Chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of the intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies.fluorescence microscopy | enzyme redesign | LplA | PRIME | chemical probe targeting F luorescent proteins are used ubiquitously in imaging, but their dim fluorescence, rapid photobleaching, and large size limit their utility. At ∼27 kDa (∼240 aa), fluorescent proteins can disrupt protein folding and trafficking or impair protein function (1, 2). Chemical fluorophores, in comparison, are typically less than 1 kDa in size, and are brighter and more photostable. These properties allow chemical fluorophores to perform better than fluorescent proteins in advanced imaging modalities such as singlemolecule tracking and superresolution microscopies (3, 4).Site-specific labeling of proteins with chemical fluorophores inside living cells is challenging because these fluorophores are not genetically encodable and therefore must be posttranslationally targeted inside a complex cellular milieu. Existing methods to achieve this targeting either require large fusion tags [such as HaloTag (5), the SNAP tag (6), and the DHFR tag (7)] or have insufficient specificity [such as biarsenical dye targeting (8) and amber codon suppression (9)]. To achieve a labeling specificity comparable to fluorescent proteins, we developed PRIME (PRobe Incorporation Mediated by Enzymes), which uses Escherichia coli lipoic acid ligase to attach small molecules to a 13-aa peptide tag (Fig. 1A) (10). To make PRIME more useful for cellular protein imaging, we sought to engineer the system for the targeting of bright chemical fluorophores. The challenge, though, is that lipoic acid ligase (LplA) has a small and fully enclosed substrate-binding pocket that even with extensive structure-guided mutagenesis has not until this point been able to accommodate large chemical structures. ResultsSynthesis of Resorufin Derivatives for PRIME. We considered fluorophores for PRIME based on the steric constraints of LplA. Far-red emitters such as Cy5 and Atto 647N, although photophysically desirable, are so bulky that binding inside LplA would require...

Objectives Initial reports indicate adequate performance of some serology-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) assays. However, additional studies are required to facilitate interpretation of results, including how antibody levels impact immunity and disease course. Methods A total of 967 subjects were tested for IgG antibodies reactive to SARS-CoV-2, including 172 suspected cases of SARS-CoV-2, 656 plasma samples from healthy donors, 49 sera from patients with rheumatic disease, and 90 specimens from individuals positive for polymerase chain reaction (PCR)–based respiratory viral panel. A subgroup of SARS-CoV-2 PCR-positive cases was tested for IgM antibodies by proteome array method. Results All specificity and cross-reactivity specimens were negative for SARS-CoV-2 IgG antibodies (0/795, 0%). Positive agreement of IgG with PCR was 83% of samples confirmed to be more than 14 days from symptom onset, with less than 100% sensitivity attributable to a case with severe immunosuppression. Virus-specific IgM was positive in a higher proportion of cases less than 3 days from symptom onset. No association was observed between mild and severe disease course with respect to IgG and IgM levels. Conclusions The studied SARS-CoV-2 IgG assay had 100% specificity and no adverse cross-reactivity. Measures of IgG and IgM antibodies did not predict disease severity in our patient population.

We present a methodology for targeting quantum dots to proteins on living cells in two steps. In the first step, E. coli lipoic acid ligase (LplA) site-specifically attaches 10-bromodecanoic acid onto a 13-amino acid peptide that can be genetically fused to a protein of interest. In the second step, quantum dots derivatized with HaloTag, a modified haloalkane dehalogenase, react with the ligated bromodecanoic acid to form a covalent adduct. We found this targeting method to be specific, fast, and fully orthogonal to a previously reported and analogous quantum dot targeting method using E. coli biotin ligase and streptavidin. We used these two methods in combination for two-color quantum dot visualization of different proteins expressed on the same cell or on neighboring cells. Both methods were also used to track single molecules of neurexin, a synaptic adhesion protein, to measure its lateral diffusion in the presence of neuroligin, its trans-synaptic adhesion partner.

Background: Initial reports indicate adequate performance of some serological-based SARS-CoV-2 assays. However, additional studies are required to facilitate interpretation of results, including how antibody levels impact immunity and disease course. Methods: In this study, a total of 968 subjects were tested for IgG antibodies reactive to SARS-CoV-2. We confirmed analytic specificity using 656 plasma samples from healthy donors, 49 sera from patients with rheumatic disease, and 90 specimens from individuals positive for PCR-based respiratory viral panel. One-hundred seventy-three cases of confirmed or suspected SARS-CoV-2 were tested for IgG. A subgroup of 37 SARS-CoV-2 PCR-positive cases was tested for nucleocapsid-specific IgM antibody using an in-house developed microarray method. Antibody levels were compared between disease severity groups. Results: All specificity specimens were negative for SARS-CoV-2 IgG antibodies (0/656, 0%). Cross reactivity was not detected in specimens with antinuclear antibodies and rheumatoid factor, or cases with previous diagnosis of viral infection including human coronavirus. Positive agreement of IgG with PCR was 83% of samples confirmed to be more than 14 days from symptom onset, with less than 100% sensitivity attributable to a case with severe immunosuppression. Virus-specific IgM was positive in a higher proportion of cases less than 3 days from symptom onset. No association was observed between mild and severe disease course with respect to IgG and IgM levels. Conclusions: The studied SARS-CoV-2 IgG assay had 100% specificity and no adverse cross-reactivity. Index values of IgG and IgM antibodies did not predict disease severity in our patient population.

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