λ-phage DNA extended in 50 nm grooves. The DNA extension is ~90% of its contour length.

The current goal of my research at McGill University is to develop a platform for linearly extending long DNA molecules for genomic analysis. We do this by confining them in nanoscale grooves etched in glass and observe them with fluorescence microscopy. It’s desirable that the DNA is completely extended without any loops in its conformation, so that its genetic sequence is arrayed linearly in space. Achieving this requires that the width and depth of the nanogrooves are less than 50 nm. This corresponds to the persistence length of DNA, which is a measure of the polymer’s stiffness. Getting DNA from solution (where it is coiled up in a high-entropy state) into grooves this thin (where it is stretch out linearly in a low-entropy state) turns out to be quite challenging. Some techniques use pressure or electric fields (DNA is charged) to drive DNA into closed nanofluidic channels. This works great for larger channels but below 50 nm, DNA breakage due to the large forces becomes a problem.

We use a technique called “Convex Lens-induced Confinement” (CLiC, invented by my supervisor Prof. Sabrina Leslie) to confine DNA into nanogrooves using tunable vertical confinement. The DNA are loaded into an imaging chamber formed by two microscope coverslips separated by a thin double-sided tape spacer with a flow channel laser-cut in it. The bottom chamber surface contains the nanogrooves. A microscope objective observes molecules inside the chamber from below. A convex lens presses on the top surface of the chamber to squeeze DNA into the nanogrooves from above. The entropy change is gradual, and the DNA don’t break in the process. I’ve used this technique to extend T4-phage DNA (166 kbp) to grooves as narrow as 27 nm.

Compressing the top surface of the imaging chamber squeezes DNA into the nanogrooves from above. Not to scale.

The CLiC device that I designed for these experiments uses a piezoelectric actuator to raise and lower the convex lens. The piezo only has 100 μm of travel, so it’s mounted to a motorized coarse-approach stage. The imaging chamber mounts on an XY stage for aligning the nanogrooves with the convex lens, so that I can precisely control where the confinement will be applied. Air pressure is used to control the flow of liquids through the chamber.

The device is useful for many types of biological experiments. Confining molecules to nanoscale slits (i.e. simply using the device without any nanogrooves) brings them all within the narrow focal plane of the microscope objective so that individual molecules can be observed at physiological concentrations for several seconds to minutes.