Dr Jon Bath

Abstract:

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 multiscale modeling with strand displacement experiments, we show that the distribution of bases within the displacement domain has a very strong effect on reaction kinetics, a feature unique to DNA–RNA hybrid strand displacement. Merely by redistributing bases within a displacement domain of fixed base composition, we are able to design sequences whose reaction rates span more than four orders of magnitude. We extensively characterize this effect in reactions involving the invasion of dsDNA by an RNA strand, as well as the invasion of a hybrid duplex by a DNA strand. In all-DNA strand displacement reactions, we find a predictable but relatively weak sequence dependence, confirming that DNA–RNA strand displacement permits far more thermodynamic and kinetic control than its all-DNA counterpart. 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 simple kinetic model for predicting strand displacement rates. On the basis of these results, we argue that base distribution effects may play an important role in natural R-loop formation and in the function of the guide RNAs that direct CRISPR-Cas systems.

Abstract:

Nucleic acid nanostructures offer unique opportunities for biomedical applications due to their sequence-programmable structures and functions, which enable the design of complex responses to molecular cues. Control of the biological activity of therapeutic cargoes based on endogenous molecular signatures holds the potential to overcome major hurdles in translational research: cell specificity and off-target effects. Endogenous microRNAs (miRNAs) can be used to profile cell type and cell state, and are ideal inputs for RNA nanodevices. Here, we present CRISPR MiRAGE (miRNA-activated genome editing), a tool comprising a dynamic single-guide RNA that senses miRNA complexed with Argonaute proteins and controls downstream CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) activity based on the detected miRNA signature. We study the operation of the miRNA-sensing single-guide RNA and attain muscle-specific activation of gene editing through CRISPR MiRAGE in models of Duchenne muscular dystrophy. By enabling RNA-controlled gene editing activity, this technology creates opportunities to advance tissue-specific CRISPR treatments for human diseases.

Abstract:

<jats:p>Construction of a molecular assembler from DNA that executes a programmed sequence of chemical reactions is a formidable challenge but worthwhile because it would allow assembly and evolution of functional...</jats:p>