Developing a DNA-based nanoplatform for extended single-molecule fluorescence measurements with potential applications for in vivo studies

Abstract

Modern-day spectroscopy is capable of providing high-resolution, non-diffraction-limited images of the cellular interior. Using multiple fluorophores, biological processes such as vesicular transport, organelle remodelling and even protein-protein interactions can be visualized. However, the single-molecule perspective, crucial for understanding the spatially and temporally heterogeneous intracellular environment, is missing. While single-molecule and single-particle tracking, including inside living cells, has become routine, a lot of chemical information is lost because of the short observation time of the least intrusive fluorescent labels: organic fluorophores. These are prone to photobleaching: irreversible reactions of the excited state that completely quench the fluorescence of a molecule. Presented here are the initial stages of the experimental investigation of a proposed platform for overcoming this limitation by replacing a spent probe in situ. This proof-of-concept nanoplatform involves a dye/quencher-labelled DNA hairpin that can be added to and removed from target in a controlled fashion. The DNA hairpin fluctuations between the ‘open’ and the ‘closed’ state when bound to target can be observed by single-molecule F¨orster-type Resonance Energy Transfer (smFRET). Demonstrating that this design works experimentally with a trackable nanoparticle as the target will open doors to carrying out extended single-molecule fluorescence + tracking experiments in vitro and potentially in vivo, allowing rare biological events to be observed.