arXiv subjects

Zhaokun Zhou

Publications and source records attributed to Zhaokun Zhou.

23 records · Page 2Linked to original sources

arXiv2017

Single-molecule techniques in biophysics: a review of the progress in methods and applications

Single-molecule biophysics has transformed our understanding of the fundamental molecular processes involved in living biological systems, but also of the fascinating physics of life. Far more exotic than a collection of exemplars of soft matter behaviour, active biological matter lives far from thermal equilibrium, and typically covers multiple length scales from the nanometre level of single molecules up several orders of magnitude to longer length scales in emergent structures of cells, tissues and organisms. Biological molecules are often characterized by an underlying instability, in that multiple metastable free energy states exist which are separated by energy levels of typically just a few multiples of the thermal energy scale of kBT, where kB is the Boltzmann constant and T the absolute temperature, implying complex, dynamic inter-conversion kinetics across this bumpy free energy landscape in the relatively hot, wet environment of real, living biological matter. The key utility of single-molecule biophysics lies in its ability to probe the underlying heterogeneity of free energy states across a population of molecules, which in general is too challenging for conventional ensemble level approaches which measure mean average properties. Parallel developments in both experimental and theoretical techniques have been key to the latest insights and are enabling the development of highly-multiplexed, correlative techniques to tackle previously intractable biological problems. Experimentally, technological developments in the sensitivity and speed of biomolecular detectors, the stability and efficiency of light sources, probes and microfluidics, have enabled and driven the study of heterogeneous behaviours both in vitro and in vivo that were previously undetectable by ensemble methods...

physics.bio-ph↗

arXiv2016

Force spectroscopy in studying infection

Biophysical force spectroscopy tools - for example optical tweezers, magnetic tweezers, atomic force microscopy, - have been used to study elastic, mechanical, conformational and dynamic properties of single biological specimens from single proteins to whole cells to reveal information not accessible by ensemble average methods such as X-ray crystallography, mass spectroscopy, gel electrophoresis and so on. Here we review the application of these tools on a range of infection-related questions from antibody-inhibited protein processivity to virus-cell adhesion. In each case we focus on how the instrumental design tailored to the biological system in question translates into the functionality suitable for that particular study. The unique insights that force spectroscopy has gained to complement knowledge learned through population averaging techniques in interrogating biomolecular details prove to be instrumental in therapeutic innovations such as those in structure-based drug design.

q-bio.BM↗

arXiv2015

Developing a new biophysical tool to combine magneto-optical tweezers with super-resolution fluorescence microscopy

We present a novel experimental setup in which magnetic and optical tweezers are combined for torque and force transduction onto single filamentous molecules in a transverse configuration to allow simultaneous mechanical measurement and manipulation. Previously we have developed a super-resolution imaging module which in conjunction with advanced imaging techniques such as Blinking assisted Localisation Microscopy (BaLM) achieves localisation precision of single fluorescent dye molecules bound to DNA of ~30 nm along the contour of the molecule; our work here describes developments in producing a system which combines tweezing and super-resolution fluorescence imaging. The instrument also features an acousto-optic deflector that temporally divides the laser beam to form multiple traps for high throughput statistics collection. Our motivation for developing the new tool is to enable direct observation of detailed molecular topological transformation and protein binding event localisation in a stretching/twisting mechanical assay that previously could hitherto only be deduced indirectly from the end-to-end length variation of DNA. Our approach is simple and robust enough for reproduction in the lab without the requirement of precise hardware engineering, yet is capable of unveiling the elastic and dynamic properties of filamentous molecules that have been hidden using traditional tools.

q-bio.BM↗

arXiv2015

Superresolution imaging of single DNA molecules using stochastic photoblinking of minor groove and intercalating dyes

As proof-of-principle for generating superresolution structural information from DNA we applied a method of localization microscopy utilizing photoblinking comparing intercalating dye YOYO-1 against minor groove binding dye SYTO-13, using a bespoke multicolor single-molecule fluorescence microscope. We used a full-length ~49 kbp λ DNA construct possessing oligo inserts at either terminus allowing conjugation of digoxigenin and biotin at opposite ends for tethering to a glass coverslip surface and paramagnetic microsphere respectively. We observed stochastic DNA-bound dye photoactivity consistent with dye photoblinking as opposed to binding/unbinding events, evidenced through both discrete simulations and continuum kinetics analysis. We analyzed dye photoblinking images of immobilized DNA molecules using superresolution reconstruction software from two existing packages, rainSTORM and QuickPALM, and compared the results against our own novel home-written software called ADEMS code. ADEMS code generated lateral localization precision values of 30-40 nm and 60-70 nm for YOYO-1 and SYTO-13 respectively at video-rate sampling, similar to rainSTORM, running more slowly than rainSTORM and QuickPALM algorithms but having a complementary capability over both in generating automated centroid distribution and cluster analyses. Our imaging system allows us to observe dynamic topological changes to single molecules of DNA in real-time, such as rapid molecular snapping events. This will facilitate visualization of fluorescently-labeled DNA molecules conjugated to a magnetic bead in future experiments involving newly developed magneto-optical tweezers combined with superresolution microscopy.

q-bio.BM↗

arXiv2015

Probing DNA interactions with proteins using a single-molecule toolbox: inside the cell, in a test tube, and in a computer

DNA-interacting proteins have roles multiple processes, many operating as molecular machines which undergo dynamic metastable transitions to bring about their biological function. To fully understand this molecular heterogeneity, DNA and the proteins that bind to it must ideally be interrogated at a single molecule level in their native in vivo environments, in a time-resolved manner fast to sample the molecular transitions across the free energy landscape. Progress has been made over the past decade in utilising cutting-edge tools of the physical sciences to address challenging biological questions concerning the function and modes of action of several different proteins which bind to DNA. These physiologically relevant assays are technically challenging, but can be complemented by powerful and often more tractable in vitro experiments which confer advantages of the chemical environment with enhanced detection single-to-noise of molecular signatures and transition events. Here, we discuss a range of techniques we have developed to monitor DNA-protein interactions in vivo, in vitro and in silico. These include bespoke single-molecule fluorescence microscopy techniques to elucidate the architecture and dynamics of the bacterial replisome and the structural maintenance of bacterial chromosomes, as well as new computational tools to extract single-molecule molecular signatures from live cells to monitor stoichiometry, spatial localization and mobility in living cells. We also discuss recent developments from our lab made in vitro, complementing these in vivo studies, which combine optical and magnetic tweezers to manipulate and image single molecules of DNA, with and without bound protein, in a new superresolution fluorescence microscope.

q-bio.BM↗