Tracking single particles for hours via continuous DNA-mediated fluorophore exchange

Florian Stehr,Christian Niederauer,Petra Schwille,Julian Bauer,Ralf Jungmann,Kristina Ganzinger,Johannes Stein

doi:10.1038/s41467-021-24223-4

Abstract

Monitoring biomolecules in single-particle tracking experiments is typically achieved by employing fixed organic dyes or fluorescent fusion proteins linked to a target of interest. However, photobleaching typically limits observation times to merely a few seconds, restricting downstream statistical analysis and observation of rare biological events. Here, we overcome this inherent limitation via continuous fluorophore exchange using DNA-PAINT, where fluorescently-labeled oligonucleotides reversibly bind to a single-stranded DNA handle attached to the target molecule. Such versatile and facile labeling allows uninterrupted monitoring of single molecules for extended durations. We demonstrate the power of our approach by observing DNA origami on membranes for tens of minutes, providing perspectives for investigating cellular processes on physiologically relevant timescales.

Highlights

Monitoring biomolecules in single-particle tracking experiments is typically achieved by employing fixed organic dyes or fluorescent fusion proteins linked to a target of interest
For quantitative comparison of single dyes and THs, we distinguish between the full trajectory (x, y, t) of a molecule and the sections where this molecular path can be visualized by detecting a fluorescence signal, leading to the recording of a multitude of trajectories per particle
In summary, our labeling strategy for fluorescence-based Single-Particle Tracking (SPT), using a 1:1 functionalization with a DNA-based TH and exploiting DNA-mediated fluorophore exchange largely decouples trajectory lengths from the photon budget of single dye molecules, allowing for observations of target particles from minutes to hours depending on the experimental conditions

Summary

Introduction

Monitoring biomolecules in single-particle tracking experiments is typically achieved by employing fixed organic dyes or fluorescent fusion proteins linked to a target of interest. Photobleaching typically limits observation times to merely a few seconds, restricting downstream statistical analysis and observation of rare biological events We overcome this inherent limitation via continuous fluorophore exchange using DNA-PAINT, where fluorescently-labeled oligonucleotides reversibly bind to a single-stranded DNA handle attached to the target molecule. Such versatile and facile labeling allows uninterrupted monitoring of single molecules for extended durations. The key principle is DNA-mediated fluorophore exchange: short fluorescently-labeled oligonucleotides (‘imagers’, 8 bp; sequence: 5′-GAGGAGGA-3′-Cy3B) transiently bind a complementary single, long DNA strand attached to the molecule of interest (‘tracking handle’ TH, 54 base pairs, Fig. 1a). This is achieved by allowing multiple (max. 6, see Supplementary Fig. 2) imagers to bind simultaneously, maximizing their association rate (kon)[17,18,19,20,21], and optimizing their dwell times (koff) (see Fig. 1b for schematic depiction)

Methods

Results

Conclusion