Biological and solid-state nanopores have emerged as viable tools for analyzing the structure and kinetics of DNA and enzymes that bind or modify DNA, at the single molecule level, and offer great promise for de novo genomic sequencing. The broad objective of the proposed research is to develop an integrated dual-nanopore instrument that will offer new modes of single molecule analysis of molecular species that bind or modify nucleic acids, and will facilitate DNA nanopore sequencing. There are two aims:
Aim 1 : (Year 1) Develop a dual-pore microfluidic chip and demonstrate capture of a single DNA in both pores using a single amplifier voltage source. In parallel, develop an integrated dual-amplifier system that will permit independent voltage control and current measurement for each pore in the dual-pore chip. Significance: The instrument provides a new method for coupling two nanopores to measure one DNA molecule. Capture of a single DNA into two pores has not been demonstrated, but has high likelihood of success for the proposed chips. We've developed an integrated amplifier that is optimized for nanopores, providing a small-footprint and low-cost module that provides a scalable means of functionalizing multiple pores in a single chip. Independent voltage control and current measurement afforded by the proposed dual-amplifier system is also a prerequisite for the dual-pore applications proposed in Aim 2.
Aim 2 : (Year 2) The dual-pore chip and dual-amplifier system will have two focused applications in parallel: (a) Measure the presence and translocation time of an enzyme through a nanopore, along a DNA captured and immobilized in both pores, at high temporal resolution. (b) Demonstrate controlled motion of a DNA through both pores, by electrophoretic tug-of-war (i.e., by competing voltages), and detection of proteins bound to the DNA at high spatial resolution. Significance: (a) As a single molecule instrument, the dual-pore setup will permit detection and measurement (at ~ 10 kHz bandwidth) of numerous enzymes that bind and move along DNA or RNA, including exonucleases and polymerases. (b) While many research groups are refining nanopore sensitivity for sequencing, controlled motion of the DNA through a nanopore remains a universal technical challenge. The proposed instrument will provide a purely electrophoretic method of motion control that provides decoupled high signal-to-noise current measurements for each pore, while achieving slow delivery of the DNA through each pore by electrophoretic "tug-of-war." The independent current measurements can be cross-correlated to identify structural variations in the DNA during controlled delivery. Detection and localization of individual proteins along a single DNA could facilitate efforts to screen for transcription factors along a genome. As an infrastructure to support nanopore sequencing, the motion control-enabling architecture can be employed for any pair of pores that can be integrated into a chip, and so can accommodate advances in pores/substrates that are optimized for single nucleotide sensitivity.

Public Health Relevance

Biological and solid-state nanopores are viable instruments for single molecule analysis of polynucleotide-binding proteins and offer great promise for inexpensive genomic sequencing. The proposed instrument will advance sequencing efforts and facilitate new modes of single molecule analysis, by providing a new method to control DNA motion and speed through two nanopores that dominates the otherwise stochastic DNA motion, provides high signal-to-noise detection currents, and is entirely electrophoretic (i.e., does not require auxiliary means of manipulating an attachment to the DNA). A dedicated integrated circuit for control of the dual-pore device will be developed, representing an advance for application-specific nanopore control instrumentation.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21HG006561-02
Application #
8458100
Study Section
Enabling Bioanalytical and Imaging Technologies Study Section (EBIT)
Program Officer
Schloss, Jeffery
Project Start
2012-04-11
Project End
2015-01-31
Budget Start
2013-02-01
Budget End
2015-01-31
Support Year
2
Fiscal Year
2013
Total Cost
$179,379
Indirect Cost
$38,629
Name
University of California Santa Cruz
Department
Biostatistics & Other Math Sci
Type
Schools of Engineering
DUNS #
125084723
City
Santa Cruz
State
CA
Country
United States
Zip Code
95064
O'Donnell, Christopher R; Wang, Hongyun; Dunbar, William B (2013) Error analysis of idealized nanopore sequencing. Electrophoresis 34:2137-44
Liu, Shuo; Yuzvinsky, Thomas D; Schmidt, Holger (2013) Effect of fabrication-dependent shape and composition of solid-state nanopores on single nanoparticle detection. ACS Nano 7:5621-7