Nucleic acid nanotechnology

Abstract:

Collagen IV is one of the main components of the basement membrane, a layer of material that lines the majority of tissues in multicellular organisms. Collagen IV molecules assemble into networks, providing stiffness and elasticity to tissues and informing cell and organ shape, especially during development. In this work, we develop two coarse-grained models for collagen IV molecules that retain biochemical bond specificity and coarse grain at different length scales. Through molecular-dynamics simulations, we test the assembly and mechanics of the resulting networks and measure their response to strain in terms of stress, microscopic alignment, and bond dynamics. Within the basement membrane, collagen IV networks rearrange by molecule turnover, which affects tissue organization and can be linked with enzyme activity. Here we explore network rearrangements via bond remodeling, the process of breaking and remaking of bonds between network molecules. We then investigate the effects of active (enzymatic) bond remodeling. We find that this nonequilibrium remodeling allows a network to keep its integrity under strain, while relaxing fully over a variety of timescales, a dynamic response that is unavailable to networks undergoing equilibrium remodeling.

Abstract:

Fundamental investigations of ice nucleation, a key process in fields from environmental science to cryobiology, require model systems with chemical and physical structures that are well defined and easily varied. DNA origami is an especially promising model because of the exquisite control that it offers over the physical geometry of the nucleating agent at the nano-scale. Here we compare ice nucleation by solutions of a rectangular DNA origami tile, formed by annealing a 2.6 kbase single-stranded DNA scaffold with ninety shorter ‘staple’ oligonucleotides, to ice nucleation when these components are mixed at the same concentrations but not annealed. Isothermal measurements show that the molecular conformation has a dramatic effect on the ice nucleating efficiency. For an array of droplets containing annealed, well-folded tiles the freezing rate is constant, whereas for unannealed DNA the freezing rate decreases with time. Despite the freezing rate measured at low temperature being higher for the annealed DNA origami samples than for a significant proportion of the unannealed ones, in slow temperature-ramp measurements the latter generally freeze at higher temperatures. We show that this behaviour is consistent with the formation of small numbers of highly efficient nucleating agents in the unannealed samples, likely through molecular aggregation.

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.