Handhold-Mediated Strand Displacement: A Nucleic Acid Based Mechanism for Generating Far-from-Equilibrium Assemblies through Templated Reactions

The use of templates is a well-established method for the production of sequence-controlled assemblies, particularly long polymers. Templating is canonically envisioned as akin to a self-assembly process, wherein sequence-specific recognition interactions between a template and a pool of monomers favor the assembly of a particular polymer sequence at equilibrium. However, during the biogenesis of sequence-controlled polymers, template recognition interactions are transient; RNA and proteins detach spontaneously from their templates to perform their biological functions and allow template reuse. Breaking template recognition interactions puts the product sequence distribution far from equilibrium, since specific product formation can no longer rely on an equilibrium dominated by selective copy-template bonds. The rewards of engineering artificial polymer systems capable of spontaneously exhibiting nonequilibrium templating are large, but fields like DNA nanotechnology lack the requisite tools; the specificity and drive of conventional DNA reactions rely on product stability at equilibrium, sequestering any recognition interaction in products. The proposed alternative is handhold-mediated strand displacement (HMSD), a DNA-based reaction mechanism suited to producing out-of-equilibrium products. HMSD decouples the drive and specificity of the reaction by introducing a transient recognition interaction, the handhold. We measure the kinetics of 98 different HMSD systems to prove that handholds can accelerate displacement by 4 orders of magnitude without being sequestered in the final product. We then use HMSD to template the selective assembly of any one product DNA duplex from an ensemble of equally stable alternatives, generating a far-from-equilibrium output. HMSD thus brings DNA nanotechnology closer to the complexity of out-of-equilibrium biological systems.

Automated summary

The use of templates for sequence-controlled assemblies, particularly in long polymers, is a well-established method. However, the transient template recognition interactions during the biogenesis of sequence-controlled polymers can disrupt the equilibrium, leading to the production of far-from-equilibrium output. Handhold-mediated strand displacement (HMSD) is proposed as an alternative mechanism to generate out-of-equilibrium products by decoupling drive and specificity in DNA-based reactions.