Rapid exchange of stably bound protein and DNA cargo on a DNA origami receptor

Abstract

Biomolecular complexes can form stable assemblies yet can also rapidly exchange their subunits to adapt to environmental changes. Simultaneously allowing for both stability and rapid exchange expands the functional capacity of biomolecular machines and enables continuous function while navigating a complex molecular world. Inspired by biology, we design and synthesize a DNA origami receptor that exploits multi-valent interactions to form stable complexes that are simultaneously capable of rapid subunit exchange. The system utilizes a mechanism first outlined in the context of the DNA replisome, known as multi-site competitive exchange, and achieves a large separation of time scales between spontaneous subunit dissociation, which requires days, and rapid subunit exchange, which occurs in minutes. In addition, we use the DNA origami receptor to demonstrate stable interactions with rapid exchange of both DNA and protein subunits, thus highlighting the applicability of our approach to arbitrary molecular cargo; an important distinction with canonical toehold exchange between single-stranded DNA. We expect this study to be the first of many that use DNA origami structures to exploit multi-valent interactions for the design and synthesis of a wide range of possible kinetic behaviors.

Bivalent Kinetics: Insights from Many Body Physics

“…More generally, and in the context of recent advances in nano-engineering [20], we believe that our work paves the way for a wide range of putative soft systemselectrically-inspired or otherwise [18]-whose kinetics and emergent spatio-temporal properties might not only be tunable, but designed a priori in a rational way.…”

“…These two mechanisms are detailed quantitatively in Appendix Sec. D. Nanoscale platforms and batons have recently been engineered via DNA origami such that single strands of DNA at either end of the baton act as tunable binding interfaces whose complementary strands can be arranged in motifs of 'primary' (circle) and 'secondary' (square) sites (panel c and [20]). A single baton interacting with a primary and secondary receptor site gives rise to a four state system whose transition rates define the principal kinetic parameters outlined in the main text (panel d).…”

“…(1). Parameters used are K2 = K1 = 10 −9 M, C eff = 10 −6 M, k on 1 = k on 2 = 10 9 M −1 s −1 , informed by experimental values in [20].…”

“…However, it is important to be broadly aware of how these quantities will change with various features of the design of the bivalent molecule. Specifically, in the case of the nano-batons in [20], the affinities of the receptor sites, characterised by K 1 and K 2 , can primarily be tuned through the sequence and length of the individual DNA strands on either end of the baton, whilst the effective concentration experienced by a vacant receptor site in the presence of a partially bound baton can primarily be tuned through the length of the baton spacer. We do, however, note the use of the separation of scales K 1 C eff which motivates the use of a small parameter expansion ε = K 1 /C eff used throughout.…”

“…In this context, and in conjunction with recent work to engineer and characterise a synthetic DNA-origami system comprising a receptor platform and bivalent 'nanobaton' ( [20] & Fig. 1c), we now revisit bivalent kinetics, and the response of effective association/dissociation rates to changes in bulk concentration.…”