High‐resolution, hybrid optical trapping methods, and their application to nucleic acid processing proteins

Chemla, Yann R.

doi:10.1002/bip.22880

Cited by 20 publications

(9 citation statements)

References 69 publications

(136 reference statements)

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“…In single molecule force experiments, stretching more than one direction simultaneously by holding more than two positions of a molecule may provide multidimensional information [94,95]. Fluorescence and force methods can also be combined to monitor multiple distances [93,96–105].…”

Section: Limitations and Future Directionsmentioning

confidence: 99%

Transition Path Times Measured by Single-Molecule Spectroscopy

Chung

2018

Journal of Molecular Biology

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The transition path is a tiny fraction of a molecular trajectory during which the free-energy barrier is crossed. It is a single-molecule property and contains all mechanistic information of folding processes of biomolecules such as proteins and nucleic acids. However, the transition path has been difficult to probe because it is short and rarely visited when transitions actually occur. Recent technical advances in single-molecule spectroscopy have made it possible to directly probe transition paths, which has opened up new theoretical and experimental approaches to investigating folding mechanisms. This article reviews recent single-molecule fluorescence and force spectroscopic measurements of transition path times and their connection to both theory and simulations.

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Section: Limitations and Future Directionsmentioning

confidence: 99%

Transition Path Times Measured by Single-Molecule Spectroscopy

Chung

2018

Journal of Molecular Biology

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“…For example, long DNA substrates are frequently used as flexible, micron-long tethers in optical and magnetic tweezers experiments 4–7 . Moreover, long DNA molecules elevate the biochemical reactions away from glass coverslips and other surfaces, reducing the potential for non-specific interactions 5–7 . DNA derived from bacteriophage λ (λ-DNA) offers several advantages for single-molecule studies.…”

Section: Introductionmentioning

confidence: 99%

Efficient modification of λ-DNA substrates for single-molecule studies

Kim

Torre

Leal

et al. 2017

Sci Rep

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Single-molecule studies of protein-nucleic acid interactions frequently require site-specific modification of long DNA substrates. The bacteriophage λ is a convenient source of high quality long (48.5 kb) DNA. However, introducing specific sequences, tertiary structures, and chemical modifications into λ-DNA remains technically challenging. Most current approaches rely on multi-step ligations with low yields and incomplete products. Here, we describe a molecular toolkit for rapid preparation of modified λ-DNA. A set of PCR cassettes facilitates the introduction of recombinant DNA sequences into the λ-phage genome with 90–100% yield. Extrahelical structures and chemical modifications can be inserted at user-defined sites via an improved nicking enzyme-based strategy. As a proof-of-principle, we explore the interactions of S. cerevisiae Proliferating Cell Nuclear Antigen (yPCNA) with modified DNA sequences and structures incorporated within λ-DNA. Our results demonstrate that S. cerevisiae Replication Factor C (yRFC) can load yPCNA onto 5′-ssDNA flaps, (CAG)13 triplet repeats, and homoduplex DNA. However, yPCNA remains trapped on the (CAG)13 structure, confirming a proposed mechanism for triplet repeat expansion. We anticipate that this molecular toolbox will be broadly useful for other studies that require site-specific modification of long DNA substrates.

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“…From the simple localization of a single fluorophore to the high-resolution detection of Förster resonance energy transfer (FRET) [83], fluorescence-based methods add visualization capabilities to the mechanical manipulation skills of OT, the result being a very powerful single-molecule technique whose potential has just begun to be explored. A few recent reviews [84,85,86] make an excellent job of detailing the earlier successes and the state of the art of such hybrid experimental setups. Here, we limit ourselves to a very concise sketch of the most promising instrumental developments and biological applications, with special attention to the latest results.…”

Section: Optical Tweezers Combined With Fluorescencementioning

confidence: 99%

Bio-Molecular Applications of Recent Developments in Optical Tweezers

et al. 2019

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In the past three decades, the ability to optically manipulate biomolecules has spurred a new era of medical and biophysical research. Optical tweezers (OT) have enabled experimenters to trap, sort, and probe cells, as well as discern the structural dynamics of proteins and nucleic acids at single molecule level. The steady improvement in OT’s resolving power has progressively pushed the envelope of their applications; there are, however, some inherent limitations that are prompting researchers to look for alternatives to the conventional techniques. To begin with, OT are restricted by their one-dimensional approach, which makes it difficult to conjure an exhaustive three-dimensional picture of biological systems. The high-intensity trapping laser can damage biological samples, a fact that restricts the feasibility of in vivo applications. Finally, direct manipulation of biological matter at nanometer scale remains a significant challenge for conventional OT. A significant amount of literature has been dedicated in the last 10 years to address the aforementioned shortcomings. Innovations in laser technology and advances in various other spheres of applied physics have been capitalized upon to evolve the next generation OT systems. In this review, we elucidate a few of these developments, with particular focus on their biological applications. The manipulation of nanoscopic objects has been achieved by means of plasmonic optical tweezers (POT), which utilize localized surface plasmons to generate optical traps with enhanced trapping potential, and photonic crystal optical tweezers (PhC OT), which attain the same goal by employing different photonic crystal geometries. Femtosecond optical tweezers (fs OT), constructed by replacing the continuous wave (cw) laser source with a femtosecond laser, promise to greatly reduce the damage to living samples. Finally, one way to transcend the one-dimensional nature of the data gained by OT is to couple them to the other large family of single molecule tools, i.e., fluorescence-based imaging techniques. We discuss the distinct advantages of the aforementioned techniques as well as the alternative experimental perspective they provide in comparison to conventional OT.

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High‐resolution, hybrid optical trapping methods, and their application to nucleic acid processing proteins

Cited by 20 publications

References 69 publications

Transition Path Times Measured by Single-Molecule Spectroscopy

Transition Path Times Measured by Single-Molecule Spectroscopy

Efficient modification of λ-DNA substrates for single-molecule studies

Bio-Molecular Applications of Recent Developments in Optical Tweezers

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