Biology subjects

Verosloff, M. S.

Publications and source records attributed to Verosloff, M. S..

3 recordsLinked to original sources

bioRxiv2024

Developing, characterizing and modelingCRISPR-based point-of-use pathogen diagnostics

Recent years have seen intense interest in the development of point-of-care nucleic acid diagnostic technologies to address the scaling limitations of laboratory-based approaches. Chief among these are combinations of isothermal amplification approaches with CRISPR-based detection and readouts of target products. Here, we contribute to the growing body of rapid, programmable point-of-care pathogen tests by developing and optimizing a one-pot NASBA-Cas13a nucleic acid detection assay. This test uses the isothermal amplification technique NASBA to amplify target viral nucleic acids, followed by Cas13a-based detection of amplified sequences. We first demonstrate an in-house formulation of NASBA that enables optimization of individual NASBA components. We then present design rules for NASBA primer sets and LbuCas13a guide RNAs for fast and sensitive detection of SARS-CoV-2 viral RNA fragments, resulting in 20 - 200 aM sensitivity without any specialized equipment. Finally, we explore the combination of high-throughput assay condition screening with mechanistic ordinary differential equation modeling of the reaction scheme to gain a deeper understanding of the NASBA-Cas13a system. This work presents a framework for developing a mechanistic understanding of reaction performance and optimization that uses both experiments and modeling, which we anticipate will be useful in developing future nucleic acid detection technologies.

synthetic biology↗

bioRxiv2020

RNA sequence and structure determinants of Pol III transcriptional termination in human cells

The precise mechanism of transcription termination of the eukaryotic RNA polymerase III (Pol III) has been a subject of considerable debate. Although previous studies have clearly shown that at the end of RNA transcripts, tracts comprised of multiple uracils are required for Pol III termination, whether upstream RNA secondary structure in the nascent transcript is necessary for robust transcriptional termination is still subject to debate. We sought to address this directly through the development of an in cellulo Pol III transcription termination assay using a synthetic biology approach. Specifically, we utilized the recently developed Tornado expression system and a stabilized Corn RNA aptamer to create a Pol III-transcribed RNA that produces a detectable fluorescent signal when transcribed in human cells. To study the effects of RNA sequence and structure on Pol III termination, we systematically varied the sequence context upstream of the aptamer and identified sequence characteristics that enhance or diminish termination. We found that in the absence of predicted secondary structure, only poly-U tracts longer than then the average length found in the human genome (4-5 nucleotides), efficiently terminate Pol III transcription. We found that shorter poly-U tracts could induce termination when placed in proximity to secondary structural elements, while secondary structure by itself was not sufficient to induce termination. These findings demonstrate a key role for sequence and structural elements within Pol III-transcribed nascent RNA for efficient transcription termination, and demonstrate a generalizable assay for characterizing Pol III transcription in human cells.

molecular biology↗

bioRxiv2019

Point-of-Use Detection of Environmental Fluoride via a Cell-Free Riboswitch-Based Biosensor

Advances in biosensor engineering have enabled the design of programmable molecular systems to detect a range of pathogens, nucleic acids, and chemicals. Here, we engineer and field-test a biosensor for fluoride, a major groundwater contaminant of global concern. The sensor consists of a cell-free system containing a DNA template that encodes a fluoride-responsive riboswitch regulating genes that produce a fluorescent or colorimetric output. Individual reactions can be lyophilized for long-term storage and detect fluoride at levels above 2 parts per million, the EPAs most stringent regulatory standard, in both laboratory and field conditions. Through onsite detection of fluoride in a real-world water source, this work provides a critical proof-of-principle for the future engineering of riboswitches and other biosensors to address challenges for global health and the environment.

synthetic biology↗