Art by Dahlia Kordit
Since the famous publication of Robert Hooke’s Micrographia in 1665, the cell has captured the imagination and fascination of innumerable scientists. Although we now know about organelles and their functions thanks to techniques like CRISPR-Cas9 and single-interference RNA, there is a barrier created by the highly dynamic nature of the inside of the cell. In other words, the cell’s inner workings are so transient that it is difficult for existing approaches to capture the full picture—often only the beginning and the end states of experiments are ascertained, not the in-between. The recent field of optogenetics is one solution to this problem.
Optogenetics is a biological technique where researchers shine light on cells to activate and control their processes with extraordinary precision. The speed and reversibility of optogenetics allow it to revolutionize the ways cell activity can be switched on and off. Living systems, such as cells, can adapt to short-term “genetic perturbations,” alterations that involve modifying genes to study their function, such as with CRISPR-Cas9. By using light to induce a change in gene function faster than genetic perturbation, the living system is given less time to adapt and can give researchers a clearer view of actual cellular processes. In particular, chemo-optogenetics, which combines genetic engineering with synthetic chemistry, has shown great promise for capturing rare insights into otherwise hidden processes.
In Sweden at Umeå University, a team of researchers led by Yaowen Wu is pioneering a chemo-optogenetic technique that allows them to control cell activity with light and observe intracellular interactions working quickly in real time. In this technique, a so-called “molecular glue” utilizes light to not only allow cells and substrates—the molecules they act on—to bind together, activate, and control their processes at a highly precise level, but also to allow them to do so reversibly.
Much like how we have photoreceptors in our eyes, cells contain proteins called opsins that detect light and function as the “on” button for many necessary internal processes. In the lab, these opsins are repurposed to produce and study new cellular interactions. However, these natural opsins come with their own set of challenges. The function of an opsin relies on the specific structure within its arrangement of the chromophore, the key light-detecting compound within the opsin. In turn, the opsins remain stable and difficult to change: a double-edged sword when it comes to experimenting with them. “If you want to do more engineering, like modifying or improving the [optogenetic] system, it’s challenging,” Wu said. “If you change one structure in the chromophores or even the protein itself, then you might lose this photoresponse, or you aren’t able to control it.”
With synthetic chemistry, light-sensitive molecules can be synthesized and structurally changed, leading to enhanced light response and control of cell function. This eliminates the limitations of regular optogenetics while also granting an element of versatility and freedom to modulate the system. Building off the concepts behind chemo-optogenetics, the Wu Lab developed photocleavable molecular glues (photoMGs) to improve the inner cell conditions that drive protein reactions and functions. However, these photoMGs only lasted one round of light manipulation, making them very short-lived and difficult to sustain during experiments. In response to this challenge, the more durable and versatile modular photoswitchable chemically induced dimerization (sCID) system was born, thanks largely to the dedication and innovation of Jun Zhang and Laura Herzog, co-first-authors of the paper published by the Wu Lab.
During key developmental phases of the sCID system, Zhang said controlling the light system was a big limitation and thus a major focus of their research. “In our earliest versions, we primarily focused on blue light and found a couple of drawbacks,” Herzog said. “Longer exposure to blue light tends to be somewhat cytotoxic and [involves] tissue penetration, so other wavelengths would be required to find different ways of delivering the light to the affected tissues.”
One of the most substantial breakthroughs with sCID is its ability to perform multiple rounds of activation and deactivation without degrading the compound. “Whereas before we [had] molecular glues that typically allowed a single round of control […] here we can switch the same compound back and forth multiple rounds,” Herzog said. This reversible capability is pivotal because it permits repeated experiments using the same cells and conditions, which minimizes variability and maximizes reliability in the data.
The research team achieved this through innovative chemical design, particularly by selecting the molecules azobenzene and diazocine as photoswitch cores. They can rapidly toggle between two states—trans and cis—when exposed to different wavelengths of light. Additionally, their robust switching efficiency ensures precise control over protein interactions.
Choosing the right linker was another crucial aspect of the molecular glue’s design. The linker bridges the photoswitch core to the proteins being manipulated. If the linker is too long, the proteins could interact unintentionally, while if it’s too short, the shape change induced by the light-activated photoswitch would not effectively control protein binding. “If the linkers are incorrect, this small space change will not affect the binding of the two other proteins [the photoswitch core and target protein],” Herzog said. To overcome this, the team utilized computer-aided rational design, known as molecular dynamics simulation, and experimented with multiple designs before identifying the optimal linker chemistry that consistently facilitated protein binding in the trans state and dissociation in the cis state.
The molecular glue system’s efficiency was validated through rigorous in vitro and cellular testing. In vitro, Herzog and Zhang measured the photoswitch’s half-life, thermal relaxation, and resistance to reduction, ensuring the compound’s stability before introducing it into living cells. They also conducted several rounds of activation and deactivation to confirm the compound’s durability. In cells, two key assays were used: a split nano luciferase complementation assay to track dimerization (a scientific test utilizing special light-producing enzymes to see if two proteins can “dimerize,” or join together, in a cell), and subcellular recruitment assays to observe protein localization changes (where special markers are used to track the motion of proteins inside cells in real-time).
This modular sCID system’s versatility is a game-changer for chemo-optogenetics. It offers an unprecedented level of control over space and time, allowing scientists to manipulate cellular processes with precision. “We illuminated parts of cell populations and upregulated a protein function in one part, but not in the other part of the population,” Zhang said, illustrating the method’s capability to target specific cellular regions.
The Wu Lab is eager to explore new photoswitchable compounds and further optimize the system for different cellular contexts. The planned applications in zebrafish and mouse models will enable a deeper understanding of the system’s efficacy and safety in organisms, paving the way for studies that translate the research into everyday life. Additionally, expanding the use of this technology to address challenges faced by conventional optogenetic systems—such as light penetration limitations and off-target effects—could meaningfully enhance its utility in complex tissues and whole organisms. By exploring bistable molecules, which can switch between two states, and alternative protein designs, their findings offer innovative solutions for precision medicine and synthetic biology. Their pioneering work demonstrates how the marriage of synthetic chemistry and genetic engineering can overcome the limitations of conventional methods, opening new avenues for biological research.