Andre Marziali

Single molecule nanopore sensing is emerging as a powerful tool for probing the properties of individual biomolecules. This is particularly true for DNA where nanopore technology is being actively developed toward ultra low-cost, high-throughput whole-genome sequencing. Most approaches to nanopore DNA sequencing require that DNA translocate a nanometer-scale pore (nanopore). Understanding and characterizing the physics of DNA translocation through nanopores is critical to the design and optimization of these methods. We sought to investigate DNA translocation dynamics and elucidate the mechanism of DNA transport through nanopores. We show that stochastic, sequence-dependent DNA-pore binding interactions play an important role in translocation and lead to subdiffusive translocation dynamics, which in the case of short DNA strands, is consistent with fractional dynamics. We characterize the sequence-dependent kinetics of DNA translocation and show that nucleotide dwell-time in the pore can potentially be used as a metric to distinguish individual nucleotides in nanopore sequencing, opening up new avenues by which to optimize nanopore sequencing technology.While development of nanopore DNA sequencing has largely dominated nanopore applications, other applications including nanopore protein analysis are of great interest as a means to explore protein conformational dynamics and structure at the single molecule level. We present methods by which to capture and trap proteins in nanopores (via asymmetric salt concentration) and analyze their complex dynamics (via Hidden Markov Model signal processing), resolving two important challenges associated with nanopore sensing of proteins. We apply these methods to characterize the kinetics and dynamics of the prion protein in a nanopore (a protein whose conversion into a misfolded isoform is responsible for the pathogenesis of prion diseases in humans and animals) as a first step towards understanding the relationship between prion protein conformational dynamics and conversion in disease. Moreover, we demonstrate the potential of nanopore technology for highly-sensitive, real-time protein and small molecule detection based on single molecule kinetics with potential application in medical diagnostics. Our methods enable studies of the long timescale conformational motions of proteins known to be critically important to protein function, at the single molecule level, making nanopore sensing a new tool for studying protein dynamics.

View record

Sequence based enrichment of nucleic acids is a critical enabling component of future nucleic acid detection methods in many fields including detection of nucleic acid tumor biomarkers in body fluids, non-invasive prenatal detection of fetal genetic abnormalities, and detection of pathogenic microorganisms. In many cases the problem of detecting the nucleic acid biomarker of interest is confounded by the presence of a large excess of nucleic acid sequences that may differ from the sequence of interest by only a single base. Consequently, existing methods are limited in sensitivity and amount of starting material to avoid overwhelming the detection methods with background nucleic acids. This limits their usefulness to a small number of applications. Techniques for enrichment of specific sequences rely on hybridization, and are generally not capable of enriching for low abundance sequences by more than 10 fold, a limit imposed by the thermodynamics of hybridization. In this dissertation I present a technique for sequence enrichment of nucleic acids based on synchronous coefficient of drag alteration (SCODA), which enables sequence specific enrichment of nucleic acids from sample volumes greater than 100 μL, with concurrent concentration of the nucleic acids to volumes appropriate for PCR detection (
View record

No abstract available.