Enzyme-less DNA base discrimination using solid-state nanopores with high-frequency integrated de- tection electronics There is strong demand for third-generation DNA sequencing systems to be single-molecule, massively- parallel, and real-time, while also reducing operating costs and supporting long read lengths. No technologies have yet met this challenge, but the most successful attempts to date have been based on methods which track the real-time operation of single enzyme molecules operating on a strand of DNA. Optical approaches to single-polymerase imaging suffer from low signal-to-noise ratios deriving from the weak photon emission from single fluorophores (< 2500 photons/sec), and thus demand both complex optics and purposely-reduced base incorporation rates (~1 Hz). Nanopore-based detection approaches offer faster detection and have been demonstrated to track polymerase activity at higher incorporation rates (~10-100 Hz), but have struggled with reliability issues and high error rates associated with the still-weak signal levels produced by popular protein nanopores. These struggles suggest that nanopore-based single-molecule sequencing techniques which do not de- pend on real-time imaging of active enzymes would have several important advantages. First and foremost, they could offer sequencing speeds even faster than a free-running polymerase molecule (which operate at ~100-1000 Hz). Second, removing active enzymes from the detection platform offers more freedom to optimize key parameters such as buffer conditions and temperatures outside the operating range of natural enzymes. Third, sequencing platforms without active enzymes may prove simpler and cheaper to operate, ship, and store. Lastly, nanopores, particularly biological ones, face reliability challenges as electronic devices, experi- encing degradation during use. In this three-year effort, we focus on the development of a multiplexed solid-state nanopore platform ena- bling a per-pore sequencing rate of at least 105 bases/sec at an error rate of less than 0.1%, leveraging inte- grated electronics and state-of-the-art solid-state nanopores based on ultra-thin membrance and delivering useful signal bandwidths in excess of 10 MHz when required. We expect to be able to delect signal levels as low as 225 pA at signal-to-noise ratios greater than 8 and bandwidth better than 3 MHz, making possible high- speed free-running single-molecule electrophoretic sequencing. This goal is pursued through three Specific Aims: the design of biomimetic solid-state nanopores with sensing geometries driven to single nanometer siz- es (Specific Aim 1), the design of electronics optimized for high-speed multiplexed detection of these na- nopores (Specific Aim 2), and data analysis of the resulting signals (Specific Aim 3).
The proposal seeks to address key obstacles that must be overcome to achieve enzyme-less nanopore-based low-cost high-speed sequencing of chromosomal length DNA molecules. Fast and low cost full genome DNA sequencing will allow, for example, major improvements in the understanding, diagnosis, treatment and pre- vention of disease, and significant advances in evolutionary research and the understanding of cellular opera- tion.
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