Directing the Self-Assembly of Multiple DNA Origami Nanostructures in a Single Reaction

DNA origami is a programmable self-assembly technique that combines a single-stranded DNA template, called a “scaffold,” with hundreds of custom DNA strands, called “staples,” to fabricate nanostructures in a bottom-up assembly process. These nanostructures are designed with unprecedented geometric precision and have promise for applications in cancer therapeutics, biosensors, nanorobotics and more. Despite these promising applications, the DNA origami self-assembly process is not well-understood, and only a few studies have explored detailed folding pathways and mechanisms. In addition, many applications require the higher order assembly of multiple structures, which is currently carried out in a multi-step assembly processes that leads to inefficient formation of the multi-structure assembly. The aim of this work is to study the competitive self-assembly of simultaneously folding two or more unique structures as a means to study detailed folding mechanisms and pathways and to establish methods for the efficient assembly of complex higher order assemblies. We tested the possibility of folding two structures with similar but distinguishable geometry, in the same folding reaction where the staples for both structures compete for the same scaffold, and we explored the effect of adjusting the staple concentration of each structure during folding. Knowing one of the two structures is more energetically favorable, we chose to keep its concentration constant while varying the concentration of the least favorable structure. By adjusting the staple concentrations we can tip the balance toward formation of either structure and adjust relative yields. Interestingly, we find that chimeras that form over a short timescale appear to revert to well folded structures over long time periods. These findings establish a foundation to assemble complex multi-structure assemblies in one step, and this work advances the field of nanomanufacturing by establishing thermodynamic and kinetic principles for the controllable and scalable self-assembly of a entire fleet of nanostructures simultaneously.