We introduce a nanoscale experimental platform that enables kinetic and equilibrium measurements of a wide range of molecular interactions by expanding the functionality of gel electrophoresis. Programmable, self-assembled DNA nanoswitches serve both as templates for positioning molecules, and as sensitive, quantitative reporters of molecular association and dissociation. We demonstrate this low cost, versatile, “lab-on-a-molecule” system by characterizing 10 different interactions, including a complex 4-body interaction with 5 discernable states.

Protein detection and quantification play critical roles in both basic research and clinical practice. Current detection platforms range from the widely used ELISA to more sophisticated, and more expensive, approaches such as digital ELISA. Despite advances, there remains a need for a method that combines the simplicity and cost-effectiveness of ELISA with the sensitivity and speed of modern approaches in a format suitable for both laboratory and rapid, point-of-care applications. Building on recent developments in DNA structural nanotechnology, we introduce the nanoswitch-linked immunosorbent assay (NLISA), a detection platform based on easily constructed DNA nanodevices that change conformation upon binding to a target protein with the results read out by gel electrophoresis. NLISA is surface-free and includes a kinetic-proofreading step for purification, enabling both enhanced sensitivity and reduced cross-reactivity. We demonstrate femtomolar-level detection of prostate-specific antigen in biological fluids, as well as reduced cross-reactivity between different serotypes of dengue and also between a single-mutation and wildtype protein. NLISA is less expensive, uses less sample volume, is more rapid, and, with no washes, includes fewer hands-on steps than ELISA, while also achieving superior sensitivity. Our approach also has the potential to enable rapid point-of-care assays, as we demonstrate by performing NLISA with an iPad/ iPhone camera for imaging.biodetection | point of care | DNA nanotechnology | molecular self-assembly P rotein quantification plays a significant role in a wide range of clinical and research applications, from detecting Zika virus infection (1), to ascertaining the presence of a heart attack (2), to finding trace amounts of allergens in food products (3). Over the years, a variety of detection platforms have emerged, from the traditional sandwich ELISA (4), to more sophisticated and costly approaches such as digital ELISA (5). However, the need remains for an approach that combines the simplicity and cost-effectiveness of ELISA, which remains widespread due to these advantages, with the improvements in sensitivity and speed of modern approaches-ideally in a format suitable for both laboratory and rapid, point-of-care applications. One promising strategy, based on recent developments in DNA structural nanotechnology, has been the development of programmable reagents capable of sensing, responding, and reporting changes in their local environments. These include tension gauge tethers to measure the forces required to activate signaling through a ligand-receptor bond (6), toehold switches for paper-based synthetic gene networks (7), nanocalipers to probe nucleosome stability (8), and nanoactuators that can propagate distance changes (9). Proof-of-principle experiments of protein detection using self-assembled DNA nanodevices that undergo changes in shape to report the binding of molecular targets have recently been demonstrated (10, 11), although widespread use of these approaches has b...

Recent methods in DNA nanotechnology are enabling the creation of intricate nanostructures through the use of programmable, bottom-up self-assembly. However, structures consisting only of DNA are limited in their ability to act on other biomolecules. Proteins, on the other hand, perform a variety of functions on biological materials, but directed control of the self-assembly process remains a challenge. While DNA-protein hybrids have the potential to provide the best-of-both-worlds, they can be difficult to create as many of the conventional techniques for linking proteins to DNA render proteins dysfunctional. We present here a sortase-based protocol for covalently coupling proteins to DNA with minimal disturbance to protein function. To accomplish this we have developed a two-step process. First, a small synthetic peptide is bioorthogonally and covalently coupled to a DNA oligo using click chemistry. Next, the DNA-peptide chimera is covalently linked to a protein of interest under protein-compatible conditions using the enzyme sortase. Our protocol allows for the simple coupling and purification of a functional DNA-protein hybrid. We use this technique to form oligos bearing cadherin-23 and protocadherin-15 protein fragments. Upon incorporation into a linear M13 scaffold, these protein-DNA hybrids serve as the gate to a binary nanoswitch. The outlined protocol is reliable and modular, facilitating the construction of libraries of oligos and proteins that can be combined to form functional DNA-protein nanostructures. These structures will enable a new class of functional nanostructures, which could be used for therapeutic and industrial processes.

The conversion of auditory and vestibular stimuli into electrical signals is initiated by force transmitted to a mechanotransduction channel through the tip link, a double stranded protein filament held together by two adhesion bonds in the middle. Although thought to form a relatively static structure, the dynamics of the tip-link connection has not been measured. Here, we biophysically characterize the strength of the tip-link connection at single-molecule resolution. We show that a single tip-link bond is more mechanically stable relative to classic cadherins, and our data indicate that the double stranded tip-link connection is stabilized by single strand rebinding facilitated by strong cis-dimerization domains. The measured lifetime of seconds suggests the tip-link is far more dynamic than previously thought. We also show how Ca2+ alters tip-link lifetime through elastic modulation and reveal the mechanical phenotype of a hereditary deafness mutation. Together, these data show how the tip link is likely to function during mechanical stimuli.