Guiding the Self-Assembly of RecA Proteinfilaments on DNA Scaffolds to Create Rationally Designed Nanostructures

Abstract

Over the past three decades DNA self-assembly has been used to create increasingly complex nanoscale structures. DNA nanotubes with tunable persistence length have provided an attractive model system for protein filaments and structures with rationally defined nanoscale shapes, made using the “DNA origami” technique are promising for applications such as drug delivery and nano-plasmonics. However, the scale-up necessary for many DNA origami applications remains prohibited by cost and less than perfect yields. We seek to combine the ability of rational design afforded by DNA origami with the cost-efficient, high yield assembly of protein filaments to enable the construction of nanostructures with new functionalities. For a protein to be used in combination with DNA nanostructures, it must interact with DNA in a controllable way. RecA protein is an ideal choice because of its ability to form rigid filaments on DNA. Here we demonstrate the construction of a nanostructures including tetrahedra, rectangular shapes as well as micrometer scale filaments from DNA origami and RecA protein. We first fold specific sections of M13 single-stranded DNA, using the DNA origami technique, to create a mechanically flexible skeleton. Unfolded sections of the M13 DNA sequence are made double-stranded using a DNA polymerase gap-filling reaction. This skeleton is then rigidified by the addition of RecA, which self-assembles into filaments of defined length at specific double-stranded DNA target sites. Our method greatly improves upon conventional DNA origami in several ways. By reducing the number of distinct input components, we not only reduce cost but also the number of possible mis-folding events, allowing for higher yield nanostructures. Furthermore, the direct incorporation of proteins with origami adds a new level of functionality, thus broadening the scope of potential origami applications.