Handhold-mediated strand displacement: a nucleic acid-based mechanism for generating far-from-equilibrium assemblies through templated reactions

Toehold-mediated strand displacement (TMSD) is a nucleic acid-based reaction wherein an invader strand ( I ) replaces an incumbent strand ( N ) in a duplex with a target strand ( T ). TMSD is driven by toeholds, overhanging single-stranded domains in T recognised by I . Although TMSD is responsible for the outstanding potential of dynamic DNA nanotechnology 1, 2 , TMSD cannot implement templating, the central mechanism by which biological systems generate complex, far-from equilibrium assemblies like RNA or proteins 3, 4 . Therefore, we introduce handhold-mediated strand displacement (HMSD). Handholds are toehold analogues located in N and capable of implementing templating. We measure the kinetics of 98 different HMSD systems to demonstrate that handholds can accelerate the rate of invader-target ( IT ) binding by more than 4 orders of magnitude. Furthermore, handholds of moderate length accelerate reactions whilst allowing detachment of the product IT from N . We are thus able to experimentally demonstrate the use of HMSD-based templating to produce highly-specific far-from-equilibrium DNA duplexes.