Spectroscopic Analysis of a Library of DNA Tension Probes for Mapping Cellular Forces at Fluid Interfaces

Abstract

Oligonucleotide-based probes offer the highest spatial resolution, force sensitivity, and molecular specificity for cellular tension sensing and have been developed to measure a variety of molecular forces mediated by individual receptors in T cells, platelets, fibroblasts, B-cells, and immortalized cancer cell lines. These fluorophore−oligonucleotide conjugate probes are designed with a stem−loop structure that engages cell receptors and reversibly unfolds due to mechanical strain. With the growth of recent work bridging molecular mechanobiology and biomaterials, there is a need for a detailed spectroscopic analysis of DNA tension probes that are used for cellular imaging. In this manuscript, we conducted an analysis of 19 DNA hairpin-based tension probe variants using molecular dynamics simulations, absorption spectroscopy, and fluorescence imaging (epifluorescence and fluorescence lifetime imaging microscopy). We find that tension probes are highly sensitive to their molecular design, including donor and acceptor proximity and pairing, DNA stem−loop structure, and conjugation chemistry. We demonstrate the impact of these design features using a supported lipid bilayer model of podosome-like adhesions. Finally, we discuss the requirements for tension imaging in various biophysical contexts and offer a series of experimental recommendations, thus providing a guide for the design and application of DNA hairpin-based molecular tension probes.

Mechanical Properties of DNA Hydrogels: Towards Highly Programmable Biomaterials

“…An alternative mechanoelectric DNA-based probe was developed for potassium detection based on the conformational transition of DNA into a G-quadruplex [132]. Inversely, DNA extension can be programmed to occur above a certain force threshold (<100 pN) and FRET reporter probes have been used to further study mechanotransduction at single cell-ECM and cell-cell adhesion sites [133][134][135][136][137].…”

All-Covalent Nuclease-Resistant and Hydrogel-Tethered DNA Hairpin Probes Map pN Cell Traction Forces

“…Following the quenching efficiency (QE) calculation, we determined the QE to be ∼87.5%, which is slightly less efficient than our previous reports (Figure 3f). 33,58 This lower quenching efficiency can be derived from the weaker stability of PS DNA HP at 37 °C, resulting in a slightly greater probability of probe breathing (Figure 2c,d). Finally, we titrated a range of DNA concentrations and incubation times to maximize probe density using the least amount of DNA HP reagent.…”

DNA Origami Tension Sensors (DOTS) to study T cell receptor mechanics at membrane junctions

“…Because the Cy3B/BHQ2 pair is subject to static quenching, we replaced Cy3B with Atto488 given its excellent imaging properties and larger t shift after being quenched by BHQ2. 22,26 We captured FLIM images of cells on the SLB before and after adding locking strand and generated a phasor plot for the FLIM datasets (Fig. 2i).…”

“…2i). From the phasor plot, we identified pixels containing fluorescence lifetimes of t = 2 ns and 4 ns corresponding to folded (tclosed) and unfolded (topen) hairpin Atto488 lifetimes (conformations), respectively, 26 and obtained photon count maps for these two lifetimes. We then examined the change of these two populations before and after adding locking strand.…”