4,886 research outputs found
Crowding-induced hybridization of single DNA hairpins
It is clear that a crowded environment influences the structure, dynamics, and interactions of biological molecules, but the complexity of this phenomenon demands the development of new experimental and theoretical approaches. Here we use two complementary single-molecule FRET techniques to show that the kinetics of DNA base pairing and unpairing, which are fundamental to both the biological role of DNA and its technological applications, are strongly modulated by a crowded environment. We directly observed single DNA hairpins, which are excellent model systems for studying hybridization, either freely diffusing in solution or immobilized on a surface under crowding conditions. The hairpins followed two-state folding dynamics with a closing rate increasing by 4-fold and the opening rate decreasing 2-fold, for only modest concentrations of crowder [10% (w/w) polyethylene glycol (PEG)]. These experiments serve both to unambiguously highlight the impact of a crowded environment on a fundamental biological process, DNA base pairing, and to illustrate the benefits of single-molecule approaches to probing the structure and dynamics of complex biomolecular systems
Single-molecule experiments in biological physics: methods and applications
I review single-molecule experiments (SME) in biological physics. Recent
technological developments have provided the tools to design and build
scientific instruments of high enough sensitivity and precision to manipulate
and visualize individual molecules and measure microscopic forces. Using SME it
is possible to: manipulate molecules one at a time and measure distributions
describing molecular properties; characterize the kinetics of biomolecular
reactions and; detect molecular intermediates. SME provide the additional
information about thermodynamics and kinetics of biomolecular processes. This
complements information obtained in traditional bulk assays. In SME it is also
possible to measure small energies and detect large Brownian deviations in
biomolecular reactions, thereby offering new methods and systems to scrutinize
the basic foundations of statistical mechanics. This review is written at a
very introductory level emphasizing the importance of SME to scientists
interested in knowing the common playground of ideas and the interdisciplinary
topics accessible by these techniques. The review discusses SME from an
experimental perspective, first exposing the most common experimental
methodologies and later presenting various molecular systems where such
techniques have been applied. I briefly discuss experimental techniques such as
atomic-force microscopy (AFM), laser optical tweezers (LOT), magnetic tweezers
(MT), biomembrane force probe (BFP) and single-molecule fluorescence (SMF). I
then present several applications of SME to the study of nucleic acids (DNA,
RNA and DNA condensation), proteins (protein-protein interactions, protein
folding and molecular motors). Finally, I discuss applications of SME to the
study of the nonequilibrium thermodynamics of small systems and the
experimental verification of fluctuation theorems. I conclude with a discussion
of open questions and future perspectives.Comment: Latex, 60 pages, 12 figures, Topical Review for J. Phys. C (Cond.
Matt
Advanced Fluorescence Microscopy Techniques-FRAP, FLIP, FLAP, FRET and FLIM
Fluorescence microscopy provides an efficient and unique approach to study fixed and living cells because of its versatility, specificity, and high sensitivity. Fluorescence microscopes can both detect the fluorescence emitted from labeled molecules in biological samples as images or photometric data from which intensities and emission spectra can be deduced. By exploiting the characteristics of fluorescence, various techniques have been developed that enable the visualization and analysis of complex dynamic events in cells, organelles, and sub-organelle components within the biological specimen. The techniques described here are fluorescence recovery after photobleaching (FRAP), the related fluorescence loss in photobleaching (FLIP), fluorescence localization after photobleaching (FLAP), Forster or fluorescence resonance energy transfer (FRET) and the different ways how to measure FRET, such as acceptor bleaching, sensitized emission, polarization anisotropy, and fluorescence lifetime imaging microscopy (FLIM). First, a brief introduction into the mechanisms underlying fluorescence as a physical phenomenon and fluorescence, confocal, and multiphoton microscopy is given. Subsequently, these advanced microscopy techniques are introduced in more detail, with a description of how these techniques are performed, what needs to be considered, and what practical advantages they can bring to cell biological research
A practical review on the measurement tools for cellular adhesion force
Cell cell and cell matrix adhesions are fundamental in all multicellular
organisms. They play a key role in cellular growth, differentiation, pattern
formation and migration. Cell-cell adhesion is substantial in the immune
response, pathogen host interactions, and tumor development. The success of
tissue engineering and stem cell implantations strongly depends on the fine
control of live cell adhesion on the surface of natural or biomimetic
scaffolds. Therefore, the quantitative and precise measurement of the adhesion
strength of living cells is critical, not only in basic research but in modern
technologies, too. Several techniques have been developed or are under
development to quantify cell adhesion. All of them have their pros and cons,
which has to be carefully considered before the experiments and interpretation
of the recorded data. Current review provides a guide to choose the appropriate
technique to answer a specific biological question or to complete a biomedical
test by measuring cell adhesion
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FRET-based dynamic structural biology: Challenges, perspectives and an appeal for open-science practices
Single-molecule FRET (smFRET) has become a mainstream technique for studying biomolecular structural dynamics. The rapid and wide adoption of smFRET experiments by an ever-increasing number of groups has generated significant progress in sample preparation, measurement procedures, data analysis, algorithms and documentation. Several labs that employ smFRET approaches have joined forces to inform the smFRET community about streamlining how to perform experiments and analyze results for obtaining quantitative information on biomolecular structure and dynamics. The recent efforts include blind tests to assess the accuracy and the precision of smFRET experiments among different labs using various procedures. These multi-lab studies have led to the development of smFRET procedures and documentation, which are important when submitting entries into the archiving system for integrative structure models, PDB-Dev. This position paper describes the current ‘state of the art’ from different perspectives, points to unresolved methodological issues for quantitative structural studies, provides a set of ‘soft recommendations’ about which an emerging consensus exists, and lists openly available resources for newcomers and seasoned practitioners. To make further progress, we strongly encourage ‘open science’ practices
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