Nanoswitch Caliper Trains for High-Throughput, High-Resolution Structural Analysis of Complex DNA Nanostructures
Abstract
RESEARCH PROBLEM AND OBJECTIVES We propose Nanoswitch Caliper Trains (NCTs) for high- throughput, angstrom-resolution, single-molecule measurements of distances between pairs of ssDNA handles displayed on the surface of ~target~ DNA nanostructures. A randomly selected pair of ~target~ handles loops out a segment of a long ssDNA ~nanoswitch caliper~ and thereby reduces the latter~s full- extension length. Here we seek to achieve angstrom resolution from single measurements and thousands to millions fold higher throughput than is possible with optical traps. We propose the key innovation of constructing each NCT as a ~T series~ ~ a covalently connected chain of six single-stranded ~T- junctions~ spaced by single-stranded DNA segments of defined length. The middle pair of T-junctions can be used for covalent ligation, or else hybridization, to target handles. The other four T-junctions can be functionalized with 40 nm gold nanoparticles (AuNPs) for tracking purposes. Then following the separation between the central pairing of AuNPs can report on changes in the length of the target which they flank, while simultaneously following the separations between the peripheral pairings of AuNPs can be used for real-time force calibration. AIM I: Construct nanoswitch caliper cars (NCCs) with different T-junction (covalently branched DNA) arrangements and functionalize with AuNPs and DNA targets. Subsequently, we will ligate multiple NCCs in series to assemble a nanoswitch caliper train (NCT). AIM II: Capture and stretch NCTs between a coverslip and immobilized microbead. We will focus on covalently attaching the calipers to the coverslip and microbead by ligation of hairpin adaptors that are covalently attached via copper click chemistry to the surfaces. We will optimize the density of the caliper attachment to achieve ~1e6 NCT microbeads per coverslip. AIM III: Measure pairwise distances between AuNPs mounted on NCTs using dark-field illumination. We will investigate TIRF microscopy to selectively illuminate AuNPs in order to increase the signal-to-noise. Measurements of the pairwise distances between tracking AuNPs of stretched NCTs will be optimized to demonstrate angstrom- resolution. Furthermore, the AuNP tracking analysis will be multiplexed to analyze ~1e6 NCTs per coverslip simultaneously. TECHNICAL APPROACHES To construct our devices, we will use DNA sequence design, enzymatic DNA synthesis and DNA purification, thermal ramps for self-assembly. To characterize our devices, we will use agarose gel electrophoresis, transmission electron microscopy, and total-internal-reflectance darkfield microscopy. EXPECTED OUTCOMES We expect to demonstrate analysis of 1e6 NCTs per coverslip with angstrom-resolution recovery of pairwise distances on labeled ssDNA and DNA-nanostructure analytes. IMPACT ON DOD CAPABILITIES DNA nanostructures could enable diverse applications [1], including the following: reconfigurable visible-light plasmonic and metamaterial devices (e.g. applications to optical computing, adaptive coloration, cloaking); structural scaffolding that adaptively strengthens in the direction of applied stress (e.g. application to wound healing); single-molecule sensing with nanopores or nanobarcodes; nanorobotic therapeutic-delivery vehicles; tools for macromolecular structure determination. The process of DNA self-assembly is error- prone, however, and experimental feedback on the structure and composition of DNA nanostructures will be required to develop reliable design principles that minimize DNA self-assembly defects, if the full potential of DNA-based nanostructure-devices is to be realized. A major roadblock is the current inability to determine the atomic-resolution structure of such objects. For example, the standard methods of imaging DNA nanostructures at nanometer resolution by atomic force microscopy or transmission electron microscopy are insufficient to resolve subtle defects. APPROVED FOR PUBLIC RELEASE.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Feb 17, 2020
- Source ID
- N000142012097
Entities
People
- William M. Shih
Organizations
- Dana–Farber Cancer Institute
- Office of Naval Research
- United States Navy