scholarly journals DNA hybridisation kinetics using single-molecule fluorescence imaging

2021 ◽  
Author(s):  
Rebecca Andrews
Keyword(s):  
Nucleic Acid ◽  
Single Molecule ◽  
Biological Processes ◽  
Single Molecule Fluorescence ◽  
Extrinsic Factors ◽  
Factors Affecting ◽  
Dna Hybridisation ◽  
Single Molecule Studies ◽  
Kinetics Of ◽  
Insight Into

Abstract Deoxyribonucleic acid (DNA) hybridisation plays a key role in many biological processes and nucleic acid biotechnologies, yet surprisingly there are many aspects about the process which are still unknown. Prior to the invention of single-molecule microscopy, DNA hybridisation experiments were conducted at the ensemble level, and thus it was impossible to directly observe individual hybridisation events and understand fully the kinetics of DNA hybridisation. In this mini-review, recent single-molecule fluorescence-based studies of DNA hybridisation are discussed, particularly for short nucleic acids, to gain more insight into the kinetics of DNA hybridisation. As well as looking at single-molecule studies of intrinsic and extrinsic factors affecting DNA hybridisation kinetics, the influence of the methods used to detect hybridisation of single DNAs is considered. Understanding the kinetics of DNA hybridisation not only gives insight into an important biological process but also allows for further advancements in the growing field of nucleic acid biotechnology.

scholarly journals Mechanisms underlying sequence-dependent DNA hybridisation rates in the absence of secondary structure

2021 ◽  
Author(s):  
Sophie Hertel ◽  
Richard Spinney ◽  
Stephanie Xu ◽  
Thomas E Ouldridge ◽  
Richard Morris ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Dna Sequences ◽  
Physical Factors ◽  
Biological Processes ◽  
Physical Mechanisms ◽  
Mechanistic Insight ◽  
Sequence Dependent ◽  
Dna Hybridisation ◽  
Kinetics Of ◽  
Insight Into

The kinetics of DNA hybridisation are fundamental to biological processes and DNA-based technologies. However, the precise physical mechanisms that determine why different DNA sequences hybridise at different rates are not well understood. Secondary structure is one predictable factor that influences hybridisation rates but is not sufficient on its own to fully explain the observed sequence-dependent variance. Consequently, to achieve a good correlation with experimental data, current prediction algorithms require many parameters that provide little mechanistic insight into DNA hybridisation. In this context, we measured hybridisation rates of 43 different DNA sequences that are not predicted to form secondary structure and present a parsimonious physically justified model to quantify their hybridisation rates. Accounting only for the combinatorics of complementary nucleating interactions and their sequence-dependent stability, the model achieves good correlation with experiment with only two free parameters, thus providing new insight into the physical factors underpinning DNA hybridisation rates.

scholarly journals Investigating the Factors Affecting the Aggregation of Alpha-Synuclein using Single Molecule Fluorescence and Fast Flow Microfluidics

2012 ◽  
Vol 102 (3) ◽  
pp. 255a
Author(s):  
Mathew H. Horrocks ◽  
Nunilo Cremades ◽  
Laura Tosatto ◽  
Tim Guilliams ◽  
Erwin De Genst ◽  
...  
Keyword(s):  
Single Molecule ◽  
Alpha Synuclein ◽  
Single Molecule Fluorescence ◽  
Factors Affecting ◽  
Fast Flow

scholarly journals Dynamics of Tpm1.8 domains on actin filaments with single molecule resolution

2020 ◽  
Author(s):  
Ilina Bareja ◽  
Hugo Wioland ◽  
Miro Janco ◽  
Philip R. Nicovich ◽  
Antoine Jégou ◽  
...  
Keyword(s):  
Actin Filament ◽  
Single Molecule ◽  
Actin Filaments ◽  
High Probability ◽  
Actin Binding ◽  
Single Molecule Level ◽  
Filament Dynamics ◽  
Kinetics Of ◽  
Insight Into

ABSTRACTTropomyosins regulate dynamics and functions of the actin cytoskeleton by forming long chains along the two strands of actin filaments that act as gatekeepers for the binding of other actin-binding proteins. The fundamental molecular interactions underlying the binding of tropomyosin to actin are still poorly understood. Using microfluidics and fluorescence microscopy, we observed the binding of fluorescently labelled tropomyosin isoform Tpm1.8 to unlabelled actin filaments in real time. This approach in conjunction with mathematical modeling enabled us to quantify the nucleation, assembly and disassembly kinetics of Tpm1.8 on single filaments and at the single molecule level. Our analysis suggests that Tpm1.8 decorates the two strands of the actin filament independently. Nucleation of a growing tropomyosin domain proceeds with high probability as soon as the first Tpm1.8 molecule is stabilised by the addition of a second molecule, ultimately leading to full decoration of the actin filament. In addition, Tpm1.8 domains are asymmetrical, with enhanced dynamics at the edge oriented towards the barbed end of the actin filament. The complete description of Tpm1.8 kinetics on actin filaments presented here provides molecular insight into actin-tropomyosin filament formation and the role of tropomyosins in regulating actin filament dynamics.

scholarly journals Deaminase-independent inhibition of HIV-1 reverse transcription by APOBEC3G

2007 ◽  
Vol 35 (21) ◽  
pp. 7096-7108 ◽  
Author(s):  
Yasumasa Iwatani ◽  
Denise S.B. Chan ◽  
F. Wang ◽  
Kristen Stewart-Maynard ◽  
Wataru Sugiura ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Single Molecule ◽  
Reverse Transcription ◽  
Cytidine Deaminase ◽  
Rnase H ◽  
Host Protein ◽  
Nucleic Acid Binding ◽  
Nucleic Acid Chaperone ◽  
Kinetics Of ◽  
Hiv 1

Abstract APOBEC3G (A3G), a host protein that inhibits HIV-1 reverse transcription and replication in the absence of Vif, displays cytidine deaminase and single-stranded (ss) nucleic acid binding activities. HIV-1 nucleocapsid protein (NC) also binds nucleic acids and has a unique property, nucleic acid chaperone activity, which is crucial for efficient reverse transcription. Here we report the interplay between A3G, NC and reverse transcriptase (RT) and the effect of highly purified A3G on individual reactions that occur during reverse transcription. We find that A3G did not affect the kinetics of NC-mediated annealing reactions, nor did it inhibit RNase H cleavage. In sharp contrast, A3G significantly inhibited all RT-catalyzed DNA elongation reactions with or without NC. In the case of ( − ) strong-stop DNA synthesis, the inhibition was independent of A3G's catalytic activity. Fluorescence anisotropy and single molecule DNA stretching analyses indicated that NC has a higher nucleic acid binding affinity than A3G, but more importantly, displays faster association/disassociation kinetics. RT binds to ssDNA with a much lower affinity than either NC or A3G. These data support a novel mechanism for deaminase-independent inhibition of reverse transcription that is determined by critical differences in the nucleic acid binding properties of A3G, NC and RT.

scholarly journals Kinetics of CrPV and HCV IRES-mediated eukaryotic translation using single-molecule fluorescence microscopy

RNA ◽  
2017 ◽  
Vol 23 (11) ◽  
pp. 1626-1635 ◽  
Author(s):  
Olivier Bugaud ◽  
Nathalie Barbier ◽  
Hélène Chommy ◽  
Nicolas Fiszman ◽  
Antoine Le Gall ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Single Molecule ◽  
Single Molecule Fluorescence ◽  
Eukaryotic Translation ◽  
Hcv Ires ◽  
Kinetics Of ◽  
Single Molecule Fluorescence Microscopy

scholarly journals Nucleic Acid Binding Kinetics of HIV-1 Nucleocapsid Proteins from Single Molecule DNA Stretching

2013 ◽  
Vol 104 (2) ◽  
pp. 254a
Author(s):  
Jialin Li ◽  
Robert J. Gorelick ◽  
Ioulia Rouzina ◽  
Mark C. Williams
Keyword(s):  
Nucleic Acid ◽  
Single Molecule ◽  
Binding Kinetics ◽  
Nucleic Acid Binding ◽  
Dna Stretching ◽  
Nucleocapsid Proteins ◽  
Kinetics Of ◽  
Hiv 1

scholarly journals Watching biological nanomotors at work : insights from single-molecule studies

2017 ◽  
Author(s):  
◽  
Nagaraju Chada
Keyword(s):  
Atomic Force Microscopy ◽  
Single Molecule ◽  
Conformational Dynamics ◽  
Halobacterium Salinarum ◽  
Biological Processes ◽  
Enzyme Dynamics ◽  
Oligomeric State ◽  
Force Microscopy ◽  
Atomic Force ◽  
Single Molecule Studies

Recent decades have seen several complimentary biophysics tools emerge to study single protein macromolecules. Most of these techniques use glass as a specimen support. The atomic force microscope, a vital tool in biophysics suited to study proteins in their near native environments, uses mica as a specimen support, as it is known for its extreme flatness and ease of use. Here we optimized glass as a specimen support for atomic force microscopy. This enables the combination of other single molecule techniques with atomic force microscopy to study the same protein macromolecular system in unison. Using bacteriorhodopsin from Halobacterium salinarum and the Sec-translocase (SecA/SecYEG) from Escherichia coli, we demonstrate that faithful images of 2D crystalline and non-crystalline membrane proteins in lipid bilayers can be obtained on common microscope cover glass following a straight-forward cleaning procedure. Repeated association and dissociation of SecA with SecYEG indicated that the proteins remain competent for biological processes on glass supports for long periods of time. This work opens the door for combining high resolution biological AFM with other powerful complementary single molecule techniques that require glass as a specimen support. In the second part of this work we studied SecA-ATP hydrolysis and catalase enzyme dynamics. Both of these protein macromolecules were observed to be highly dynamic during catalytic turnover. Single molecule studies of catalase indicated that the enzyme undergoes significant dynamics including oligomeric state changes when exposed to H2O2. Conformational dynamics of the SecA-ATPase was visualized at the single molecule level and the protein macromolecule was observed to flicker between a compact and expanded state in the presence of ATP, indicating reversible conformational changes. Future studies in the lab will shed more light onto these important biological processes.

Nucleic acid amplification-integrated single-molecule fluorescence imaging for in vitro and in vivo biosensing

2021 ◽  
Author(s):  
Fei Ma ◽  
Chen-Chen Li ◽  
Chun-Yang Zhang
Keyword(s):  
Nucleic Acid ◽  
Fluorescence Imaging ◽  
Single Molecule ◽  
High Sensitivity ◽  
Nucleic Acid Amplification ◽  
Single Molecule Fluorescence

Single-molecule fluorescence imaging is among the most advanced analytical technologies and has been widely adopted for biosensing due to its distinct advantages of simplicity, rapidity, high sensitivity, low sample consumption,...

Kinetics of Diffusion-Mediated DNA Hybridization in Lipid Monolayer Films Determined by Single-Molecule Fluorescence Spectroscopy

ACS Nano ◽  
2012 ◽  
Vol 7 (1) ◽  
pp. 308-315 ◽  
Author(s):  
Jonas K. Hannestad ◽  
Ralf Brune ◽  
Ilja Czolkos ◽  
Aldo Jesorka ◽  
Afaf H. El-Sagheer ◽  
...  
Keyword(s):  
Fluorescence Spectroscopy ◽  
Single Molecule ◽  
Dna Hybridization ◽  
Lipid Monolayer ◽  
Single Molecule Fluorescence ◽  
Monolayer Films ◽  
Single Molecule Fluorescence Spectroscopy ◽  
Kinetics Of
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