNA unwinding, we use optical tweezers to measure directly the stepping behavior of UvrD as it processes a DNA hairpin and show that UvrD exhibits a variable step size averaging ~3 base pairs. Analyzing stepping kinetics across ATP reveals the type and number of catalytic events that occur with different step sizes. These single-molecule data reveal a mechanism in which UvrD moves one base pair at a time but sequesters the nascent single strands, releasing them non-uniformly after a variable number of catalytic cycles. Molecular dynamics simulations point to a structural basis for this behavior, identifying the protein-DNA interactions responsible for strand sequestration. Based on structural and sequence alignment data, we propose that this stepping mechanism may be conserved among other non-hexameric helicases

Statistical Analysis of Molecular Signal Recording

A molecular device that records time-varying signals would enable new approaches in neuroscience. We have recently proposed such a device, termed a “molecular ticker tape”, in which an engineered DNA polymerase (DNAP) writes time-varying signals into DNA in the form of nucleotide misincorporation patterns. Here, we define a theoretical framework quantifying the expected capabilities of molecular ticker tapes as a function of experimental parameters. We present a decoding algorithm for estimating time-dependent input signals, and DNAP kinetic parameters, directly from misincorporation rates as determined by sequencing. We explore the requirements for accurate signal decoding, particularly the constraints on (1) the polymerase biochemical parameters, and (2) the amplitude, temporal resolution, and duration of the time-varying input signals. Our results suggest that molecular recording devices with kinetic properties similar to natural polymerases could be used to perform experiments in which neural activity is compared across several experimental conditions, and that devices engineered by combining favorable biochemical properties from multiple known polymerases could potentially measure faster phenomena such as slow synchronization of neuronal oscillations. Sophisticated engineering of DNAPs is likely required to achieve molecular recording of neuronal activity with single-spike temporal resolution over experimentally relevant timescales.United States. Defense Advanced Research Projects Agency. Living Foundries ProgramGoogle (Firm)New York Stem Cell Foundation. Robertson Neuroscience Investigator AwardNational Institutes of Health (U.S.) (EUREKA Award 1R01NS075421)National Institutes of Health (U.S.) (Transformative R01 1R01GM104948)National Institutes of Health (U.S.) (Single Cell Grant 1 R01 EY023173)National Institutes of Health (U.S.) (Grant 1R01DA029639)National Institutes of Health (U.S.) (Grant 1R01NS067199)National Science Foundation (U.S.) (CAREER Award CBET 1053233)National Science Foundation (U.S.) (Grant EFRI0835878)National Science Foundation (U.S.) (Grant DMS1042134)Paul G. Allen Family Foundation (Distinguished Investigator in Neuroscience Award

Broadband THz absorption spectrometer based on excitonic nonlinear optical effects

A broadly tunable THz source is realized via difference frequency generation, in which an enhancement to χ (3) that is obtained via resonant excitation of III–V semiconductor quantum well excitons is utilized. The symmetry of the quantum wells (QWs) is broken by utilizing the built-in electric-field across a p–i–n junction to produce effective χ (2) processes, which are derived from the high χ (3) . This χ (2) media exhibits an onset of nonlinear processes at ~4 W cm −2 , thereby enabling area (and, hence, power) scaling of the THz emitter. Phase matching is realized laterally through normal incidence excitation. Using two collimated 130 mW continuous wave (CW) semiconductor lasers with ~1-mm beam diameters, we realize monochromatic THz emission that is tunable from 0.75 to 3 THz and demonstrate the possibility that this may span 0.2–6 THz with linewidths of ~20 GHz and efficiencies of ~1 × 10 –5 , thereby realizing ~800 nW of THz power. Then, transmission spectroscopy of atmospheric features is demonstrated, thereby opening the way for compact, low-cost, swept-wavelength THz spectroscopy

Quantitatively probing the magnetic behavior of individual nanoparticles by an AC field-modulated magnetic force microscopy

Despite decades of advances in magnetic imaging, obtaining direct, quantitative information with nanometer scale spatial resolution remains an outstanding challenge. Current approaches, for example, Hall micromagnetometer and nitrogen-vacancy magnetometer, are limited by highly complex experimental apparatus and a dedicated sample preparation process. Here we present a new AC field-modulated magnetic force microscopy (MFM) and report the local and quantitative measurements of the magnetic information of individual magnetic nanoparticles (MNPs), which is one of the most iconic objects of nanomagnetism. This technique provides simultaneously a direct visualization of the magnetization process of the individual MNPs, with spatial resolution and magnetic sensitivity of about 4.8 nm and 1.85 × 10(−20) A m(2), respectively, enabling us to separately estimate the distributions of the dipolar fields and the local switching fields of individual MNPs. Moreover, we demonstrate that quantitative magnetization moment of individual MNPs can be routinely obtained using MFM signals. Therefore, it underscores the power of the AC field-modulated MFM for biological and biomedical applications of MNPs and opens up the possibility for directly and quantitatively probing the weak magnetic stray fields from nanoscale magnetic systems with superior spatial resolution