Functional Studies of DNA-Protein Interactions Using FRET Techniques

Abstract

Protein-DNA interactions underpin life and play key roles in all cellular processes and functions including DNA transcription, packaging, replication, and repair. Identifying and examining the nature of these interactions is therefore a crucial prerequisite to understand the molecular basis of how these fundamental processes take place. The application of fluorescence techniques and in particular fluorescence resonance energy transfer (FRET) to provide structural and kinetic information has experienced a stunning growth during the past decade. This has been mostly promoted by new advances in the preparation of dye-labeled nucleic acids and proteins and in optical sensitivity, where its implementation at the level of individual molecules has opened a new biophysical frontier. Nowadays, the application of FRET-based techniques to the analysis of protein-DNA interactions spans from the classical steady-state and time-resolved methods averaging over large ensembles to the analysis of distances, conformational changes, and enzymatic reactions in individual Protein-DNA complexes. This chapter introduces the practical aspects of applying these methods for the study of Protein-DNA interactions.

High-affinity RNA binding by a hyperthermophilic single-stranded DNA-binding protein

“…2d). FRET has extensively been used as a molecular ruler to monitor conformational changes within proteins and DNA–protein interactions (Blouin et al 2009). The RNA hairpin was labelled with a Cy3–Cy5 FRET pair and the change in end-to-end distance was investigated as a function of added protein (Fig.…”

“…Single-molecule techniques are emerging as unique tools to unravel the dynamics of protein–DNA interactions (Morten et al 2015; Blouin et al 2009; Craggs et al 2014) and they have been used extensively to investigate single-strand binding proteins, such as Eco SSB and RPA (Zhou and Ha 2012). To compared the dynamic properties of Sso SSB monomers binding to ssRNA and ssDNA, Sso SSB was labelled with an Alexa647 acceptor dye and a 12 C ssRNA or a 12 C ssDNA was doubly labelled with a biotin group at the 5′ end for surface immobilization to streptavidin coated microscope slides and with a Cy3 FRET donor at the 3′ end.…”

“…Surface-immobilized oligonucleotides labelled with a donor Cy3 dye were exposed to Sso SSB labelled with the acceptor dye Alexa 647 as previously described (Morten et al 2015). Quartz slides were passivated using a PEG surface and biotin/neutravidin interactions head groups were exploited to immobilise C12 ssDNA Cy3 and C12 RNA Cy3 (Blouin et al 2009). The sample was excited by a 532 nm laser (Crystalaser, USA) and the fluorescence from the donor and acceptor was collected using an electron-multiplying CCD camera (Ixon, Andor).…”

“…Apparent FRET efficiencies after background corrections were calculated using ( I A /( I A  +  I D )), where I A and I D represent the intensities of the acceptor and donor, respectively. Single-molecule FRET histograms were generated using the first 15 frames of each trajectory as previously reported (Morten et al 2015; Bluoin et al 2009; McCluskey et al 2013). Single-molecule dwell-time histograms were calculated manually after filtering for blinking and photobleaching effects and fitted to a monoexponential decay curve to extract the corresponding transition rate.…”

An integrated perspective on RNA aptamer ligand-recognition models: clearing muddy waters

“…By strategically labeling the receptor, the ligand, or both, and resolving the dynamics of individual molecules via FRET rather than relying on the ensemble average, the ligand dependence of the rate of each individual step can be measured explicitly. 73,74 While k eff increasing with ligand concentration is not a reliable signature of IF, microscopic k fwd doing the same is unambiguous proof that ligand interactions enhance the folding rate. Likewise for the stabilization of the folded state by the ligand, leading to a decrease in microscopic k rev .…”