Research Techniques

Slide Diagram

A zoom-in of slide topography, with labels for the lipid bilayer, barriers and pedestals for DNA tethering, and DNA strands.

Assay Mechanism

Figures representing assay conditions with and without buffer flow (bottom and top, respectively), and images visualizing DNA position using GFP-labeled protein.

DNA Curtains

This single-molecule optical microscopy technique is used to study fundamental interactions between proteins and nucleic acids, using a non-traditional combination of biochemistry, physics, and nano-scale technology. It allows us to observe individual protein molecules and protein complexes as they interact with their DNA substrates. The goal is to reveal which proteins bind to DNA, where they bind, how they move, and how they influence other components of the system in real-time, on a single-reaction scale.

Our lab developed the DNA curtain technique to overcome limitations on a single-reaction scale in other TIRFM-based biochemistry. The technology uses fluid lipid bilayers to render surfaces inert to biological molecules, and micro- and nano-scale materials engineering to construct parallel arrays containing individual DNA molecules with user-defined positions, orientations, tensions, and topologies. These DNA arrays allow for parallel processing of hundreds to thousands of individual protein-nucleic acid interactions in a single TIRFM experiment and serve as powerful tool for single-molecule research.

cryo-EM

Cryo-electron microscopy (cryo-EM) is a TEM-based technique for modeling molecular structure. It relies on rapid freezing of the target molecule, then compiling vast image libraries, and computationally generating a 3D model at near-atomic resolution.

By freezing the molecule, instead of crystalizing it, cryo-EM allows us to see molecules in their native states, i.e. how they exist in cells. In our lab, we generate protein models which we use to identify regions of interest for further study, for example sites of protomer-protomer binding, protein-nucleotide binding, and protein-protein binding.

Deep Mutational Scanning (DMS)

DMS, or deep mutagenesis, is high-throughput technique used to analyze the function of single-amino-acid-substitution mutations in proteins. This technique relies on random mutation of the codon of the amino acid of interest to every other codon, thereby generating a vast library of mutations. This library is introduced into a model organism and subjected to selective pressure. The frequency of each amino acid residue is identified before and after selective pressure using next-gen sequencing, which allows us to observe the relative impact of the mutations to the selected phenotype.