Research Overview

Genomic DNA acts as the blueprint for life and all organisms have evolved complex protein machines that faithfully maintain our genetic material. Genomic instability, which arises from defects in these proteins, is a defining feature of most cancers. Elucidating the mechanisms of DNA maintenance is therefore fundamental to our understanding of the molecular basis of many cancer types.

Our interdisciplinary research program combines aspects of single-molecule biophysics, molecular biology and micro-/nano-scale engineering to understand how organisms are able to maintain their genomic integrity. To increase our understanding of this essential problem, we develop new techniques that allow us to directly observe, in real time, the key biochemical reactions as they occur on DNA.

Fluorescence Microscopy

Our lab uses Total Internal Reflection Fluorescence Microscopy (TIRFM) as the primary tool for single-molecule fluorescence imaging. For TIRFM, laser excitation is limited to a shallow penetration depth near the surface of a microfluidic flowcell (100 nanometers for most applications). Molecules of interest are immobilized on a passivated flowcell surface, thus eliminating spurious background signals. Images are collected with a microscope objective and recorded using a back-illuminated charge-coupled device (CCD) with on-chip signal amplification. The CCD has >90% quantum efficiency over much of the visible spectrum and can be used with an image-splitter containing a dichroic mirror to separate the multicolor fluorescence signal for simultaneous multi-color imaging.

Figure 1. (A) Schematic of prism-type TIRFM. A choice of lasers and emission filters allows simultaneous multi-fluorophore imaging. (B) The evanescent waves decays exponentially, limiting excitation to molecules held near the surface.

DNA Curtains

Collecting statistically relevant datasets is a major intrinsic challenge for experiments designed to observe individual reactions. We employ a high-throughput approach called “DNA curtains” that incorporates elements of nanofabrication and microfluidics to construct aligned arrays of surface-tethered DNA molecules. DNA curtains allow us to observe hundreds of biochemical reactions simultaneously at the single molecule level with excellent signal to noise and a wide choice of excitation and emission options. Furthermore, by nanofabricating various DNA curtain geometries, we can control critical experimental parameters such as DNA orientation, spacing, density and tension.

Figure 2. (A) An optical image of a 2×3 array of chrome nanobarriers deposited onto fused silica. Panels (B) and (C) show images of YOYO1-stained λ-DNA curtains assembled at the barriers. The DNA molecules are imaged at 60× magnification. There are over ~800 DNA molecules in this single image. After stopping buffer flow, the DNA rapidly retracts from the surface, leaving only their tethered ends within the evanescent field (C).