Probing DNA Dynamics: Stacking‐Induced Fluorescence Increase (SIFI) versus FRET

Michael J Morten,Steven W Magennis,I Emilie Steinmark

doi:10.1002/cptc.202000069

Abstract

AbstractStacking‐induced fluorescence increase (SIFI) was introduced recently as a method to probe DNA structure and dynamics using only a single fluorescent label. Here we show that the same DNA hairpin dynamics can be recovered, at the single‐molecule level, using either SIFI (with Cy3 as the label) or FRET (with Cy3 as donor and Cy5 as acceptor). We also measured FRET using a donor that cannot undergo SIFI, Cy3B, in the presence and absence of a molecular crowding agent (PEG). Although crowding increases hairpin hybridisation to the same extent with either Cy3 or Cy3B as the donor, the absolute rates are affected by the choice of donor dye. This work shows that SIFI can be used to measure single‐molecule dynamics, which could offer advantages over FRET in some cases. It also illustrates how local dye interactions can influence biomolecular dynamics, which should be considered when designing experiments.

Highlights

Förster resonance energy transfer (FRET) using a donor that cannot undergo stackinginduced fluorescence increase (SIFI), Cy3B, in the presence and absence of a molecular crowding agent (PEG)
Crowding increases hairpin hybridisation to the same extent with either Cy3 or Cy3B as the donor, the absolute rates are affected by the choice of donor dye
The extension to single-molecule FRET has produced a step-change in the information attainable.[1b,2] FRET requires the presence of two labels, a fluorescent donor and an acceptor, which is normally fluorescent; it requires inter-dye distances in the 2–10 nm range

Summary

Introduction

FRET using a donor that cannot undergo SIFI, Cy3B, in the presence and absence of a molecular crowding agent (PEG). We previously used SIFI to detect hybridisation of a DNA hairpin at the single-molecule level, and to sense a long-range perturbation in the form of an abasic site up to 20 base pairs (bp) from the site of dye stacking.[3] Here we directly compare SIFI and FRET (using Cy3 as the donor and Cy5 as the acceptor) at the single-molecule level (termed smSIFI and smFRET, respectively) as tools for probing the dynamics of DNA.

Objectives

Results

Conclusion