scholarly journals Stable end-sealed DNA as robust nano-rulers for in vivo single-molecule fluorescence

2016 ◽  
Vol 7 (7) ◽  
pp. 4418-4422 ◽  
Author(s):  
A. Plochowietz ◽  
A. H. El-Sagheer ◽  
T. Brown ◽  
A. N. Kapanidis
Keyword(s):  
Single Molecule ◽  
Single Molecule Fluorescence

Protected DNA standards with chemically linked ends were synthesized as robust in vivo nano-rulers for smFRET studies.

scholarly journals Mechanically switching single-molecule fluorescence of GFP by unfolding and refolding

2017 ◽  
Vol 114 (42) ◽  
pp. 11052-11056 ◽  
Author(s):  
Ziad Ganim ◽  
Matthias Rief
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Optical Sensors ◽  
Structural Integrity ◽  
Fluorescent Protein ◽  
Single Molecule Fluorescence ◽  
Extended States ◽  
Green Fluorescent ◽  
Ensemble Experiments

Green fluorescent protein (GFP) variants are widely used as genetically encoded fluorescent fusion tags, and there is an increasing interest in engineering their structure to develop in vivo optical sensors, such as for optogenetics and force transduction. Ensemble experiments have shown that the fluorescence of GFP is quenched upon denaturation. Here we study the dependence of fluorescence on protein structure by driving single molecules of GFP into different conformational states with optical tweezers and simultaneously probing the chromophore with fluorescence. Our results show that fluorescence is lost during the earliest events in unfolding, 3.5 ms before secondary structure is disrupted. No fluorescence is observed from the unfolding intermediates or the ensemble of compact and extended states populated during refolding. We further demonstrate that GFP can be mechanically switched between emissive and dark states. These data definitively establish that complete structural integrity is necessary to observe single-molecule fluorescence of GFP.

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,...

scholarly journals Inferring quantity and qualities of superimposed reaction rates in single molecule survival time distributions

2019 ◽  
Author(s):  
Matthias Reisser ◽  
Johannes Hettich ◽  
Timo Kuhn ◽  
J. Christof M. Gebhardt
Keyword(s):  
Survival Time ◽  
Single Molecule ◽  
Rate Constants ◽  
Reaction Rates ◽  
Decay Rates ◽  
Single Molecule Fluorescence ◽  
Inverse Laplace Transformation ◽  
Dense Grid ◽  
Reaction Processes

Actions of molecular species, for example binding of transcription factors to chromatin, are intrinsically stochastic and may comprise several mutually exclusive pathways. Inverse Laplace transformation in principle resolves the rate constants and frequencies of superimposed reaction processes, however current approaches are challenged by single molecule fluorescence time series prone to photobleaching. Here, we present a genuine rate identification method (GRID) that infers the quantity, rates and frequencies of dissociation processes from single molecule fluorescence survival time distributions using a dense grid of possible decay rates. In particular, GRID is able to resolve broad clusters of rate constants not accessible to common models of one to three exponential decay rates. We validate GRID by simulations and apply it to the problem of in-vivo TF-DNA dissociation, which recently gained interest due to novel single molecule imaging technologies. We consider dissociation of the transcription factor CDX2 from chromatin. GRID resolves distinct, decay rates and identifies residence time classes overlooked by other methods. We confirm that such sparsely distributed decay rates are compatible with common models of TF sliding on DNA.

scholarly journals Single-molecule biophysics: at the interface of biology, physics and chemistry

2007 ◽  
Vol 5 (18) ◽  
pp. 15-45 ◽  
Author(s):  
Ashok A Deniz ◽  
Samrat Mukhopadhyay ◽  
Edward A Lemke
Keyword(s):  
Single Molecule ◽  
Biological Molecules ◽  
Single Molecule Fluorescence ◽  
Complex Behaviour ◽  
Single Molecule Biophysics ◽  
Single Molecule Studies ◽  
Dynamics And Function ◽  
And Function ◽  
Ensemble Experiments

Single-molecule methods have matured into powerful and popular tools to probe the complex behaviour of biological molecules, due to their unique abilities to probe molecular structure, dynamics and function, unhindered by the averaging inherent in ensemble experiments. This review presents an overview of the burgeoning field of single-molecule biophysics, discussing key highlights and selected examples from its genesis to our projections for its future. Following brief introductions to a few popular single-molecule fluorescence and manipulation methods, we discuss novel insights gained from single-molecule studies in key biological areas ranging from biological folding to experiments performed in vivo .

scholarly journals In Vivo Investigation of DNA Replication in Escherichia Coli using Single-Molecule Fluorescence Microscopy

2012 ◽  
Vol 102 (3) ◽  
pp. 284a
Author(s):  
Charl Moolman ◽  
Sriram Tiruvadi Krishnan ◽  
Serge Donkers ◽  
Rodrigo Reyes-Lamothe ◽  
David Sherratt ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Fluorescence Microscopy ◽  
Single Molecule ◽  
Single Molecule Fluorescence ◽  
Single Molecule Fluorescence Microscopy

Nitroxyl Modified Tobacco Mosaic Virus as a Metal-Free High-Relaxivity MRI and EPR Active Superoxide Sensor

2018 ◽  
Author(s):  
Madushani Dharmarwardana ◽  
André F. Martins ◽  
Zhuo Chen ◽  
Philip M. Palacios ◽  
Chance M. Nowak ◽  
...  
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Single Molecule ◽  
Biological Fluids ◽  
Cellular Respiration ◽  
Organic Radical ◽  
Paramagnetic Resonance ◽  
Metal Free ◽  
Disease States

Superoxide overproduction is known to occur in multiple disease states requiring critical care yet non-invasive detection of superoxide in deep tissue remains a challenge. Herein, we report a metal-free magnetic resonance imaging (MRI) and electron paramagnetic resonance (EPR) active contrast agent prepared by “click conjugating” paramagnetic organic radical contrast agents (ORCAs) to the surface of tobacco mosaic virus (TMV). While ORCAs are known to be reduced <i>in vivo</i> to an MRI/EPR silent state, their oxidation is facilitated specifically by reactive oxygen species—in particular superoxide—and are largely unaffected by peroxides and molecular oxygen. Unfortunately, single molecule ORCAs typically offer weak MRI contrast. In contrast, our data confirm that the macromolecular ORCA-TMV conjugates show marked enhancement for <i>T<sub>1</sub></i> contrast at low field (<3.0 T), and <i>T<sub>2</sub></i> contrast at high field (9.4 T). Additionally, we demonstrated that the unique topology of TMV allows for “quenchless fluorescent” bimodal probe for concurrent fluorescence and MRI/EPR imaging, which was made possible by exploiting the unique inner and outer surface of the TMV nanoparticle. <a>Finally, we show TMV-ORCAs do not respond to normal cellular respiration, minimizing the likelihood for background, yet still respond to enzymatically produced superoxide in complicated biological fluids like serum.</a>

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