Biology subjects

Goertz, J. P.

Publications and source records attributed to Goertz, J. P..

3 recordsLinked to original sources

bioRxiv2023

Strong sequence dependence in RNA/DNA hybrid strand displacement kinetics

Strand displacement reactions underlie dynamic nucleic acid nanotechnology. The kinetic and thermodynamic features of DNA-based displacement reactions are well understood and well predicted by current computational models. By contrast, understanding of RNA/DNA hybrid strand displacement kinetics is limited, restricting the design of increasingly complex RNA/DNA hybrid reaction networks with more tightly regulated dynamics. Given the importance of RNA as a diagnostic biomarker, and its critical role in intracellular processes, this shortfall is particularly limiting for the development of strand displacement-based therapeutics and diagnostics. Herein, we characterise 22 RNA/DNA hybrid strand displacement systems, systematically varying several common design parameters including toehold length and branch migration domain length. We observe the differences in stability between RNA-DNA hybrids and DNA-DNA duplexes have large effects on strand displacement rates, with rates for equivalent sequences differing by up to 3 orders of magnitude. Crucially, however, this effect is strongly sequence-dependent, with RNA invaders strongly favoured in a system with RNA strands of high purine content, and disfavoured in a system when the RNA strands have low purine content. These results lay the groundwork for more general design principles, allowing for creation of de novo reaction networks with novel complexity while maintaining predictable reaction kinetics.

synthetic biology↗

bioRxiv2023

Competitive Amplification Networks enable molecular pattern recognition with PCR

Gene expression has great potential to be used as a clinical diagnostic tool. However, despite the progress in identifying these gene expression signatures, clinical translation has been hampered by a lack of purpose-built. readily deployable testing platforms. We have developed Competitive Amplification Networks. CANs to enable analysis of an entire gene expression signature in a single PCR reaction. CANs consist of natural and synthetic amplicons that compete for shared primers during amplification, forming a reaction network that leverages the molecular machinery of PCR. These reaction components are tuned such that the final fluorescent signal from the assay is exactly calibrated to the conclusion of a statistical model. In essence, the reaction acts as a biological computer, simultaneously detecting the RNA targets, interpreting their level in the context of the gene expression signature, and aggregating their contributions to the final diagnosis. We illustrate the clinical validity of this technique, demonstrating perfect diagnostic agreement with the gold-standard approach of measuring each gene independently. Crucially, CAN assays are compatible with existing qPCR instruments and workflows. CANs hold the potential to enable rapid deployment and massive scalability of gene expression analysis to clinical laboratories around the world, in highly developed and low-resource J settings alike. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=162 SRC="FIGDIR/small/546934v3_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@79d1c5org.highwire.dtl.DTLVardef@1bafb87org.highwire.dtl.DTLVardef@d78e31org.highwire.dtl.DTLVardef@1b85125_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2018

Multi-stage chemical heating for instrument-free biosensing

Improving the portability of diagnostic medicine is crucial to alleviating global access-to-care deficiencies. This requires not only designing devices that are small and lightweight but also autonomous and independent of electricity. Here, we present a strategy for conducting automated multi-step diagnostic assays using chemically generated, passively regulated heat. Ligation and polymerization reagents for Rolling Circle Amplification of nucleic acids are separated by melt-able phase-change partitions, thus replacing precise manual reagent additions with automated partition melting. To actuate these barriers and individually initiate the various steps of the reaction, field ration heaters exothermically generate heat in a thermos while fatty acids embedded in a carbonaceous matrix passively buffer the temperature around their melting points. Achieving multi-stage temperature profiles extends the capability of instrument-free diagnostic devices and improves the portability of reaction automation systems built around phase-change partitions.

bioengineering↗