Passivating blunt-ended helices to control monodispersity and multi-subunit assembly of DNA origami structures

Abstract

DNA origami facilitates the synthesis of bespoke nanoscale structures suitable for a wide range of applications. Effective design requires prevention of uncontrolled aggregation, while still permitting directed assembly of multi-subunit superstructures. Uncontrolled aggregation can be caused by base-stacking interactions between arrays of blunt-ended helices on different structures, which are routinely passivated by incorporating disordered regions as either scaffold loops or poly-nucleotide brushes (usually poly-thymine) at the end of DNA helices. Such disordered regions are ubiquitous in DNA origami structures yet their exact design requirements in different chemical environments are ill defined. In this study, we systematically examine the use of scaffold loops and poly-nucleotide brushes for passivation and for controlling multi-subunit assembly. We assess the dependence of length and sequence for preventing aggregation amidst a titration of MgCl2 concentrations and the suitability of each strategy for enabling controlled multi-subunit assembly. We then introduce a novel strategy where double-stranded DNA helices run orthogonal to arrays of blunt-ended DNA helices forming a steric shield that prevents base stacking. The results define the limitations of each method and important design considerations for achieving monodispersity. For example, poly-thymine brushes are most effective for achieving monodispersity in the broadest conditions whereas scaffold loops can facilitate directed multi-subunit assembly. Finally, orthogonal DNA helices remove the need for disordered regions altogether, prevent aggregation in a broad range of MgCl2 concentrations and facilitate directed multi-subunit assembly. This study expands the design tools available and enables a more informed approach for achieving control of monodispersity and multi-subunit assembly in DNA origami structures.