BME PhD Dissertation Defense - Allison Squires
- 10:00 am on Monday, December 2, 2013
- 44 Cummington Mall, Room 203
Mark Grinstaff (BU, BME & Chemistry) *Chair
Allison Dennis (BU, BME & MSE)
Shyam Erramilli (BU, Physics)
Michael Smith (BU, BME)
Amit Meller (Advisor, BU, BME & Physics)
Title: "Engineering Solid-State Nanopores for Detection of Single Transcription Factors Bound to DNA"
Detection and characterization of nucleic acid-protein interactions, particularly those involving DNA and transcription factors, remain significant barriers to our understanding of genetic regulation. Solid-state nanopores are extremely sensitive single molecule sensors with the capability to map local chemical and structural characteristics along the length of a biopolymer, providing label-free detection for a wide range of analyte lengths and sizes. Previous studies have utilized solid-state nanopores to detect complexes of DNA bound to many large proteins, but improvements to the sensing resolution of the nanopore platform are necessary for detection of single small transcription factors bound to DNA. This project encompasses two novel nanopore modifications that enhance output signal quality and time resolution in nanopores, and establishes solid-state nanopores as a platform for direct measurement of transcription factor-DNA complexes.
First, a novel fabrication process was developed to create locally thinned SiN membranes on a full-wafer scale. These modified nanopore chips provide several advantages over conventional solid-state nanopores, including improved signal-to-background ratio, higher probability of functionality, and clearly marked pore locations for re-imaging and array fabrication. Next, the volume outside the nanopore was modified by electrospinning a sparse, hydrophobic co-polymer nanofiber mesh (NFM) directly onto the nanopore chip. The NFM interacts with analyte molecules as they translocate through the pore, increasing residence time in the sensing volume and improving resolution by more than two orders of magnitude for a chemically optimized blend of poly(ε-caprolactone) and poly(glycerol-co-ε-caprolactone). Finally, modified nanopores were used for direct, label-free detection of single transcription factors bound to DNA. Translocations of these complexes reveal a combination of two possible sensing modalities; either the complex passes unhindered through the pore, causing a transient drop in current at the location of the bound protein, or the protein is unable to translocate and is removed as the DNA is electrophoretically driven through the nanopore. The DNA-binding domain of the transcription factor Early Growth Response Protein 1 (EGR1), known as zif268, is presented as a model system for this research. EGR1 activates genes that control cell differentiation and mitogenesis, and participates in many regulatory processes including wound response, tumor suppression, and neuronal plasticity.