Statistical physics and mesoscopic modeling to interpret tethered particle motion experiments

Manoel Manghi, Nicolas Destainville, Annaël Brunet

Tethered particle motion experiments are versatile single-molecule techniques enabling one to address in vitro the molecular properties of DNA and its interactions with various partners involved in genetic regulations. These techniques provide raw data such as the tracked particle amplitude of movement, from which relevant information about DNA conformations or states must be recovered. Solving this inverse problem appeals to specific theoretical tools that have been designed in the two last decades, together with the data pre-processing procedures that ought to be implemented to avoid biases inherent to these experimental techniques. These statistical tools and models are reviewed in this paper.

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Multi-parameter measurements of conformational dynamics in nucleic acids and nucleoprotein complexes

Ivan E. Ivanov, Zev Bryant

Biological macromolecules undergo dynamic conformational changes. Single-molecule methods can track such structural rearrangements in real time. However, while the structure of large macromolecules may change along many degrees of freedom, single-molecule techniques only monitor a limited number of these axes of motion. Advanced single-molecule methods are being developed to track multiple degrees of freedom in nucleic acids and nucleoprotein complexes at high resolution, to enable better manipulation and control of the system under investigation, and to collect measurements in massively parallel fashion. Combining complementary single-molecule methods within the same assay also provides unique measurement opportunities. Implementations of magnetic and optical tweezers combined with fluorescence and FRET have demonstrated results unattainable by either technique alone. Augmenting other advanced single-molecule methods with fluorescence detection will allow us to better capture the multidimensional dynamics of nucleic acids and nucleoprotein complexes central to biology.

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MspA nanopore as a single-molecule tool: From sequencing to SPRNT

Andrew H. Laszlo, Ian M. Derrington, Jens H. Gundlach

Single-molecule picometer resolution nanopore tweezers (SPRNT) is a new tool for analyzing the motion of nucleic acids through molecular motors. With SPRNT, individual enzymatic motions along DNA as small as 40 pm can be resolved on sub-millisecond time scales. Additionally, SPRNT reveals an enzyme’s exact location with respect to a DNA strand’s nucleotide sequence, enabling identification of sequence-specific behaviors. SPRNT is enabled by a mutant version of the biological nanopore formed by Mycobacterium smegmatis porin A (MspA). SPRNT is strongly rooted in nanopore sequencing and therefore requires a solid understanding of basic principles of nanopore sequencing. Furthermore, SPRNT shares tools developed for nanopore sequencing and extends them to analysis of single-molecule kinetics. As such, this review begins with a brief history of our work developing the nanopore MspA for nanopore sequencing. We then describe the underlying principles of SPRNT, how it works in detail, and propose some potential future uses. We close with a comparison of SPRNT to other techniques and we present the methods that will enable others to use SPRNT.

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Construction of a system for single-cell transgene induction in Caenorhabditis elegans using a pulsed infrared laser

Matthew A. Churgin, Liping He, John I. Murray, Christopher Fang-Yen

The spatial and temporal control of transgene expression is an important tool in Caenorhabditis elegans biology. We previously described a method for evoking gene expression in arbitrary cells by using a focused pulsed infrared laser to induce a heat shock response (Churgin et al., 2013) [1]. Here we describe detailed methods for building and testing a system for performing single-cell heat shock. Steps include setting up the laser and associated components, coupling the laser beam to a microscope, and testing heat shock protocols. All steps can be carried out using readily available off-the-shelf components.

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Brownian nanoimaging of interface dynamics and ligand–receptor binding at cell surfaces in 3-D

Igor R. Kuznetsov, Evan A. Evans
We describe a method for nanoimaging interfacial dynamics and ligand–receptor binding at surfaces of live cells in 3-D. The imaging probe is a 1-μm diameter glass bead confined by a soft laser trap to create a “cloud” of fluctuating states. Using a facile on-line method of video image analysis, the probe displacements are reported at ∼10 ms intervals with bare precisions (±SD) of 4–6 nm along the optical axis (elevation) and 2 nm in the transverse directions. We demonstrate how the Brownian distributions are analyzed to characterize the free energy potential of each small probe in 3-D taking into account the blur effect of its motions during CCD image capture. Then, using the approach to image interactions of a labeled probe with lamellae of leukocytic cells spreading on cover-glass substrates, we show that deformations of the soft distribution in probe elevations provide both a sensitive long-range sensor for defining the steric topography of a cell lamella and a fast telemetry for reporting rare events of probe binding with its surface receptors. Invoking established principles of Brownian physics and statistical thermodynamics, we describe an off-line method of super resolution that improves precision of probe separations from a non-reactive steric boundary to ∼1 nm.
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Direct physical study of kinetochore–microtubule interactions by reconstitution and interrogation with an optical force clamp

Andrew D. Franck, Andrew F. Powers, Daniel R. Gestaut, Trisha N. Davis and Charles L. Asbury

We detail our use of computer-controlled optical traps to study interactions between kinetochore components and dynamic microtubules. Over the last two decades optical traps have helped uncover the working principles of conventional molecular motors, such as kinesin and dynein, but only recently have they been applied to study kinetochore function. The most useful traps combine sensitive position detectors and servo-control, allowing them to be operated as force clamps that maintain constant loads on objects as they move. Our instrument, which is among the simplest designs that permits force clamping, relies on a computer-controlled piezoelectric stage and a single laser for trapping and position detection. We apply it in motility assays where beads coated with pure microtubule-binding kinetochore components are attached to the tips of individual dynamic microtubules. Like kinetochores in vivo, the beads remain tip-attached, undergoing movements coupled to filament assembly and disassembly. The force clamp provides many benefits over instruments that lack feedback control. It allows tension to be applied continuously during both assembly- and disassembly-driven movement, providing a close match to the physiological situation. It also enables tracking with high resolution, and simplifies data interpretation by eliminating artifacts due to molecular compliance. The formation of persistent, load-bearing attachments to dynamic microtubule tips is fundamental to all kinetochore activities. Our direct, physical study of kinetochore–microtubule coupling may therefore furnish insights into many vital kinetochore functions, including correction of aberrant attachments and generation of the ‘wait-anaphase’ signals that delay mitosis until all kinetochores are properly attached.

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Single molecule transcription elongation

Eric A. Galburt, Stephan W. Grill and Carlos Bustamante

Single molecule optical trapping assays have now been applied to a great number of macromolecular systems including DNA, RNA, cargo motors, restriction enzymes, DNA helicases, chromosome remodelers, DNA polymerases and both viral and bacterial RNA polymerases. The advantages of the technique are the ability to observe dynamic, unsynchronized molecular processes, to determine the distributions of experimental quantities and to apply force to the system while monitoring the response over time. Here, we describe the application of these powerful techniques to study the dynamics of transcription elongation by RNA polymerase II from Saccharomyces cerevisiae.

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