Contents 1. Introduction 2 2. Post-selection sequence analysis 3 2.1 Clustering 5 2.2 Motif searching 6 2.3 Mutagenesis 8 3. High-throughput characterization methods 10 3.1 On-chip analysis 12 4. Conclusions 14 References 14

The SARS-CoV-2 pandemic has brought to light the need for expedient diagnostic testing. Cost and availability of large-scale testing capacity has led to a lag in turnaround time and hindered contact tracing efforts, resulting in a further spread of SARS-CoV-2. To increase the speed and frequency of testing, we developed a cost-effective single-tube approach for collection, denaturation, and analysis of clinical samples. The approach utilizes 1 µL microbiological inoculation loops to collect saliva, sodium dodecyl sulfate (SDS) to inactivate and release viral genomic RNA, and a diagnostic reaction mix containing polysorbate 80 (Tween 80). In the same tube, the SDS-denatured clinical samples are introduced to the mixtures containing all components for nucleic acids detection and Tween 80 micelles to absorb the SDS and allow enzymatic reactions to proceed, obviating the need for further handling of the samples. The samples can be collected by the tested individuals, further decreasing the need for trained personnel to administer the test. We validated this single-tube sample-to-assay method with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) and discovered little-to-no difference between Tween- and SDS-containing reaction mixtures, compared to control reactions. This approach reduces the logistical burden of traditional large-scale testing and provides a method of deployable point-of-care diagnostics to increase testing frequency.

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The SARS-CoV-2 pandemic has brought to light the need for expedient diagnostic testing. Cost and availability of large-scale testing capacity has led to a lag in turnaround time and hindered contact tracing efforts, resulting in a further spread of SARS-CoV-2. To increase the speed and frequency of testing, we developed a cost-effective single-tube approach for collection, denaturation, and analysis of clinical samples. The approach utilizes 1 μL microbiological inoculation loops to collect saliva, sodium dodecyl sulfate (SDS) to inactivate and release viral genomic RNA, and a diagnostic reaction mix containing polysorbate 80 (Tween 80). In the same tube, the SDS-denatured clinical samples are introduced to the mixtures containing all components for nucleic acids detection and Tween 80 micelles to absorb the SDS and allow enzymatic reactions to proceed, obviating the need for further handling of the samples. The samples can be collected by the tested individuals, further decreasing the need for trained personnel to administer the test. We validated this single-tube sample-to-assay method with RT-qPCR and RT-LAMP and discovered little-to-no difference between Tween- and SDS-containing reaction mixtures, compared to CDC-approved reagents. This approach significantly reduces the logistical burden of traditional large-scale testing and provides a method of deployable point-of-care diagnostics to increase testing frequency.

Fluorescent probes have been used for decades to illuminate RNA dynamics in cells and transparent organisms. Serial imaging remains challenging, though, owing to toxicities associated with the required excitation light. Applications in live animals are also limited due to difficulties in delivering external light to target tissues. To circumvent these issues, we developed an alternative method for visualizing RNAs that relies on bioluminescence. Bioluminescence involves a chemical reaction between luciferase enzymes and luciferin small molecules that produces photons. Since no excitation light is required, bioluminescent probes do not exhibit phototoxicity or photobleaching effects. Additionally, little to no background signal is produced, providing high signal-to-noise ratios and enabling sensitive detection in vivo--an important consideration for visualizing low-copy transcripts. We engineered a unique RNA sequence that recruits bioluminescent molecules upon transcription. We further optimized this system to modularly tag and visualize RNAs in a variety of contexts. These results provide the foundation for visualizing RNA dynamics in vivo.