Single-particle FRET microscopy of immobilized nucleosomes: Technique development

Abstract

Mononucleosomes formed by histone octamer and short DNA is an advanced model system for investigation of RNA polymerase (RNAP) transcription and its modulation with various transcription factors. Recent achievements of fluorescent microscopy allow one to complement these studies with single-particle Forster resonance energy transfer (spFRET) analysis. FRET efficiency between Cy3 and Cy5 dyes introduced in the neighboring coils of nucleosomal DNA is a sensor of structural changes caused by DNA unwrapping or looping, or restoration of DNA-histone interactions. Here we report on experimental setup and protocols for spFRET microscopy of immobilized nucleosomes. Using confocal laser scanning and total internal reflection fluorescence microscopy we demonstrated preservation of the nucleosome structure during immobilization and long-term (>100 s, ca. 140 ms/frame) spFRET kinetic measurements. The effect of ionic strength on nucleosome structure was studied. Applications of spFRET microscopy of immobilized nucleosomes include analysis of their structural dynamics (DNA “breathing”), kinetics of formation/dissociation of DNA-protein complexes, formation and structure of stalled elongation complexes with RNAP, as well as conformational transitions in nucleosome structure in the course of transcription.