Periodic Reporting for period 4 - HDPROBES (Photoactivatable Sensors and Blinking Dyes for Live-Cell, Single-Molecule Localization Microscopy)
Reporting period: 2023-08-01 to 2024-12-31
Fluorescence microscopy is one of the most widely used methods in cell biology. The popularity of this method is due to its non-invasiveness and molecular precision, which allow for the observation of biological processes in real time. A salient problem of fluorescence microscopy is its resolution, which does not allow for the direct observation of individual molecules. In the last 15 years, methods to surpass this resolution limit have been developed, and the concept of single-molecule localization microscopy (SMLM) has emerged. This technique relies on molecular labels that emit light discontinuously; a phenomenon often referred to as “blinking”. The main goal of this action is to develop new molecular labels that blink in several different ways, allowing for various cellular processes to be observed, with single-molecule resolution, in living cells.
We are specifically interested in two main uses of blinking molecules: As sensors for the activity of enzymes and the presence of reactive signaling molecules and as labels to observe proteins for a very long time. The first part is being addressed by coupling the ability of molecules to blink to specific reactions with biologically important partners, such as enzymes. For this purpose, we are exploring photochemical reactions of various functional groups before and after they have been exposed to biologically relevant reactive oxygen and nitrogen species (i. e., hydrogen peroxide, nitroxyl, etc.). This combination of ground-state and excited-state reactivity has already given us a few probes that can be used to track the behavior of single molecules in living cells. We are also exploring molecules that can blink spontaneously. This characteristic would enable timelapse imaging of single molecules for very long periods of time because the spontaneous blinking decreases the amount of light needed for imaging and thus minimizes toxicity to the cells.
These studies are important for society because health and disease are dictated to a large extent by how proteins behave in our cells. By providing new methods to study proteins and their activities in living cells, we will contribute to understanding fundamental biological mechanisms behind disease and, potentially, contribute to the development of novel therapies.
We are specifically interested in two main uses of blinking molecules: As sensors for the activity of enzymes and the presence of reactive signaling molecules and as labels to observe proteins for a very long time. The first part is being addressed by coupling the ability of molecules to blink to specific reactions with biologically important partners, such as enzymes. For this purpose, we are exploring photochemical reactions of various functional groups before and after they have been exposed to biologically relevant reactive oxygen and nitrogen species (i. e., hydrogen peroxide, nitroxyl, etc.). This combination of ground-state and excited-state reactivity has already given us a few probes that can be used to track the behavior of single molecules in living cells. We are also exploring molecules that can blink spontaneously. This characteristic would enable timelapse imaging of single molecules for very long periods of time because the spontaneous blinking decreases the amount of light needed for imaging and thus minimizes toxicity to the cells.
These studies are important for society because health and disease are dictated to a large extent by how proteins behave in our cells. By providing new methods to study proteins and their activities in living cells, we will contribute to understanding fundamental biological mechanisms behind disease and, potentially, contribute to the development of novel therapies.
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).
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).
We can already count several advances that this action has brought about, some of them with genuinely transformative potential in various fields:
1. Protein/small-molecule hybrid sensors. This new kind of probe, developed during the action, allows for very selective imaging of reactive biological molecules and enzymes in any subcellular location. This concept will enable accurate counting of reactive molecules in specific compartments within the cell, allowing for an unprecedented level of resolution.
2. New scaffolds for SMLM. We recently developed completely new scaffolds (beyond the rhodamine standard) for SMLM. A crucial advance of these molecules is that they are significantly brighter than state-of-the-art dyes. They are highly tunable and can be easily extended into very long wavelengths. This last feature will enable super-resolution microscopy in samples thicker than isolated cells. Furthermore, these dyes will allow us to develop ligation methods that won’t require large self-labeling proteins, which will open up opportunities to label very small proteins and delicate protein complexes that do not tolerate large tags (Nat. Chem. 2024, 16, 28). Furthermore, a patent application has been submitted (EP23153834.9). Further advances have been recently accomplished through protein engineering efforts using state-of-the-art deep learning methods (manuscript in preparation).
3. Blinking patterns and their information content. After developing several spontaneously blinking dyes, we are exploring further applications of these tools beyond SMLM. We have identified an exciting opportunity, which is the topic of a recent publication (J. Am. Chem. Soc. 2023, 145, 2, 1441), and newer results show that this principle could be applied to develop novel methods for supersensitive protein detection. Furthermore, this work has is also the topic of a patent application (EP 22210671.8).
1. Protein/small-molecule hybrid sensors. This new kind of probe, developed during the action, allows for very selective imaging of reactive biological molecules and enzymes in any subcellular location. This concept will enable accurate counting of reactive molecules in specific compartments within the cell, allowing for an unprecedented level of resolution.
2. New scaffolds for SMLM. We recently developed completely new scaffolds (beyond the rhodamine standard) for SMLM. A crucial advance of these molecules is that they are significantly brighter than state-of-the-art dyes. They are highly tunable and can be easily extended into very long wavelengths. This last feature will enable super-resolution microscopy in samples thicker than isolated cells. Furthermore, these dyes will allow us to develop ligation methods that won’t require large self-labeling proteins, which will open up opportunities to label very small proteins and delicate protein complexes that do not tolerate large tags (Nat. Chem. 2024, 16, 28). Furthermore, a patent application has been submitted (EP23153834.9). Further advances have been recently accomplished through protein engineering efforts using state-of-the-art deep learning methods (manuscript in preparation).
3. Blinking patterns and their information content. After developing several spontaneously blinking dyes, we are exploring further applications of these tools beyond SMLM. We have identified an exciting opportunity, which is the topic of a recent publication (J. Am. Chem. Soc. 2023, 145, 2, 1441), and newer results show that this principle could be applied to develop novel methods for supersensitive protein detection. Furthermore, this work has is also the topic of a patent application (EP 22210671.8).