Biology subjects

Turberfield, A. J.

Publications and source records attributed to Turberfield, A. J..

3 recordsLinked to original sources

bioRxiv2024

Controlling DNA-RNA strand displacement kinetics with base distribution

DNA-RNA hybrid strand displacement underpins the function of many natural and engineered systems. Understanding and controlling factors affecting DNA-RNA strand displacement reactions is necessary to enable control of processes such as CRISPR-Cas9 gene editing. By combining multi-scale modelling with strand displacement experiments we show that the distribution of bases along the displacement domain of an invading strand has a very strong effect on reaction kinetics. Merely by redistributing bases within a displacement domain of fixed base composition, we are able to design sequences whose reaction rates span more than two orders of magnitude. We characterize this effect in reactions involving the invasion of dsDNA by an RNA strand and invasion of a hybrid duplex by a DNA strand. We show that oxNA, a recently introduced coarse-grained model of DNA-RNA hybrids, can reproduce trends in experimentally observed reaction rates. We also develop a kinetic model for predicting strand displacement rates. On the basis of these results, we argue that base distribution effects are likely to play an important role in the function of the guide RNAs that direct CRISPR-Cas systems.

biophysics↗

bioRxiv2021

A DNA-based optical force sensor for live-cell applications

Mechanical forces are relevant for many biological processes, from wound healing or tumour formation to cell migration and differentiation. Cytoskeletal actin is largely responsible for responding to forces and transmitting them in cells, while also maintaining cell shape and integrity. Here, we describe a novel approach to employ a FRET-based DNA force sensor in vitro and in cellulo for non-invasive optical monitoring of intracellular mechanical forces. We use fluorescence lifetime imaging to determine the FRET efficiency of the sensor, which makes the measurement robust against intensity variations. We demonstrate the applicability of the sensor by monitoring cross-linking activity in in vitro actin networks by bulk rheology and confocal microscopy. We further demonstrate that the sensor readily attaches to stress fibers in living cells which opens up the possibility of live-cell force measurements.

biophysics↗

bioRxiv2018

Rational design of hidden thermodynamic driving through DNA mismatch repair

Recent years have seen great advances in the development of synthetic self-assembling molecular systems. Designing out-of-equilibrium architectures, however, requires a more subtle control over the thermodynamics and kinetics of reactions. We propose a new mechanism for enhancing thermodynamic drive of DNA strand displacement reactions whilst barely perturbing forward reaction rates - introducing mismatches in an internal location within the initial duplex. Through a combination of experiment and simulation, we demonstrate that displacement rates are strongly sensitive to mismatch location and can be tuned by rational design. By placing mismatches away from duplex ends, the thermodynamic drive for a strand-displacement reaction can be varied without significantly affecting the forward reaction rate. This hidden thermodynamic driving motif is ideal for the engineering of nonequilibrium systems that rely on catalytic control and must be robust to leak reactions.

synthetic biology↗