Sequential Pull-Down Purification of DNA Origami Superstructures.

Abstract

Higher-order superstructures of individual DNA origami building blocks are frequently used in DNA nanotechnology in order to increase the structure dimensions and complexity. Here, a purification method is presented to specifically enrich a fully assembled superstructure out of an excess of substructures. The approach is based on pull-down reactions with magnetic beads, where superstructures are captured via an anchor strand on a specific terminus and then become separated from terminus-free structures. By carrying out several pull-down reactions sequentially on different termini, the full superstructures that possess all termini become finally enriched. The approach is demonstrated by purifying linear origami superstructures with up to nine monomers by two-sided pull-down reactions and a T-shaped superstructure in a three-sided pull-down reaction. In all cases, high recovery yields and purities are obtained. A crucial prerequisite for the sequential pull-down scheme is the establishment of highly specific, orthogonal sequence sets for capture, and anchor strands. It is expected that the introduced approach provides a useful and universal method to purify complex DNA origami superstructures with high specificity and yield and this way allows the massive parallel fabrication of nanostructures at high homogeneity.