The Fluorescent Biosensor Revolution Begins

A major step forward is the high-contrast “binding-activated fluorescent biosensors” (nanosensors) that glow only when they bind to their target molecule. However, creating such nanosensors is difficult because an effective target binder and a fluorescence switch must be combined in a small molecular package that can also be efficiently delivered to a variety of sample types and economically produced on a large scale.

Now, a research team working with the Wyss Institute at Harvard University, Harvard Medical School (HMS), MIT, and the University of Edinburgh in the United Kingdom has developed a synthetic biology platform to facilitate the discovery, molecular evolution, and cost-effective production of small, high-throughput nanosensors that can detect specific proteins, peptides, and small molecules by increasing their fluorescence by up to 100-fold in less than a second. The platform uses novel fluorogenic amino acids (FgAAs) as a key component, which can be encoded into the target-binding sequences of small proteins using an innovative methodology that enables in vitro expansion of the genetic code. Through a process of high-throughput sensor screening, validation, and directed evolution, the platform enables the rapid and cost-effective transformation of protein bonds into high-contrast nanosensors for a wide range of applications in fundamental research, environmental monitoring, medical diagnostics, and extended therapy. The results were published in Nature communication.

“We have long been working to expand the genetic code of cells to give them new capabilities that will enable research, biotechnology and medicine in various fields. This study is a very promising extension of this work.” in vitro“ said George Church, Ph.D., a Wyss faculty member who led the study. “This novel synthetic biology platform addresses many of the barriers that have prevented us from improving proteins with new chemistries, such as more efficient, immediate biosensors, and is poised to impact many biomedical areas.” Church is the leader of the Wyss Institute’s Synthetic Biology Platform, as well as the Robert Winthrop Professor of Genetics at HMS and a professor of health sciences and technology at Harvard University and MIT.

Protein plus scaffold equals nanosensor

The team, led by co-senior author and co-corresponding author Erkin Kuru, Ph.D., from Church’s group, built on an earlier discovery that FgAAs can convert known binding proteins into optical sensors that fluoresce when their FgAA is sandwiched between the binding sequence and the target molecule. The Wyss scientists collaborated on the study with co-corresponding author Marc Vendrell, Ph.D., a professor at the University of Edinburgh and an expert in translational chemistry and biomedical imaging, with whom Kuru shared an early interest in FgAAs.

Starting with the pandemic, the team first envisioned “instant diagnostics for COVID-19” and focused on a miniature, engineered antibody (nanobody) that binds to the SARS-CoV-2 Spike protein on the virus’s surface. They created hundreds of linker variants, essentially assembling FgAA by chemically linking cysteine ​​or lysine amino acids that were genetically introduced to positions known to be in close contact with the Spike target to one of 20 different fluorogenic chemical scaffolds. Using a simple binding assay, they selected the fluorogenic variants that produced the largest increase in fluorescence within milliseconds of binding to the target.

They then used the same process to construct nanosensors made of nanobodies, or mini-proteins, that bind to different SARS-CoV-2 target sites, as well as a range of other molecular targets, including the cancer-relevant cell growth factor receptor EGFR, the alpha-tag peptide used by cell biologists to track proteins in cells, and the stress hormone cortisol. Importantly, the nanosensors also effectively signaled the presence of their targets in human cells and living bacteria under a microscope, demonstrating their usefulness as effective imaging tools.

Evolution of Nano-Sensors

Despite its potential, the first version of the platform was limited by a laborious and time-consuming process involving multiple purification steps of the generated binding sequences. “We wanted to significantly expand our molecular design space by increasing the platform’s high-throughput capabilities,” Kuru said. “To achieve this, we allowed the ribosome, which naturally synthesizes all proteins in cells, to do most of the work in an engineered cell-free process.”

In version 2.0 of their platform, the team prefabricated a so-called “synthetic amino acid” with a fluorogenic scaffold already pre-attached to it. Synthetic amino acids have already proven their worth in therapies such as the weight-loss drug Ozempic; however, they cannot be easily incorporated into protein sequences because there is no natural mechanism for the ribosome to handle them. “To overcome this obstacle, we reassigned a rarely used codon in the universal genetic code using novel genetic expansion chemistry so that it could encode synthetic amino acids such as our prefabricated custom FgAA. We essentially adapted the protein synthesis process to build binding-activated fluorescent nanosensors,” said first author Jonathan Rittichier, Ph.D., who co-developed the method.

Their new process not only allowed the researchers to produce millions of nanosensor candidates at once, but also helped speed up subsequent nanosensor testing because the entire synthesis mix could be directly coupled to the target molecule or added to living cells without additional purification. They could now screen for hundreds of variants in a day, rather than a few dozen in a few weeks. Underscoring the power of the advanced platform, they found a specific position encoding their FgAA in the original SARS-CoV-2 nanoparticle binding site, which unexpectedly resulted in a nanosensor with higher affinity than their original COVID-19 nanosensor when exposed to the Spike target protein.

Finally, because this would greatly increase the potential for creating better nanosensors, the team used their platform to optimize the nanobody sequence itself. They used a classic synthetic biology process known as “directed evolution,” in which proteins are optimized through iterative design-build-test cycles that use the best versions of the protein identified in one cycle as the basis for finding even better ones in the next. Starting with the best nanosensor, which they had previously designed to immediately detect the Spike protein of the original SARS-CoV-2 strain, Kuru, Rittichier, and team created extensive libraries of nanobodies that included variants that maintained the nonstandard FgAA in the original position but had many of the standard amino acids in other critical positions structurally replaced. Evolution of the best ones led them to new nanosensors with orders of magnitude higher binding affinities for the Spike protein. Interestingly, using a modified version of this directed evolution system, the researchers discovered nanosensors that were able to selectively detect various newer Omicron variants.

“This is a major step forward in our ability to rapidly design inexpensive fluorescent biosensors for real-time disease monitoring with enormous potential for diagnostics and precision medicine,” Vendrell said. Kuru added, “we can also incorporate synthetic amino acids with many other functionalities into all sorts of proteins to create new therapies and a much broader range of research tools.” Indeed, Kuru and co-authors Helena de Puig, Ph.D., and Allison Flores, along with Church and senior author and Wyss Senior Faculty Member James Collins, Ph.D., also launched the Wyss Institute’s AminoX project, which is using the platform to develop new therapies.

“This highly innovative work, enabling a new and more efficient generation of binding-activated biosensors, demonstrates the extraordinary potential of synthetic biology. The Wyss team has successfully developed a fundamental biological process into a platform with enormous potential to ultimately solve many diagnostic and therapeutic challenges,” said Donald Ingber, MD, Ph.D., founding director of Wyss, who is also Judah Folkman Professor of Vascular Biology at HMS and Boston Children’s Hospital, as well Hansjörg Wyss Professor of Bio-Inspired Engineering in SEAS.

Additional authors on the study are Subhrajit Rout, Isaac Han, Abigail Reese, Thomas Bartlett, Fabio De Moliner, Sylvie Bernier, Jason Galpin, Jorge Marchand, William Bedell, Lindsay Robinson-McCarthy, Christopher Ahern, Thomas Bernhardt, and David Rudner. The study was supported by a Wyss Institute Validation Project, a US Department of Energy Grant (award no. DE-FG02-02ER63445) and an ERC Consolidator Grant (award no. DYNAFLUORS, 771443), and a Life Science Research Foundation Fellowship to Kuru.