Biology subjects

Thachuk, C.

Publications and source records attributed to Thachuk, C..

3 recordsLinked to original sources

bioRxiv2025

Systematic non-natural base-pairs for strand displacement circuits in complex environments

Nucleic acid strand displacement (SD) circuits have enabled impressive realizations of molecular computing in vitro, but operating them in contention with competing reaction pathways can cause signal leak and circuit failure. Further, operating SD circuits in complex environments, ranging from pools of nucleic acids to living organisms, can exacerbate these challenges. To address these issues, we explore the incorporation of isoC and isoG non-natural bases in single- and multi-step SD cascades and started by studying their promiscuity. We combined the non-natural bases with a mismatch strategy, showing them to be fully compatible. The synthesis we show between strategic placement of non-natural bases and systematic mismatch optimization of SD circuits offers a design space for future circuits that are fast, robust, and biologically compatible. Comparing circuit slowdowns by random pool backgrounds in the presence and absence of non-natural base pairs reveals a significant difference. Using isoC:isoG base pairs in the toehold region shows the greatest effect, with some further positive effects caused by additional non-natural bases in the branch migration domain. Such insights will pave the way for developing SD circuits that operate in complex environments, expanding the applications of molecular computing in a wider range of scenarios.

bioengineering↗

bioRxiv2022

Digital nanoreactors for control over absolute stoichiometry and spatiotemporal behavior of receptors within lipid bilayers

Interactions between membrane proteins are essential for cell survival and proper function, but the structural and mechanistic details of these interactions are often poorly understood. Even the biologically functional ratio of protein components within a multi-subunit membrane complex--the native stoichiometry--is difficult to establish. We have demonstrated digital nanoreactors that can control interactions between lipid-bound molecular receptors along three key dimensions: stoichiometric, spatial, and temporal. Each nanoreactor is based on a DNA origami ring, which both templates the synthesis of a liposome and provides tethering sites for DNA-based receptors. Receptors are released into the liposomal membrane using strand displacement and a DNA logic gate measures receptor heterodimer formation. High-efficiency tethering of receptors enables the kinetics of receptors in 1:1 and 2:2 absolute stoichiometries to be observed by bulk fluorescence in a plate reader which in principle is generalizable to any ratio. Similar single molecule in bulk experiments using DNA-linked membrane proteins could determine native stoichiometry and the kinetics of membrane protein interactions for applications ranging from signalling research to drug discovery.

bioengineering↗

bioRxiv2022

Classifying enthalpy-neutral DNA strand displacement cascades: trading off desired and undesired signal propagation

Molecular control circuits embedded within chemical systems to direct molecular events have transformative applications in synthetic biology, medicine, and other fields. However, it is challenging to understand the collective behavior of components due to the combinatorial complexity of possible interactions. Some of the largest engineered molecular systems to date have been constructed from DNA strand displacement reactions, in which signals can be propagated without a net change in base pairs. For linear chains of such enthalpy-neutral displacement reactions, we develop a rigorous framework to reason about interactions between regions that must be complementary. We then analyze desired and undesired properties affecting speed and correctness of such systems, including the spurious release of output (leak) and reversible unproductive binding (toehold occlusion), and experimentally confirm the predictions. Our approach, analogous to the rigorous proofs of algorithm correctness in computer science, can guide engineering of robust and efficient molecular algorithms.

bioengineering↗