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Boronic acid-containing fluorescent molecules have been widely used to sense hydrogen peroxide and peroxynitrite, which are important reactive oxygen and nitrogen species in biological systems. However, it has been challenging to gain specificity. Our previous studies developed genetically encoded, green fluorescent peroxynitrite biosensors by genetically incorporating a boronic acid-containing noncanonical amino acid (ncAA), p-boronophenylalanine (pBoF), into the chromophore of circularly permuted green fluorescent proteins (cpGFPs). In this work, we introduced pBoF to amino acid residues spatially close to the chromophore of an enhanced circularly permuted red fluorescent protein (ecpApple). Our effort has resulted in two responsive ecpApple mutants: one bestows reactivity toward both peroxynitrite and hydrogen peroxide, while the other, namely, pnRFP, is a selective red fluorescent peroxynitrite biosensor. We characterized pnRFP in vitroand in live mammalian cells. We further studied the structure and sensing mechanism of pnRFP using X-ray crystallography, 11B-NMR, and computational methods. The boron atom in pnRFP adopts an sp2-hybridization geometry in a hydrophobic pocket, and the reaction of pnRFP with peroxynitrite generates a product with a twisted chromophore, corroborating the observed “turn-off” fluorescence response. Thus, this study extends the color palette of genetically encoded peroxynitrite biosensors, provides insight into the response mechanism of the new biosensor, and demonstrates the versatility of using protein scaffolds to modulate chemoreactivity.

Naturally occurring protein switches have been repurposed for the development of biosensors and reporters for cellular and clinical applications1. However, the number of such switches is limited, and reengineering them is challenging. Here we show that a general class of protein-based biosensors can be created by inverting the flow of information through de novo designed protein switches in which the binding of a peptide key triggers biological outputs of interest2. The designed sensors are modular molecular devices with a closed dark state and an open luminescent state; analyte binding drives the switch from the closed to the open state. Because the sensor is based on the thermodynamic coupling of analyte binding to sensor activation, only one target binding domain is required, which simplifies sensor design and allows direct readout in solution. We create biosensors that can sensitively detect the anti-apoptosis protein BCL-2, the IgG1 Fc domain, the HER2 receptor, and Botulinum neurotoxin B, as well as biosensors for cardiac troponin I and an anti-hepatitis B virus antibody with the high sensitivity required to detect these molecules clinically. Given the need for diagnostic tools to track the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)3, we used the approach to design sensors for the SARS-CoV-2 spike protein and antibodies against the membrane and nucleocapsid proteins. The former, which incorporates a de novo designed spike receptor binding domain (RBD) binder4, has a limit of detection of 15 pM and a luminescence signal 50-fold higher than the background level. The modularity and sensitivity of the platform should enable the rapid construction of sensors for a wide range of analytes, and highlights the power of de novo protein design to create multi-state protein systems with new and useful functions.

Coelenterazine (CTZ)-utilizing marine luciferases and their derivatives have attracted significant attention because of their ATP-independency, fast enzymatic turnover, and high bioluminescence brightness. However, marine luciferases typically emit blue photons and their substrates, including CTZ and the recently developed diphenylterazine (DTZ), have poor water solubility, hindering their in vivoapplications. Herein, we report a family of pyridyl CTZ and DTZ analogs that exhibit spectrally shifted emission and improved water solubility. Through directed evolution, we engineered a LumiLuc luciferase with broad substrate specificity. In the presence of corresponding pyridyl substrates (i.e., pyCTZ, 6pyDTZ, or 8pyDTZ), LumiLuc generates highly bright blue, teal, or yellow bioluminescence. We compared our LumiLuc–8pyDTZ pair with several benchmark reporters in a tumor xenograft mouse model. Our new pair, which does not need organic cosolvents for in vivoadministration, surpasses other reporters by detecting early tumors. We further fused LumiLuc to a red fluorescent protein, resulting in a LumiScarlet reporter with further red-shifted emission and enhanced tissue penetration. LumiScarlet–8pyDTZ was comparable to Akaluc–AkaLumine, the brightest ATP-dependent luciferase–luciferin pair, for detecting cells in deep tissues of mice. In summary, we have engineered a new family of ATP-independent bioluminescent reporters, which will have broad applications because of their ATP-independency, excellent biocompatibility, and superior in vivosensitivity.

In vivobioluminescence imaging (BLI) has become a standard, non-invasive imaging modality for following gene expression or the fate of proteins and cells in living animals. Currently, bioluminescent reporters used in laboratories are mostly derivatives of two major luciferase families: ATP-dependent insect luciferases and ATP-independent marine luciferases. Inconsistent results of experiments using different bioluminescent reporters, such as Akaluc and Antareas2, have been reported. Herein, we re-examined the inconsistency in several experimental settings and identified the factors, such as ATP dependency, stability in serum, and molecular sizes of luciferases, that contributed to the observed differences. We expect this study will make the research community aware of these factors and facilitate more accurate interpretation of BLI data by considering the nature of each bioluminescent reporter.

Red-shifted luciferins and corresponding mutants of NanoLuc enable brighter bioluminescence imaging in vitro, in cells, and in deep tissues of living mice alone and in the context of the newly developed Antares2 BRET reporter.

New variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to arise and prolong the coronavirus disease 2019 (COVID-19) pandemic. Here, we used a cell-free expression workflow to rapidly screen and optimize constructs containing multiple computationally designed miniprotein inhibitors of SARS-CoV-2. We found the broadest efficacy was achieved with a homotrimeric version of the 75-residue angiotensin-converting enzyme 2 (ACE2) mimic AHB2 (TRI2-2) designed to geometrically match the trimeric spike architecture. Consistent with the design model, in the cryo-electron microscopy structure TRI2-2 forms a tripod at the apex of the spike protein that engaged all three receptor binding domains simultaneously. TRI2-2 neutralized Omicron (B.1.1.529), Delta (B.1.617.2), and all other variants tested with greater potency than the monoclonal antibodies used clinically for the treatment of COVID-19. TRI2-2 also conferred prophylactic and therapeutic protection against SARS-CoV-2 challenge when administered intranasally in mice. Designed miniprotein receptor mimics geometrically arrayed to match pathogen receptor binding sites could be a widely applicable antiviral therapeutic strategy with advantages over antibodies in greater resistance to viral escape and antigenic drift, and advantages over native receptor traps in lower chances of autoimmune responses.