Single molecule fluorescence methodologies for investigating transcription factor binding kinetics to nucleosomes and DNA

Luo, Yi; North, Justin A.; Poirier, Michael G.

doi:10.1016/j.ymeth.2014.09.011

Cited by 35 publications

(28 citation statements)

References 54 publications

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“…To prepare the structures for actuation, each system had to be fixed to the surface in a channel. Nonspecific binding was reduced by initially cleaning the coverslips with piranha solution and coating with unmodified and biotin-modified PEG (10% biotin-PEG) following previously described protocols 51 . 20 μL of free streptavidin (0.1 mg∙mL −1 ) mixed with BSA (0.1 mg∙mL −1 ) was added to the channel and incubated for 5 min to allow for attachment of Streptavidin to the biotin-PEG on the surface.…”

Section: Methodsmentioning

confidence: 99%

Real-time magnetic actuation of DNA nanodevices via modular integration with stiff micro-levers

et al. 2018

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DNA nanotechnology has enabled complex nanodevices, but the ability to directly manipulate systems with fast response times remains a key challenge. Current methods of actuation are relatively slow and only direct devices into one or two target configurations. Here we report an approach to control DNA origami assemblies via externally applied magnetic fields using a low-cost platform that enables actuation into many distinct configurations with sub-second response times. The nanodevices in these assemblies are manipulated via mechanically stiff micron-scale lever arms, which rigidly couple movement of a micron size magnetic bead to reconfiguration of the nanodevice while also enabling direct visualization of the conformation. We demonstrate control of three assemblies—a rod, rotor, and hinge—at frequencies up to several Hz and the ability to actuate into many conformations. This level of spatiotemporal control over DNA devices can serve as a foundation for real-time manipulation of molecular and atomic systems.

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Section: Methodsmentioning

confidence: 99%

Real-time magnetic actuation of DNA nanodevices via modular integration with stiff micro-levers

et al. 2018

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“…DNA can also be labeled internally, by using mutated bases at strategic positions. We will not discuss the method for internal DNA labeling here but more information can be obtained from (Luo, North, & Poirier, 2014). …”

Section: Chromatin Reconstitution Strategiesmentioning

confidence: 99%

In Vitro Chromatin Assembly

Muthurajan

Mattiroli

Bergeron

et al. 2016

Methods in Enzymology

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Chromatin accessibility is modulated by structural transitions that provide timely access to the genetic and epigenetic information during many essential nuclear processes. These transitions are orchestrated by regulatory proteins that coordinate intricate structural modifications and signalling pathways. In vitro reconstituted chromatin samples from defined components are instrumental in defining the mechanistic details of such processes. The bottleneck to appropriate in vitro analysis is the production of high quality, and quality-controlled, chromatin substrates. In this chapter we describe methods for in vitro chromatin reconstitution and quality control. We highlight the strengths and weaknesses of various approaches, and emphasize quality control steps that ensure reconstitution of a bona fide homogenous chromatin preparation. This is essential for optimal reproducibility and reliability of ensuing experiments using chromatin substrates.

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“…Single-molecule fluorescence approaches offer several advantages in the study of the structure and dynamics of the complex and often heterogeneous NCPs (3)(4)(5)9,(13)(14)(15)(29)(30)(31)(32)(33). Single-pair Förster resonance energy transfer (FRET) provides high-resolution information about conformations and distribution of conformations.…”

Section: Introductionmentioning

confidence: 99%

Nucleosome Core Particle Disassembly and Assembly Kinetics Studied Using Single-Molecule Fluorescence

Hazan

Tomov

Tsukanov

et al. 2015

Biophysical Journal

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The stability of the nucleosome core particle (NCP) is believed to play a major role in regulation of gene expression. To understand the mechanisms that influence NCP stability, we studied stability and dissociation and association kinetics under different histone protein (NCP) and NaCl concentrations using single-pair Förster resonance energy transfer and alternating laser excitation techniques. The method enables distinction between folded, unfolded, and intermediate NCP states and enables measurements at picomolar to nanomolar NCP concentrations where dissociation and association reactions can be directly observed. We reproduced the previously observed nonmonotonic dependence of NCP stability on NaCl concentration, and we suggest that this rather unexpected behavior is a result of interplay between repulsive and attractive forces within positively charged histones and between the histones and the negatively charged DNA. Higher NaCl concentrations decrease the attractive force between the histone proteins and the DNA but also stabilize H2A/H2B histone dimers, and possibly (H3/H4)2 tetramers. An intermediate state in which one DNA arm is unwrapped, previously observed at high NaCl concentrations, is also explained by this salt-induced stabilization. The strong dependence of NCP stability on ion and histone concentrations, and possibly on other charged macromolecules, may play a role in chromosomal morphology.

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Single molecule fluorescence methodologies for investigating transcription factor binding kinetics to nucleosomes and DNA

Cited by 35 publications

References 54 publications

Real-time magnetic actuation of DNA nanodevices via modular integration with stiff micro-levers

Real-time magnetic actuation of DNA nanodevices via modular integration with stiff micro-levers

In Vitro Chromatin Assembly

Nucleosome Core Particle Disassembly and Assembly Kinetics Studied Using Single-Molecule Fluorescence

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