Single-Molecule Analysis Tools
Molecular identification methods can either be affinity-based (e.g. ELISA) or non-affinity-based (e.g. mass spectrometry). We are pursuing extension of affinity and non-affinity identification and characterization to single-molecule analysis. Such tools will offer greater sensitivity and ability to resolve heterogeneity within molecular populations.
Crisscross detection
The main impediments for affinity-based single-molecule identification assays boil down to two challenges of signal-to-noise: (i) perfect discrimination between cognate and near cognate, and (ii) zero-background amplification after each molecular identification of a cognate particle. With crisscross polymerization — a general tiling strategy that enables robust and rapid seed-dependent polymerization of multi-micrometer DNA ribbons and tubes without any observable spurious nucleation — we believe we have developed a foundation for solving this second challenge in a low-cost, enzyme-free fashion.
Animation credit Anastasia Ershova
DNA nanoswitch calipers
In collaboration with the group of Wesley Wong from BCH and Wyss, we have been developing DNA nanoswitch calipers for massively parallel single-molecule identification, structure determination, and mechanical characterization. Our goal is to read protein structures tomorrow as easily as we read gene sequences today. We believe our calipers will usher in a new era for single-cell proteomics, structural biology, and synthetic-biology design of novel macromolecular devices.
Image credit Prakash Shrestha
Calipers are stretched between two optically trapped beads and actuated by force to measure the lengths of ssDNA targets.
Animation credit Anastasia Ershova
DNA-corralled nanodiscs
In collaboration with the groups of Mahmoud Nasr at BWH and Gerhard Wagner at HMS, we're constructing DNA corrals that direct the reconstitution of multiple 10 nm MSP lipid nanodiscs into larger ones that are roughly 60 nm in diameter. DNA corrals act as bumper cases to prevent unwanted aggregation and can enable control over stoichiometry, geometry, and orientation of inserted guests through tethering to the corral. We're investigating clusters of these nanodiscs for capture for a high density of guest membrane proteins for cryoEM analysis.
Key publications:
Zhao Z, Zhang M, Hogle JM, Shih WM, Wagner G, Nasr ML. DNA-Corralled Nanodiscs for the Structural and Functional Characterization of Membrane Proteins and Viral Entry. J Am Chem Soc. 140, 10639–10643, 2018. [zip]
Minev D*, Wintersinger CM*, Ershova A, Shih WM. Robust Nucleation Control via Crisscross Polymerization of Highly Coordinated DNA Slats. Nat Commun. 12, 1741, 2021. [zip]