Enzymeless, controlled electrostatic ratcheting in solid-state nanopores

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Enzymeless, controlled electrostatic ratcheting in solid-state nanopores 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. 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, particu- larly biological ones, face reliability challenges as electronic devices, experiencing degradation during use. In this four-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, leveraging integrated electronics and state-of-the- art solid-state nanopores based on ultra-thin membranes of layered two-dimensional materials and delivering useful signal bandwidths in excess of 10 MHz when required. We expect to be able to detect signal levels as low as 50 pA at signal-to-noise ratios greater than 8 and bandwidth better than 2 MHz, making possible high- speed free-running single-molecule electrophoretic sequencing if the translocation rate and diffusive motion of the translocating DNA can be controlled. This is accomplished through electrostatic control through gate elec- trodes in the pore itself and closed-loop feedback. This goal is pursued through three Specific Aims: the de- sign of solid-state nanopores based on layered two-dimensional materials include hexagonal boron nitride (h- BN) and graphene or transition metal dichalcogenides and application of these pores to translocating DNA (Specific Aim 1); the design of electronics optimized for high-speed multiplexed detection of these nanopores and closed-loop electronic control of the gates within the pore (Specific Aim 2); and application of this system to controlling translocation rates for sequencing (Specific Aim 3).