scholarly journals DNA Dynamics and Single-Molecule Biology

2014 ◽  
Vol 114 (6) ◽  
pp. 3072-3086 ◽  
Author(s):  
Daniel Duzdevich ◽  
Sy Redding ◽  
Eric C. Greene
Keyword(s):  
Single Molecule ◽  
Dna Dynamics

scholarly journals Bridging the spatiotemporal scales of macromolecular transport in crowded biomimetic systems

2018 ◽  
Author(s):  
Kathryn Regan ◽  
Devynn Wulstein ◽  
Hannah Rasmussen ◽  
Ryan McGorty ◽  
Rae M. Robertson-Anderson
Keyword(s):  
Single Molecule ◽  
Biological Molecules ◽  
Biological Macromolecules ◽  
Dna Dynamics ◽  
Dna Transport ◽  
Macromolecular Transport ◽  
Dna Compaction ◽  
Biomimetic Systems ◽  
Conformational Properties ◽  
Spatiotemporal Scales

AbstractCrowding plays a key role in the transport and conformations of biological macromolecules. Gene therapy, viral infection and transfection require DNA to traverse the crowded cytoplasm, including a heterogeneous cytoskeleton of filamentous proteins. Given the complexity of cellular crowding, the dynamics of biological molecules can be highly dependent on the spatiotemporal scale probed. We present a powerful platform that spans molecular and cellular scales by coupling single-molecule conformational tracking (SMCT) and selective-plane illumination differential dynamic microscopy (SPIDDM). We elucidate the transport and conformational properties of large DNA, crowded by custom-designed networks of actin and microtubules, to link single-molecule conformations with ensemble DNA transport and cytoskeleton structure. We show that actin crowding leads to DNA compaction and suppression of fluctuations, combined with anomalous subdiffusion and heterogeneous transport, whereas microtubules have much more subdued impact across all scales. Interestingly, in composite networks of both filaments, microtubules primarily govern single-molecule DNA dynamics whereas actin governs ensemble transport.

scholarly journals Influence of the Experimental Set-Up on Single Molecule DNA Dynamics When Analyzed by Tethered Particle Motion

2010 ◽  
Vol 98 (3) ◽  
pp. 184a
Author(s):  
Catherine Tardin ◽  
Manoel Manghi ◽  
Julien Baglio ◽  
Laurence Salome ◽  
Nicolas Destainville
Keyword(s):  
Single Molecule ◽  
Particle Motion ◽  
Dna Dynamics ◽  
Tethered Particle Motion ◽  
Set Up

scholarly journals A molecular view of DNA flexibility

2021 ◽  
Vol 54 ◽  
Author(s):  
Alberto Marin-Gonzalez ◽  
J. G. Vilhena ◽  
Ruben Perez ◽  
Fernando Moreno-Herrero
Keyword(s):  
Mechanical Properties ◽  
Single Molecule ◽  
Base Pair ◽  
Molecular Mechanisms ◽  
Cytosine Methylation ◽  
Persistence Length ◽  
Dna Dynamics ◽  
Dna Mechanics ◽  
Future Design ◽  
Pair Level

Abstract DNA dynamics can only be understood by taking into account its complex mechanical behavior at different length scales. At the micrometer level, the mechanical properties of single DNA molecules have been well-characterized by polymer models and are commonly quantified by a persistence length of 50 nm (~150 bp). However, at the base pair level (~3.4 Å), the dynamics of DNA involves complex molecular mechanisms that are still being deciphered. Here, we review recent single-molecule experiments and molecular dynamics simulations that are providing novel insights into DNA mechanics from such a molecular perspective. We first discuss recent findings on sequence-dependent DNA mechanical properties, including sequences that resist mechanical stress and sequences that can accommodate strong deformations. We then comment on the intricate effects of cytosine methylation and DNA mismatches on DNA mechanics. Finally, we review recently reported differences in the mechanical properties of DNA and double-stranded RNA, the other double-helical carrier of genetic information. A thorough examination of the recent single-molecule literature permits establishing a set of general ‘rules’ that reasonably explain the mechanics of nucleic acids at the base pair level. These simple rules offer an improved description of certain biological systems and might serve as valuable guidelines for future design of DNA and RNA nanostructures.

scholarly journals Sequence-Dependent Dynamics of Synthetic and Endogenous RSSs in V(D)J Recombination

2019 ◽  
Author(s):  
Soichi Hirokawa ◽  
Griffin Chure ◽  
Nathan M. Belliveau ◽  
Geoffrey A. Lovely ◽  
Michael Anaya ◽  
...  
Keyword(s):  
Single Molecule ◽  
Large Scale ◽  
Antigen Receptor ◽  
Quantitative Information ◽  
Dna Dynamics ◽  
Endogenous Gene ◽  
Quantitative Studies ◽  
Recombination Signal Sequences ◽  
Sequence Dependent ◽  
Dna Nicking

Developing lymphocytes in the immune system of jawed vertebrates assemble antigen-receptor genes by undergoing large-scale reorganization of spatially separated V, D, and J gene segments through a process known as V(D)J recombination. The RAG protein initiates this process by binding and cutting recombination signal sequences (RSSs) composed of conserved heptamer and nonamer sequences flanking less well-conserved 12- or 23-bp spacers. Little quantitative information is known about the contributions of individual RSS positions over the course of the RAG-RSS interaction. We employ a single-molecule method known as tethered particle motion to quantify the formation, stability, and cleavage of the RAG-12RSS-23RSS paired complex (PC) for numerous synthetic and endogenous 12RSSs. We thoroughly investigate the sequence space around a RSS by making 40 different single-bp changes and characterizing the reaction dynamics. We reveal that single-bp changes affect RAG function based on their position: loss of cleavage function (first three positions of the heptamer); reduced propensity for forming the PC (the nonamer and last four bp of the heptamer); or variable effects on PC formation (spacer). We find that the rare usage of some endogenous gene segments can be mapped directly to their adjacent 12RSSs to which RAG binds weakly. The 12RSS, however, cannot explain the high-frequency usage of other gene segments. Finally, we find that RSS nicking, while not required for PC formation, substantially stabilizes the PC. Our findings provide detailed insights into the contribution of individual RSS positions to steps of the RAG-RSS re-action that previously have been difficult to assess quantitatively.SummaryV(D)J recombination is a genomic cut-and-paste process for generating diverse antigen-receptor repertoires. The RAG enzyme brings separate gene segments together by binding the neighboring sequences called RSSs, forming a paired complex (PC) before cutting the DNA. There are limited quantitative studies of the sequence-dependent dynamics of the crucial inter-mediate steps of PC formation and cleavage. Here, we quantify individual RAG-DNA dynamics for various RSSs. While RSSs of frequently-used segments do not comparatively enhance PC formation or cleavage, the rare use of some segments can be explained by their neighboring RSSs crippling PC formation and/or cleavage. Furthermore, PC lifetimes reveal DNA-nicking is not required for forming the PC, but PCs with nicks are more stable.

scholarly journals The relevance of nonlinear stacking interactions in simple models of double-stranded DNA

2006 ◽  
Vol 3 (10) ◽  
pp. 655-667 ◽  
Author(s):  
Giuseppe Saccomandi ◽  
Ivonne Sgura
Keyword(s):  
Single Molecule ◽  
Thermal Denaturation ◽  
Stacking Interactions ◽  
Solitary Wave Solutions ◽  
Dna Dynamics ◽  
Sharp Transition ◽  
Classical Domain ◽  
Double Stranded Dna ◽  
Dna Thermal Denaturation ◽  
Type Potential

Single molecule DNA experiments provide interesting data that allow a better understanding of the mechanical interactions between the strands and the nucleotides of this molecule. In some sense, these experiments complement the classical ones about DNA thermal denaturation. It is well known that the original Peyrard–Bishop (PB) model by means of a harmonic stacking potential and a nonlinear substrate potential has been able to predict the existence of a critical temperature of full denaturation of the molecule. In the present paper, driven by the findings of single molecule experiments, we substitute the original harmonic intra-strand stacking potential with a Duffing type potential. By elementary and analytical arguments, we show that with this choice it is possible to obtain a sharp transition in the classical domain wall solution of the PB model and the compactification of the classical solitary wave solutions of other models for the dynamics of DNA. We discuss why these solutions may improve our knowledge of the DNA dynamics in several directions.

scholarly journals Single molecule dynamics of DNA receptor ComEA, membrane permease ComEC and taken up DNA in competent Bacillus subtilis cells

2021 ◽  
Author(s):  
Marie Burghard-Schrod ◽  
Alexandra Kilb ◽  
Kai Krämer ◽  
Peter L. Graumann
Keyword(s):  
Bacillus Subtilis ◽  
Cell Membrane ◽  
Single Molecule ◽  
Metal Binding ◽  
Outer Cell ◽  
Dna Uptake ◽  
Dna Dynamics ◽  
Cell Pole ◽  
C Terminus ◽  
Capture Mechanism

In competent Gram-negative and Gram-positive bacteria, double stranded DNA is taken up through the outer cell membrane and/or the cell wall, and is bound by ComEA, which in Bacillus subtilis is a membrane protein. DNA is converted to single stranded DNA, and transported through the cell membrane via ComEC. We show that in Bacillus subtilis , the C-terminus of ComEC, thought to act as a nuclease, is not only important for DNA uptake, as judged from a loss of transformability, but also for the localization of ComEC to the cell pole and its mobility within the cell membrane. Using single molecule tracking, we show that only 13% of ComEC molecules are statically localised at the pole, while 87% move throughout the cell membrane. These experiments suggest that recruitment of ComEC to the cell pole is mediated by a diffusion/capture mechanism. Mutation of a conserved aspartate residue in the C-terminus, likely affecting metal binding, strongly impairs transformation efficiency, suggesting that this periplasmic domain of ComEC could indeed serve a catalytic function as nuclease. By tracking fluorescently labeled DNA, we show that taken up DNA has a similar mobility as a protein, in spite of being a large polymer. DNA dynamics are similar within the periplasm as those of ComEA, suggesting that most taken up molecules are bound to ComEA. We show that DNA can be highly mobile within the periplasm, indicating that this subcellular space can act as reservoir for taken up DNA, before its entry into the cytosol. Importance Bacteria can take up DNA from the environment and incorporate it into their chromosome, termed “natural competence” that can result in the uptake of novel genetic information. We show that fluorescently labelled DNA moves within the periplasm of competent Bacillus subtilis cells, with similar dynamics as DNA receptor ComEA. This indicates that DNA can accumulate in the periplasm, likely bound by ComEA, and thus can be stored before uptake at the cell pole, via integral membrane DNA permease ComEC. Assembly of the latter assembles at the cell pole likely occurs by a diffusion-capture mechanism. DNA uptake into cells thus takes a detour through the entire periplasm, and involves a high degree of free diffusion along and within the cell membrane.

The method of replication affects metal film granularity and resolution

1989 ◽  
Vol 47 ◽  
pp. 708-709
Author(s):  
George C. Ruben
Keyword(s):  
Electron Beam ◽  
High Resolution ◽  
Film Thickness ◽  
Single Molecule ◽  
Metal Film ◽  
Vertical Surface ◽  
High Resolution Imaging ◽  
Controllable Factors ◽  
Resolution Imaging

Single molecule resolution in electron beam sensitive, uncoated, noncrystalline materials has been impossible except in thin Pt-C replicas ≤ 150Å) which are resistant to the electron beam destruction. Previously the granularity of metal film replicas limited their resolution to ≥ 20Å. This paper demonstrates that Pt-C film granularity and resolution are a function of the method of replication and other controllable factors. Low angle 20° rotary , 45° unidirectional and vertical 9.7±1 Å Pt-C films deposited on mica under the same conditions were compared in Fig. 1. Vertical replication had a 5A granularity (Fig. 1c), the highest resolution (table), and coated the whole surface. 45° replication had a 9Å granulartiy (Fig. 1b), a slightly poorer resolution (table) and did not coat the whole surface. 20° rotary replication was unsuitable for high resolution imaging with 20-25Å granularity (Fig. 1a) and resolution 2-3 times poorer (table). Resolution is defined here as the greatest distance for which the metal coat on two opposing faces just grow together, that is, two times the apparent film thickness on a single vertical surface.

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