Formatting and ligating biopolymers using adjustable nanoconfinement

Abstract

Sensitive visualization and conformational control of long, delicate biopolymers present critical challenges to emerging biotechnologies and biophysical studies. Next-generation nanofluidic manipulation platforms strive to maintain the structural integrity of genomic DNA prior to analysis but can face challenges in device clogging, molecular breakage, and single-label detection. We address these challenges by integrating the Convex Lens-induced Confinement (CLiC) technique with a suite of nanotopographies embedded within thin-glass nanofluidic chambers. We gently load DNA polymers into open-face nanogrooves in linear, concentric circular, and ring array formats and perform imaging with single-fluorophore sensitivity. We use ring-shaped nanogrooves to access and visualize confinement-enhanced self-ligation of long DNA polymers. We use concentric circular nanogrooves to enable hour-long observations of polymers at constant confinement in a geometry which eliminates the confinement gradient which causes drift and can alter molecular conformations and interactions. Taken together, this work opens doors to myriad biophysical studies and biotechnologies which operate on the nanoscale. Published by AIP Publishing.

Visualizing structure-mediated interactions in supercoiled DNA molecules

“… CLiC nanopit microscopy and binding events. ( A ) Schematic of the CLiC method confining plasmids and labeled oligo-probes to pits made using micro/nano lithography techniques ( 28 ). The circle in the front-center pit demonstrates a pit containing a bound probe–plasmid complex.…”

Visualizing structure-mediated interactions in supercoiled DNA molecules

“…Here, we demonstrate a direct, single-molecule approach to studying topology-mediated dynamics and interaction kinetics of biomolecules in solution while simultaneously leaving them free to explore all possible structural conformations. Our method combines the Convex Lens-induced Confinement (CLiC) technique (26,27,28) with a nanofabricated array of pits that are etched in the bottom surface of a deformable glass flow-cell. In our experiments, we load and trap DNA oligo-probes and plasmids in individual pits so they are free to diffuse, fluctuate, and interact.…”

“…The DNA was sufficiently underwound to drive local strand-separation, CLiC nanopit microscopy and binding events. a) Schematic of the CLiC method confining plasmids and labeled oligo-probes to pits made using micro/nano lithography techniques (28). The circle in the front-center pit demonstrates a pit containing a bound probe-plasmid complex.…”

Single-cell Analysis for Drug Development Using Convex Lens-Induced Confinement Imaging

“…The principle of CLiC is as follows: using a lens-rod perpendicular to the focal plane, pressure is applied downward directly onto the flow cell. The resulting deformation of the flow cell confines molecules between the smooth glass surfaces and can load them into features such as pits, which are embedded in the surfaces ( Figure 1B) [8,9]. This technique had been shown to be able to successfully confine and isolate diffusing [5] and interacting molecules [10][11][12] for long periods and improve fluorescent signal detection [7].…”