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How molecules interact governs how they move. Single-molecule tracking (SMT) thus provides a unique window into the dynamic interactions of biomolecules within live cells. Using transcription regulation as a case study, we describe how SMT works, what it can tell us about molecular biology, and how it has changed our perspective on the inner workings of the nucleus. We also describe what SMT cannot yet tell us and how new technical advances seek to overcome its limitations. This ongoing progress will be imperative to address outstanding questions about how dynamic molecular machines function in live cells.

ObjectivesTo identify incident SARS-CoV-2 infections and inform effective mitigation strategies in university settings, we piloted an integrated symptom and exposure monitoring and testing system among a cohort of university students and employees.DesignProspective cohort study.SettingA public university in California from June to August 2020.Participants2180 university students and 738 university employees.Primary outcome measuresAt baseline and endline, we tested participants for active SARS-CoV-2 infection via quantitative PCR (qPCR) test and collected blood samples for antibody testing. Participants received notifications to complete additional qPCR tests throughout the study if they reported symptoms or exposures in daily surveys or were selected for surveillance testing. Viral whole genome sequencing was performed on positive qPCR samples, and phylogenetic trees were constructed with these genomes and external genomes.ResultsOver the study period, 57 students (2.6%) and 3 employees (0.4%) were diagnosed with SARS-CoV-2 infection via qPCR test. Phylogenetic analyses revealed that a super-spreader event among undergraduates in congregate housing accounted for at least 48% of cases among study participants but did not spread beyond campus. Test positivity was higher among participants who self-reported symptoms (incidence rate ratio (IRR) 12.7; 95% CI 7.4 to 21.8) or had household exposures (IRR 10.3; 95% CI 4.8 to 22.0) that triggered notifications to test. Most (91%) participants with newly identified antibodies at endline had been diagnosed with incident infection via qPCR test during the study.ConclusionsOur findings suggest that integrated monitoring systems can successfully identify and link at-risk students to SARS-CoV-2 testing. As the study took place before the evolution of highly transmissible variants and widespread availability of vaccines and rapid antigen tests, further research is necessary to adapt and evaluate similar systems in the present context.

SARS coronavirus 2 (SARS-CoV-2) has caused an ongoing global pandemic with significant mortality and morbidity. At this time, the only FDA-approved therapeutic for COVID-19 is remdesivir, a broad-spectrum antiviral nucleoside analog. Efficacy is only moderate, and improved treatment strategies are urgently needed. To accomplish this goal, we devised a strategy to identify compounds that act synergistically with remdesivir in preventing SARS-CoV-2 replication. We conducted combinatorial high-throughput screening in the presence of submaximal remdesivir concentrations, using a human lung epithelial cell line infected with a clinical isolate of SARS-CoV-2. This identified 20 approved drugs that act synergistically with remdesivir, many with favorable pharmacokinetic and safety profiles. Strongest effects were observed with established antivirals, Hepatitis C virus nonstructural protein 5A (HCV NS5A) inhibitors velpatasvir and elbasvir. Combination with their partner drugs sofosbuvir and grazoprevir further increased efficacy, increasing remdesivir's apparent potency > 25-fold. We report that HCV NS5A inhibitors act on the SARS-CoV-2 exonuclease proofreader, providing a possible explanation for the synergy observed with nucleoside analog remdesivir. FDA-approved Hepatitis C therapeutics Epclusa® (velpatasvir/sofosbuvir) and Zepatier® (elbasvir/grazoprevir) could be further optimized to achieve potency and pharmacokinetic properties that support clinical evaluation in combination with remdesivir.

Single-molecule imaging provides a powerful way to study biochemical processes in live cells, yet it remains challenging to track single molecules while simultaneously detecting their interactions. Here, we describe a novel property of rhodamine dyes, proximity-assisted photoactivation (PAPA), in which one fluorophore (the 'sender') can reactivate a second fluorophore (the 'receiver') from a dark state. PAPA requires proximity between the two fluorophores, yet it operates at a longer average intermolecular distance than Förster resonance energy transfer (FRET). We show that PAPA can be used in live cells both to detect protein-protein interactions and to highlight a subpopulation of labeled protein complexes in which two different labels are in proximity. In proof-of-concept experiments, PAPA detected the expected correlation between androgen receptor self-association and chromatin binding at the single-cell level. These results establish a new way in which a photophysical property of fluorophores can be harnessed to study molecular interactions in single-molecule imaging of live cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has emerged as a major global health threat. The COVID-19 pandemic has resulted in over 168 million cases and 3.4 million deaths to date, while the number of cases continues to rise. With limited therapeutic options, the identification of safe and effective therapeutics is urgently needed. The repurposing of known clinical compounds holds the potential for rapid identification of drugs effective against SARS-CoV-2. Here, we utilized a library of FDA-approved and well-studied preclinical and clinical compounds to screen for antivirals against SARS-CoV-2 in human pulmonary epithelial cells. We identified 13 compounds that exhibit potent antiviral activity across multiple orthogonal assays. Hits include known antivirals, compounds with anti-inflammatory activity, and compounds targeting host pathways such as kinases and proteases critical for SARS-CoV-2 replication. We identified seven compounds not previously reported to have activity against SARS-CoV-2, including B02, a human RAD51 inhibitor. We further demonstrated that B02 exhibits synergy with remdesivir, the only antiviral approved by the FDA to treat COVID-19, highlighting the potential for combination therapy. Taken together, our comparative compound screening strategy highlights the potential of drug repurposing screens to identify novel starting points for development of effective antiviral mono- or combination therapies to treat COVID-19.

The most common method for RNA detection involves reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR) analysis. Commercial one-step master mixes-which include both a reverse transcriptase and a thermostable polymerase and thus allow performing both the RT and qPCR steps consecutively in a sealed well-are key reagents for SARS-CoV-2 diagnostic testing; yet, these are typically expensive and have been affected by supply shortages in periods of high demand. As an alternative, we describe here how to express and purify Taq polymerase and M-MLV reverse transcriptase and assemble a homemade one-step RT-qPCR master mix. This mix can be easily assembled from scratch in any laboratory equipped for protein purification. We also describe two simple alternative methods to prepare clinical swab samples for SARS-CoV-2 RNA detection by RT-qPCR: heat-inactivation for direct addition, and concentration of RNA by isopropanol precipitation. Finally, we describe how to perform RT-qPCR using the homemade master mix, how to prepare in vitro-transcribed RNA standards, and how to use a fluorescence imager for endpoint detection of RT-PCR amplification in the absence of a qPCR machine In addition to being useful for diagnostics, these versatile protocols may be adapted for nucleic acid quantification in basic research. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of a one-step RT-qPCR master mix using homemade enzymes Basic Protocol 2: Preparation of swab samples for direct RT-PCR Alternate Protocol 1: Concentration of RNA from swab samples by isopropanol precipitation Basic Protocol 3: One-step RT-qPCR of RNA samples using a real-time thermocycler Support Protocol: Preparation of RNA concentration standards by in vitro transcription Alternate Protocol 2: One-step RT-PCR using endpoint fluorescence detection.

Re-opening of communities in the midst of the ongoing COVID-19 pandemic has ignited new waves of infections in many places around the world. Mitigating the risk of reopening will require widespread SARS-CoV-2 testing, which would be greatly facilitated by simple, rapid, and inexpensive testing methods. This study evaluates several protocols for RNA extraction and RT-qPCR that are simpler and less expensive than prevailing methods. First, isopropanol precipitation is shown to provide an effective means of RNA extraction from nasopharyngeal (NP) swab samples. Second, direct addition of NP swab samples to RT-qPCRs is evaluated without an RNA extraction step. A simple, inexpensive swab collection solution suitable for direct addition is validated using contrived swab samples. Third, an open-source master mix for RT-qPCR is described that permits detection of viral RNA in NP swab samples with a limit of detection of approximately 50 RNA copies per reaction. Quantification cycle (Cq) values for purified RNA from 30 known positive clinical samples showed a strong correlation (r2 = 0.98) between this homemade master mix and commercial TaqPath master mix. Lastly, end-point fluorescence imaging is found to provide an accurate diagnostic readout without requiring a qPCR thermocycler. Adoption of these simple, open-source methods has the potential to reduce the time and expense of COVID-19 testing.

Re-opening of communities in the midst of the ongoing COVID-19 pandemic has ignited a second wave of infections in many places around the world. Mitigating the risk of reopening will require widespread SARS-CoV-2 testing, which would be greatly facilitated by simple, rapid, and inexpensive testing methods. To this end, we evaluated several protocols for RNA extraction and RT-qPCR that are simpler and less expensive than prevailing methods. First, we show that isopropanol precipitation provides an effective means of RNA extraction from nasopharyngeal (NP) swab samples. Second, we evaluate direct addition of NP swab samples to RT-qPCR reactions without an RNA extraction step. We describe a simple, inexpensive swab collection solution suitable for direct addition, which we validate using contrived swab samples. Third, we describe an open-source master mix for RT-qPCR and show that it permits detection of viral RNA in NP swab samples. Lastly, we show that an end-point fluorescence measurement provides an accurate diagnostic readout without requiring a qPCR thermocycler. Adoption of these simple, inexpensive methods has the potential to significantly reduce the time and expense of COVID-19 testing.