Poster Z7 - DNA-based shear stress nanoreporters

Introduction: Cells biologically respond to flow induced hydrodynamic forces. Decades of research has shown that shear stress alters key signaling pathways including those involved with growth, inflammation, extracellular matrix deposition, and more.1 Understanding shear stress is of paramount importance since shear can induce cellular dysfunction that is implicated in the pathophysiology of many cell-tissue interfaces. However, the key barrier to further progress has been the lack of direct, easy to use fluorescent reporters that measure the multiaxial and dynamic shear flows that occur in vivo across an entire surface of interest, where shear can be directly correlated to physiological changes in cells in real time. To that end, we present an innovative DNA bionanomechanical reporter to measure shear stress. Each reporter can be likened to a kite and contains: 1) an antibody-based anchor targeted to a ligand of interest, 2) a DNA or protein-based optical force transducer that fluoresces when unfolded at a threshold force, and 3) a mechanical amplifier (kite) that increases the total force on the transducer (Fig. 1A). In flow, a bead (kite) generates tensile force as a function of fluidic drag force and bead size. As the tensile force increases, an optical force reporter, which consists of a series of DNA hairpins with fluor-quencher pairs, unfolds and fluoresces.

Materials and

Methods: 20 DNA hairpin force probes with fluorophores and quenchers are hybridized with a long single stranded DNA scaffold. Digoxigenin and biotin-modified sequences at 3’ and 5’ primer of the scaffold, are combined with the surfaces and microbeads. To test successful assembly, all DNA strands were mixed in a salted TE buffer and assembled overnight in a thermocycler. After purification Atom Force Microscopy (AFM) was conducted to characterize DNA sensors. To investigate feasibility, we incubated the purified sensors overnight with streptavidin coated silica microbeads. We then tested them in a simple microfluidic device by anchoring sensors on a glass surface via digoxigenin. Total Internal Reflectance Fluorescence (TIRF) microscopy was performed and resulting images were analyzed via Image J.

Results, Conclusions, and Discussions: Our results confirm that our nanoreporters successfully measure complex shear stress in microfluidics with a tunable threshold shear stress. The nanoreporter opened and fluoresced immediately when a shear of 25 dynes/cm2 was applied to a bead size of 1 micron in diameter (Fig. 1B). Quantification of this fluorescence yields a sigmoid curve of the hairpin assembly’s active range. Using this, we modeled the F1/2 and compared our results to other methods for quantification (Fig. 1C). The average fluorescence intensities versus flow rates show a similar curve as modeled (Fig. 1D).

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