Passivating Blunt‐Ended Helices to Control Monodispersity and Multi‐Subunit Assembly of DNA Origami Structures

• Published in: Small structures 2024-04, Vol.5 (4), p.n/a
• Main Authors: Berengut, Jonathan F., Berg, Willi R., Rizzuto, Felix J., Lee, Lawrence K.
• Format: Article
• Language: English
• Subjects: aggregation; Assembly; Brushes; Deoxyribonucleic acid; DNA; DNA nanotechnology; DNA origami; Helices; Magnesium chloride; Nucleotides; passivation; Scaffolds; Thymine
• ISSN: 2688-4062; 2688-4062
• DOI: 10.1002/sstr.202300441

Deoxyribonucleic acid (DNA) origami enables the synthesis of bespoke nanoscale structures suitable for diverse applications. Effective design requires preventing uncontrolled aggregation, while still facilitating directed multi‐subunit assembly. Uncontrolled aggregation is often caused by base‐stacking interactions between arrays of blunt‐ended helices, a problem which is typically mitigated by incorporating disordered single‐stranded DNA (ssDNA) (like scaffold loops or poly‐thymine brushes) at the end of double‐stranded DNA (dsDNA) helices. Such disordered regions are ubiquitous in DNA origami structures yet their optimal design requirements in various chemical environments remains unclear. This study systematically investigates scaffold loops and poly‐nucleotide brushes for aggregation prevention and control of multi‐subunit assembly, examining length, sequence, and MgCl2 concentration dependencies. Additionally, a novel strategy is introduced using orthogonal double‐stranded DNA helices to create a steric shield against base stacking. The results reveal key considerations. Poly‐thymine brushes excel in achieving monodispersity across diverse conditions whereas scaffold loops aid directed multi‐subunit assembly. Orthogonal DNA helices remove the need for disordered regions altogether, preventing aggregation over a broad range of MgCl2 concentrations and facilitating controlled multi‐subunit assembly. This study expands the toolbox for DNA origami design and enables a more informed approach for achieving control of monodispersity and multi‐subunit assembly in DNA origami structures. Deoxyribonucleic acid (DNA) origami nanostructures often stick together due to blunt‐end stacking of double‐stranded DNA (dsDNA). This problem is commonly addressed by incorporating single‐stranded DNA (ssDNA) at the structures' ends. A systematic exploration of the parameters (ssDNA length, sequence) and ramifications (aggregation, assembly) of these strategies is presented, along with a new method, orthogonal end‐capping, which doesn't rely on disordered ssDNA domains.