Coarse-Grained Model of RNA Provides Insight into RNA Strand Displacement Reaction

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The cover image of Biophysical Journal (March 10, 2015 [Volume 108, Issue 5]) shows an RNA toehold mediated strand displacement reaction, as represented by the coarse-grained oxRNA model. The invading strand (shown in blue) attaches to a single stranded overhang (the “toehold”) of the substrate strand (shown in red) and will eventually replace the incumbent strand (shown in green) because it forms the more stable complex. This process “catalyzes” the detachment of the incumbent strand, which is then available for other reactions.

RNA shows great promise as a material for nanotechnology. Like DNA, it has a four-letter alphabet that facilitates the design of stable, three-dimensional structures with near-atomic precision. Moreover, in vivo, it not only stores genetic material, as DNA does, but also acts as a structural element and can exhibit catalytic activity, much like proteins do. This versatility makes the prospect of using RNA nanotechnology for sophisticated biomedical applications, both in vitro and in vivo, particularly appealing.

Toehold-mediated strand displacement has long been an essential component for designing active DNA machines, because it allows kinetic control over the rates and ordering of key reactions. By combining multiple strand displacement reactions, complex logic operations and computation have been realized. Such success naturally raises the question of how this process could be used to create new applications in RNA nanotechnology. It also suggests that nature may exploit this relatively simple reaction inside the cell.

To unravel the underlying biophysics of strand displacement for RNA, we employed a recently derived nucleotide level coarse-grained model of RNA called oxRNA. Coarse-grained models are necessary to describe such processes that rely on rare events since the relevant time and length-scales are typically not accessible to more detailed atomistic models. OxRNA shares many features with the previously derived oxDNA model, which has successfully been used to describe many processes that are fundamental to DNA nanotechnology. In particular, these processes have reproduced experimentally measured relative rates for DNA displacement reactions with near quantitative accuracy.

We find that RNA displacement reaction rates are dominated by a complex interplay of enthalpic and entropic effects at the junction between the invading and incumbent strands. These processes cannot be captured by simple secondary structure models, and so a fully three-dimensional model such as oxRNA is necessary. We predict up to six orders of magnitude speedup between the rate for a toehold of length 1 and the saturated maximum speed for a toehold of length 5 and more. However, in contrast with DNA systems, we find that the displacement reaction is faster (by about a factor of two to nine) depending on which end of the substrate (3′ or 5′) the toehold is placed, with the 5′ toehold being faster. This difference arises from the asymmetry of the A-form helix adopted by RNA duplexes, which results in bigger stabilization of an invading strand at the 5′ end of the incumbent-substrate duplex. We also study the displacement rate at different temperatures, and find that for longer toeholds, the displacement slows down with increasing temperature.

Our results provide new insight into the fundamental biophysics of the RNA strand displacement reaction, which can be exploited to modulate reaction rates. Thus, our results improve the accuracy and flexibility of RNA nanotechnology design.

Further information about oxRNA, including a publicly accessible code and instructions for its use, can be found at http://dna.physics.ox.ac.uk.

– Petr Sulc, Thomas Ouldridge, Flavio Romano, Jonathan Doye, and Ard Louis

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