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Monday, June 18, 2018

Quantifying the stability of oxidatively damaged DNA by single-molecule DNA stretching

Micah J McCauley Leah Furman Catherine A Dietrich Ioulia Rouzina Megan E Núñez Mark C Williams

One of the most common DNA lesions is created when reactive oxygen alters guanine. 8-oxo-guanine may bind in the anti-conformation with an opposing cytosine or in the syn-conformation with an opposing adenine paired by transversion, and both conformations may alter DNA stability. Here we use optical tweezers to measure the stability of DNA hairpins containing 8-oxoguanine (8oxoG) lesions, comparing the results to predictive models of base-pair energies in the absence of the lesion. Contrasted with either a canonical guanine-cytosine or adenine-thymine pair, an 8oxoG-cytosine base pair shows significant destabilization of several kBT. The magnitude of destabilization is comparable to guanine-thymine ‘wobble’ and cytosine-thymine mismatches. Furthermore, the measured energy of 8oxoG-adenine corresponds to theoretical predictions for guanine-adenine pairs, indicating that oxidative damage does not further destabilize this mismatch in our experiments, in contrast to some previous observations. These results support the hypothesis that oxidative damage to guanine subtly alters the direction of the guanine dipole, base stacking interactions, the local backbone conformation, and the hydration of the modified base. This localized destabilization under stress provides additional support for proposed mechanisms of enzyme repair.

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PLANT: A Method for Detecting Changes of Slope in Noisy Trajectories

Alberto Sosa-Costa, Izabela K. Piechocka, Lucia Gardini, Francesco S. Pavone, Marco Capitanio, Maria F. Garcia-Parajo, Carlo Manzo

Time traces obtained from a variety of biophysical experiments contain valuable information on underlying processes occurring at the molecular level. Accurate quantification of these data can help explain the details of the complex dynamics of biological systems. Here, we describe PLANT (Piecewise Linear Approximation of Noisy Trajectories), a segmentation algorithm that allows the reconstruction of time-trace data with constant noise as consecutive straight lines, from which changes of slopes and their respective durations can be extracted. We present a general description of the algorithm and perform extensive simulations to characterize its strengths and limitations, providing a rationale for the performance of the algorithm in the different conditions tested. We further apply the algorithm to experimental data obtained from tracking the centroid position of lymphocytes migrating under the effect of a laminar flow and from single myosin molecules interacting with actin in a dual-trap force-clamp configuration.

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Optoelectronic forces with quantum wells for cavity optomechanics in GaAs/AlAs semiconductor microcavities

V. Villafañe, P. Sesin, P. Soubelet, S. Anguiano, A. E. Bruchhausen, G. Rozas, C. Gomez Carbonell, A. Lemaître, and A. Fainstein

Radiation pressure, electrostriction, and photothermal forces have been investigated to evidence backaction, nonlinearities, and quantum phenomena in cavity optomechanics. We show here through a detailed study of the relative intensity of the cavity mechanical modes observed when exciting with pulsed lasers close to the GaAs optical gap that optoelectronic forces involving real carrier excitation and deformation potential interaction are the strongest mechanism of light-to-sound transduction in semiconductor GaAs/AlAs distributed Bragg reflector optomechanical resonators. We demonstrate that the ultrafast spatial redistribution of the photoexcited carriers in microcavities with massive GaAs spacers leads to an enhanced coupling to the fundamental 20-GHz vertically polarized mechanical breathing mode. The carrier diffusion along the growth axis of the device can be enhanced by increasing the laser power, or limited by embedding GaAs quantum wells in the cavity spacer, a strategy used here to prove and engineer the optoelectronic forces in phonon generation with real carriers. The wavelength dependence of the observed phenomena provide further proof of the role of optoelectronic forces. The optical forces associated with the different intervening mechanisms and their relevance for dynamical backaction in optomechanics are evaluated using finite-element methods. The results presented open the path to the study of hitherto seldom investigated dynamical backaction in optomechanical solid-state resonators in the presence of optoelectronic forces.

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Single-Stranded Condensation Stochastically Blocks G-Quadruplex Assembly in Human Telomeric RNA

Irene Gutiérrez, Miguel Garavís, Sara de Lorenzo, Alfredo Villasante, Carlos González, and J. Ricardo Arias-Gonzalez

TERRA is an RNA molecule transcribed from human subtelomeric regions toward chromosome ends potentially involved in regulation of heterochromatin stability, semiconservative replication, and telomerase inhibition, among others. TERRA contains tandem repeats of the sequence GGGUUA, with a strong tendency to fold into a four-stranded arrangement known as a parallel G-quadruplex. Here, we demonstrate by using single-molecule force spectroscopy that this potential is limited by the inherent capacity of RNA to self-associate randomly and further condense into entropically more favorable structures. We stretched RNA constructions with more than four and less than eight hexanucleotide repeats, thus unable to form several G-quadruplexes in tandem, flanked by non-G-rich overhangs of random sequence by optical tweezers on a one by one basis. We found that condensed RNA stochastically blocks G-quadruplex folding pathways with a near 20% probability, a behavior that is not found in DNA analogous molecules.

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KIF15 nanomechanics and kinesin inhibitors, with implications for cancer chemotherapeutics

Bojan Milic, Anirban Chakraborty, Kyuho Han, Michael C. Bassik, and Steven M. Block

Eg5, a mitotic kinesin, has been a target for anticancer drug development. Clinical trials of small-molecule inhibitors of Eg5 have been stymied by the development of resistance, attributable to mitotic rescue by a different endogenous kinesin, KIF15. Compared with Eg5, relatively little is known about the properties of the KIF15 motor. Here, we employed single-molecule optical-trapping techniques to define the KIF15 mechanochemical cycle. We also studied the inhibitory effects of KIF15-IN-1, an uncharacterized, commercially available, small-molecule inhibitor, on KIF15 motility. To explore the complementary behaviors of KIF15 and Eg5, we also scored the effects of small-molecule inhibitors on admixtures of both motors, using both a microtubule (MT)-gliding assay and an assay for cancer cell viability. We found that (i) KIF15 motility differs significantly from Eg5; (ii) KIF15-IN-1 is a potent inhibitor of KIF15 motility; (iii) MT gliding powered by KIF15 and Eg5 only ceases when both motors are inhibited; and (iv) pairing KIF15-IN-1 with Eg5 inhibitors synergistically reduces cancer cell growth. Taken together, our results lend support to the notion that a combination drug therapy employing both inhibitors may be a viable strategy for overcoming chemotherapeutic resistance.

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Machine-learning techniques for fast and accurate feature localization in holograms of colloidal particles

Mark D. Hannel, Aidan Abdulali, Michael O’Brien, and David G. Grier

Holograms of colloidal particles can be analyzed with the Lorenz-Mie theory of light scattering to measure individual particles’ three-dimensional positions with nanometer precision while simultaneously estimating their sizes and refractive indexes. Extracting this wealth of information begins by detecting and localizing features of interest within individual holograms. Conventionally approached with heuristic algorithms, this image analysis problem can be solved faster and more generally with machine-learning techniques. We demonstrate that two popular machine-learning algorithms, cascade classifiers and deep convolutional neural networks (CNN), can solve the feature-localization problem orders of magnitude faster than current state-of-the-art techniques. Our CNN implementation localizes holographic features precisely enough to bootstrap more detailed analyses based on the Lorenz-Mie theory of light scattering. The wavelet-based Haar cascade proves to be less precise, but is so computationally efficient that it creates new opportunities for applications that emphasize speed and low cost. We demonstrate its use as a real-time targeting system for holographic optical trapping.

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Friday, June 15, 2018

Microtubules soften due to cross-sectional flattening

Edvin Memet, Feodor Hilitsk, Margaret A Morris, Walter J Schwenger, Zvonimir Dogic, L Mahadevan

We use optical trapping to continuously bend an isolated microtubule while simultaneously measuring the applied force and the resulting filament strain, thus allowing us to determine its elastic properties over a wide range of applied strains. We find that, while in the low-strain regime, microtubules may be quantitatively described in terms of the classical Euler-Bernoulli elastic filament, above a critical strain they deviate from this simple elastic model, showing a softening response with increasing deformations. A three-dimensional thin-shell model, in which the increased mechanical compliance is caused by flattening and eventual buckling of the filament cross-section, captures this softening effect in the high strain regime and yields quantitative values of the effective mechanical properties of microtubules. Our results demonstrate that properties of microtubules are highly dependent on the magnitude of the applied strain and offer a new interpretation for the large variety in microtubule mechanical data measured by different methods.

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High-Resolution Large-Ensemble Nanoparticle Trapping with Multifunctional Thermoplasmonic Nanohole Metasurface

Justus C. Ndukaife, Yi Xuan, Agbai George Agwu Nnanna, Alexander V. Kildishev, Vladimir M. Shalaev, Steven T. Wereley, and Alexandra Boltasseva

The intrinsic loss in a plasmonic metasurface is usually considered to be detrimental for device applications. Using plasmonic loss to our advantage, we introduce a thermoplasmonic metasurface that enables high-throughput large-ensemble nanoparticle assembly in a lab-on-a-chip platform. In our work, an array of subwavelength nanoholes in a metal film is used as a plasmonic metasurface that supports the excitation of localized surface plasmon and Bloch surface plasmon polariton waves upon optical illumination and provides a platform for molding both optical and thermal landscapes to achieve a tunable many-particle assembling process. The demonstrated many-particle trapping occurs against gravity in an inverted configuration where the light beam first passes through the nanoparticle suspension before illuminating the thermoplasmonic metasurface, a feat previously thought to be impossible. We also report an extraordinarily enhanced electrothermoplasmonic flow in the region of the thermoplasmonic nanohole metasurface, with comparatively larger transport velocities in comparison to the unpatterned region. This thermoplasmonic metasurface could enable possibilities for myriad applications in molecular analysis, quantum photonics, and self-assembly and creates a versatile platform for exploring nonequilibrium physics.

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Direct Particle Tracking Observation and Brownian Dynamics Simulations of a Single Nanoparticle Optically Trapped by a Plasmonic Nanoaperture

Zhe Xu, Wuzhou Song, and Kenneth B. Crozier

Optical trapping using plasmonic nanoapertures has proven to be an effective means for the contactless manipulation of nanometer-sized particles under low optical intensities. These particles have included polystyrene and silica nanospheres, proteins, coated quantum dots and magnetic nanoparticles. Here we employ fluorescence microscopy to directly observe the optical trapping process, tracking the position of a polystyrene nanosphere (20 nm diameter) trapped in water by a double nanohole (DNH) aperture in a gold film. We show that position distribution in the plane of the film has an elliptical shape. Comprehensive simulations are performed to gain insight into the trapping process, including of the distributions of the electric field, temperature, fluid velocity, optical force, and potential energy. These simulations are combined with stochastic Brownian diffusion to directly model the dynamics of the trapping process, that is, particle trajectories. We anticipate that the combination of direct particle tracking experiments with Brownian motion simulations will be valuable tool for the better understanding of fundamental mechanisms underlying nanostructure-based trapping. It could thus be helpful in the development of the future novel optical trapping devices.

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Laser trap ionization for identification of human erythrocytes with variable hemoglobin quantitation

Michele Kelley; James Cooper; Daniel Devito; Robert Mushi; Maria del Pilar Aguinaga; Daniel B. Erenso

An approach to an established technique that is potentially applicable for a more comprehensive understanding of the electrical properties of red blood cells (RBCs) is presented. Using a high-intensity gradient laser trap, RBCs can be singly trapped and consequentially ionized. The subsequent dynamics of the ionized cell allows one to calculate the charge developed and the ionization energy (IE) through a Newtonian-based analysis. RBCs with two different hemoglobin (Hb) types were ionized. The first sample was identified as carrying Hb HbAA (normal Hb) and the second one was identified as carrying HbAC (HbC trait). By analyzing the charge developed on each cell and several other related factors, we were able to discern a difference between the main Hb types contained within the individual RBC, independent of cell size. A relationship between the charge developed and the IE of the cell was also established based on the electrical properties of RBCs. Thus, we present this laser trapping technique as a study of the electrical properties of RBCs and as possible biomedical tool to be used for the differentiation of Hb types.

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