Thioredoxin (Trx) is one of the two major thiol antioxidants playing essential roles in redox homeostasis and signaling. Despite the importance, there is a lack of method in monitoring Trx redox dynamics in live cells, hindering a better understanding of physiological and pathological roles of the Trx redox system. In this work, we developed the first genetically encoded fluorescent biosensor for Trx redox by engineering a redox relay between the active-site cysteines of human Trx1 and rxRFP1—a redox-sensitive red fluorescent protein. We utilized the resultant biosensor—TrxRFP1—to selectively monitor perturbations of Trx redox in various mammalian cell lines. We subcellularly localized TrxRFP1 to image compartmentalized Trx redox changes. We further combined TrxRFP1 with a green fluorescent Grx1-roGFP2 biosensor to simultaneously monitor Trx and glutathione redox dynamics in live cells in response to chemical and physiologically relevant stimuli.

Redox signaling and homeostasis are important for all forms of life on Earth. There has been great interest in monitoring redox dynamics in living cells and organisms as a mean to better understand redox biology in physiological and pathological conditions. Herein we report our recent results on the development of a genetically encoded redox-sensitive red fluorescent protein (rxRFP). We first identified a circularly permuted RFP (cpRFP) scaffold, which maintained its autocatalytic fluorescence, from a red fluorescent Ca2+ sensor, R-GECO1. We then introduced cysteine residue pairs to the N- and C- termini of the cpRFP scaffold, and subsequently optimized the length and composition of the sequences adjacent to the cysteine residues. From these libraries, we identified rxRFP, showing up to a 4-fold fluorescence increase in the oxidized state compared to the reduced state at pH 7.4. We thoroughly characterized rxRFP in vitro, and expressed it in living mammalian cells to monitor redox dynamics. With its excitation peak at 576 nm and emission peak at 600 nm, rxRFP is one of the first genetically encoded red fluorescent probes that can sense general redox states.

The RNA transcription complex (RTC) from the virus, SARS-CoV-2, is responsible for recognizing and processing RNA for two principal purposes. The RTC copies viral RNA for propagation into new virus and for ribosomal transcription of viral proteins. To accomplish these activities the RTC mechanism must also conform to a large number of imperatives including RNA over DNA base recognition, base pairing, distinguishing viral and host RNA, production of mRNA that conforms to host ribosome conventions, interface with error checking machinery and evading host immune responses. In addition, the RTC will discontinuously transcribe specific sections of viral RNA to amplify certain proteins over others. Central to SARS-CoV-2 viability, the RTC is therefore dynamic and sophisticated. We have conducted a systematic structural investigation of three components that make up the RTC: Nsp7, Nsp8 and Nsp12 (also known as RNA dependent RNA polymerase (RdRp)). We have solved high resolution crystal structures of the Nsp7/8 complex providing insight into the interaction between the proteins. We have used small angle X-ray and neutron solution scattering (SAXS and SANS) on each component individually as pairs and higher order complexes and with and without RNA. Using size exclusion chromatography and multi-angle light scattering coupled SAXS (SEC-MALS-SAXS) we defined which combination of components form transient or stable complexes. We used contrast matching neutron scattering to mask specific complex forming components to test whether components change conformation upon complexation. Altogether, we find that individual Nsp7, Nsp8 and Nsp12 structures vary based on whether other proteins in their complex are present. Combining our crystal structure, atomic coordinates reported elsewhere, SAXS, SANS and other biophysical techniques we provide greater insight into the RTC assembly, mechanism and potential avenues for disruption of the complex and its functions.

Hydrogen sulfide (HS) is an important gasotransmitter. Although a large number of fluorescent probes for cellular HS have been reported, only a few can detect HS in mitochondria, a cellular organelle connecting HS with mitochondrial function and metabolic pathways. We hereby describe a novel near-infrared fluorescent probe, nimazide, by introducing sulfonyl azide to the core structure of a QSY-21 dark quencher. Nimazide responded quickly to HS, resulting in robust fluorescence turn-off changes. This conversion displayed high specificity and fast kinetics. More impressively, we observed a robust fluorescence decrease in live cells loaded with mitochondrial nimazide in response to extracellular addition of nanomolar HS, and successfully imaged biologically generated mitochondrial HS in live mammalian cells. Nimazide is one of the most sensitive fluorescent probes for mitochondrial HS.

We recently reported a redox-sensitive red fluorescent protein, rxRFP1, which is one of the first genetically encoded red-fluorescent probes for general redox states in living cells. As individual cellular compartments have different basal redox potentials, we hereby describe a group of rxRFP1 mutants, showing different midpoint redox potentials for detection of redox dynamics in various subcellular domains, such as mitochondria, the cell nucleus, and endoplasmic reticulum (ER). When these redox probes were expressed and subcellularly localized in human embryonic kidney (HEK) 293 T cells, they responded to membrane-permeable oxidants and reductants. In addition, a mitochondrially localized rxRFP1 mutant, Mito-rxRFP1.1, was used to detect mitochondrial oxidative stress induced by doxorubicin-a widely used cancer chemotherapy drug. Our work has expanded the fluorescent protein toolkit with new research tools for studying compartmentalized redox dynamics and oxidative stress under various pathophysiological conditions.

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There is great interest in developing boronolectins, which are synthetic lectin mimics containing a boronic acid functional group for reversible recognition of diol-containing molecules, such as glycans and ribonucleotides. However, it remains a significant challenge to gain specificity. Here, we present a genetically encoded boronolectin, which is a hybrid protein consisting of a noncanonical amino acid (ncAA) p-boronophenylalanine (pBoF), natural-lectin-derived peptide sequences, and a circularly permuted red fluorescent protein (cpRFP). The genetic encodability permitted a straightforward protein engineering process to derive a red fluorescent biosensor that can specifically bind uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), an important nucleotide sugar involved in metabolic sensing and cell signaling. We further characterized the resultant boronic acid-and peptide-assisted UDP-GlcNAc sensor (bapaUGAc) both in vitro and in live mammalian cells. Because UDP-GlcNAc in the endoplasmic reticulum (ER) and Golgi apparatus plays essential roles in glycosylating biomolecules in the secretory pathway, we genetically expressed bapaUGAc in the ER and Golgi and validated the sensor for its responses to metabolic disruption and pharmacological inhibition. In addition, we combined bapaUGAc with UGAcS, a recently reported green fluorescent UDP-GlcNAc sensor based on an alternative sensing mechanism, to monitor UDP-GlcNAc level changes in the ER and cytosol simultaneously. We expect our work to facilitate the future development of specific boronolectins for carbohydrates. In addition, this newly developed genetically encoded bapaUGAc sensor will be a valuable tool for studying UDP-GlcNAc and glycobiology.

There is great interest in developing boronolectins that are synthetic lectin mimics containing a boronic acid functional group for reversible recognition of diol-containing molecules, such as glycans and ribonucleotides. However, it remains a significant challenge to gain specificity. Here, we present a genetically encoded boronolectin which is a hybrid protein consisting of a noncanonical amino acid (ncAA) p-boronophenylalanine (pBoF), natural-lectin-derived peptide sequences, and a circularly permuted red fluorescent protein (cpRFP). The genetic encodability permitted a straightforward protein engineering process to derive a red fluorescent biosensor that can specifically bind uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), an important nucleotide sugar involved in metabolic sensing and cell signaling. We further characterized the resultant boronic acid- and peptide-assisted UDP-GlcNAc sensor (bapaUGAc) both in vitro and in live mammalian cells. Because UDP-GlcNAc in the endoplasmic reticulum (ER) and Golgi apparatus plays essential roles in glycosylating biomolecules in the secretory pathway, we genetically expressed bapaUGAc in the ER and Golgi and validated the sensor for its responses to metabolic disruption and pharmacological inhibition. In addition, we combined bapaUGAc with UGAcS, a recently reported green fluorescent UDP-GlcNAc sensor based on an alternative sensing mechanism, to monitor UDP-GlcNAc level changes in the ER and cytosol simultaneously. We expect our work to facilitate the future development of specific boronolectins for carbohydrates. In addition, this newly developed genetically encoded bapaUGAc sensor will be a valuable tool for studying UDP-GlcNAc and glycobiology.