Single-pair FRET experiments on nucleosome conformational dynamics

Preparation of Mononucleosomal Templates for Analysis of Transcription with RNA Polymerase Using spFRET

“…4. Besides home-build experimental installations (24, 34, 35) any commercially available spectrometer for fluorescence correlation spectroscopy (FCS) or confocal microscope with the module for FCS can be used for spFRET measurements. FCS modules are usually equipped with avalanche-photodiodes (APDs), which surpass conventional photomultipliers installed in commercial confocal microscopes in sensitivity and suit ideally for spFRET measurements.…”

“…Advanced insight into nucleosome structure and dynamics during various intranuclear processes can be achieved with modern experimental approaches based on single particle Forster resonance energy transfer (spFRET) analysis (24, 25). Thus spFRET analysis is informative for analysis of alterations in nucleosome structure induced by modification (methylation, acetylation) of DNA or histones (24, 26–28).…”

“…Thus spFRET analysis is informative for analysis of alterations in nucleosome structure induced by modification (methylation, acetylation) of DNA or histones (24, 26–28). spFRET records data from thousands nucleosomes, and each nucleosome is characterized individually.…”

The role of DNA sequence in nucleosome breathing

“…[17,18] were published, mainly based on FRET, which has enabled more direct probing of the dynamics of site exposure (for a review, see also [44]). However, the interpretation of data gathered through this technique has proved less straightforward [45] than that of the older experiments employing restriction enzymes [17][18][19][46][47][48].…”

Principles and practice of nucleosome positioningin vitro

“…Single-pair FRET (spFRET) methods use through-space energy transfer between a specific pair of donor and acceptor dyes to measure spatial separation when tethered to locations on the nucleosome (Buning and van Noort 2010). This FRET response is highly sensitive over a window around the characteristic Förster radius of the dye pair, which is typically in the range 10-100 Å. spFRET can be performed in solution (Tóth et al 2001), or particles can be spread on an imaging surface and the relative signal of the FRET dyes observed for each single molecule spot simultaneously for thousands of spots with high time resolution (Koopmans et al 2007).…”