Extending Single Molecule F\"orster Resonance Energy Transfer (FRET) Range Beyond 10 Nanometers in Zero-Mode Waveguides

TL;DR

This paper demonstrates that zero-mode waveguides can extend smFRET measurement range beyond 10 nm, enabling studies of larger biomolecules with improved efficiency and brightness.

Contribution

The study introduces an optimized ZMW structure that allows smFRET at distances up to 13.6 nm and combines ZMWs with multi-acceptor constructs for enhanced FRET performance.

Findings

smFRET up to 13.6 nm using ZMWs

Enhanced FRET efficiency with multiple acceptors

Guidelines for quantitative smFRET in ZMWs

Abstract

Single molecule F\"orster resonance energy transfer (smFRET) is widely used to monitor conformations and interactions dynamics at the molecular level. However, conventional smFRET measurements are ineffective at donor-acceptor distances exceeding 10 nm, impeding the studies on biomolecules of larger size. Here, we show that zero-mode waveguide (ZMW) apertures can be used to overcome the 10 nm barrier in smFRET. Using an optimized ZMW structure, we demonstrate smFRET between standard commercial fluorophores up to 13.6 nm distance with a significantly improved FRET efficiency. To further break into the classical FRET range limit, ZMWs are combined with molecular constructs featuring multiple acceptor dyes to achieve high FRET efficiencies together with high fluorescence count rates. As we discuss general guidelines for quantitative smFRET measurements inside ZMWs, the technique can be readily applied for monitoring conformations and interactions on large molecular complexes with enhanced brightness.