Before the beginning of the action, we had identified a way to measure the activity of esterases, a class of enzymes involved in metabolism, with single-molecule sensitivity. Early in the action, we showed that these tools could be used to label immune cells and track them in a whole animal with excellent selectivity and sensitivity. These results have been published (ACS Chem. Biol. 2020, 15, 6, 1613).
During the investigation of probes for esterases, we also noticed that small changes in the structure of the molecules could render them useful to sense the polarity and viscosity of the environment with super-resolution. This kind of probe is unprecedented, and we recently demonstrated that it could be used to measure the physical properties of phase-separated compartments within the nucleus of living cells. These results have been published (ACS Chem. Biol. 2023, 18, 1066)
As proposed, we have been exploring how small-molecule probes can be enhanced by using self-labeling proteins such as SNAP-tag and HaloTag (Angew. Chem. Int. Ed. 2020, 59, 7669). Early on in the action, we identified an opportunity to enhance the properties of spontaneously blinking probes. Our idea was to combine spontaneous blinking with photoactivation to create probes that are mild to cells, but that can be controlled better than spontaneously blinking dyes. This work has led to the development of a fluorescent label that, combined with HaloTag, can be used to image single molecules of proteins for unprecedentedly long times (Chem. Eur. J. 2022, 28, e202202832).
During this project, we also discovered that spontaneous blinking is exquisitely sensitive to the environment of the probe. This observation led us to hypothesize that spontaneously blinking patterns could be used to identify macromolecules, e.g. protein and peptides, without having to sequence them. Using a combination of single-molecule imaging and deep learning, we tested this hypothesis and showed that we can identify the primary sequence and presence of post-translational modifications in peptides (J. Am. Chem. Soc. 2023, 145, 2, 1441). More recently, we also showed that full-length, folded proteins can be fingerprinted and identified using this method (manuscript in preparation).
We also identified completely novel scaffolds for SMLM and beyond (Nat. Chem. 2024, 16, 28). An advantage of these molecules is that they cover a vast range of the electromagnetic spectrum, including the near-infrared. Using these probes, we are also developing a single-molecule FRET method applicable in intact cells, potentially providing a much-needed approach to study protein-protein interactions in their native environment. Importantly, the exploitation of these novel probes is reflected in a recent patent application (EP23153834.9) and the dissemination of the probes will be made possible through a licensing deal with Spirochrome (spirochrome.com).
