Programmable Multivalent DNA-Origami Tension Probes for Reporting Cellular Traction Forces

Abstract

Mechanical forces are central to most, if not all, biological processes, including cell development, immune recognition, and metastasis. Because the cellular machinery mediating mechano-sensing and force generation is dependent on the nanoscale organization and geometry of protein assemblies, a current need in the field is the development of force-sensing probes that can be customized at the nanometer-length scale. In this work, we describe a DNA origami tension sensor that maps the piconewton (pN) forces generated by living cells. As a proof-of-concept, we engineered a novel library of six-helix-bundle DNA-origami tension probes (DOTPs) with a tailorable number of tension-reporting hairpins (each with their own tunable tension response threshold) and a tunable number of cell-receptor ligands. We used single-molecule force spectroscopy to determine the probes' tension response thresholds and used computational modeling to show that hairpin unfolding is semi-cooperative and orientation-dependent. Finally, we use our DOTP library to map the forces applied by human blood platelets during initial adhesion and activation. We find that the total tension signal exhibited by platelets on DOTP-functionalized surfaces increases with the number of ligands per DOTP, likely due to increased total ligand density, and decreases exponentially with the DOTP's force-response threshold. This work opens the door to applications for understanding and regulating biophysical processes involving cooperativity and multivalency.

Cholesterol Anchors Enable Efficient Binding and Intracellular Uptake of DNA Nanostructures

“…DNA nanostructures are a powerful tool-set for cell biology and biomedicine. To enhance the interaction with bilayer membranes, DNA nanostructures have been equipped with lipid anchors and used for a variety of applications such as to shape 50,54,57,78 or puncture bilayers 55,60,64,[79][80][81] for bio-sensing, [82][83][84][85] to attain controlled drug release, 55 to probe membrane interaction forces, 86 or to alter membrane composition. 87 Yet, lipidmodified DNA nanostructures have not been examined with cells to explore uptake and internalization.…”

DNA Nanotechnology as an Emerging Tool to Study Mechanotransduction in Living Systems

“…Given the intimate connection between receptor oligomerization, mechanotransduction and signaling, we and the Ke lab recently reported a new generation of origami‐based force probes. These probes allow presentation of multiple ligands in a distance defined manner and also with more tunable F 1/2 values . This probe utilized a six‐helix bundle DNA origami as the “body.” The top part of this origami body was functionalized with RGD ligands at an intermolecular spacing of ≈6 nm, while the bottom part of the body was linked to one, two or three DNA hairpin force probes ( Figure ).…”

“…B) RICM and fluorescence imaging reveal human platelets pull on origami force probes with increasing F unfolding . Reproduced with permission . Copyright 2018, American Chemical Society.…”

Dynamic DNA Origami Devices<strong> </strong>

“…The sensitive device equipped with (Cy3)-donor/(Cy5)acceptor FRET-pair is able to characterize a movement of four-way Holliday junction between different states and also protein-induced DNA bending. Very recently, related to above-mentioned force spectroscopy, Dutta and co-workers [101] reported a DNA origami tension probe (DOTP) capable of depicting the traction forces generated by living cells. Various DOTP combinations were employed to map the forces applied by human blood platelets during initial adhesion and activation.…”