Project Summary To improve DNA sequencing capabilities with respect to accuracy, robustness and speed and to develop practical methods of RNA sequencing, this NIH R43 Phase I project focuses on using multilayer-design solid-state pore sensors on low-noise glass chips, towards DNA sequencing and direct RNA sequencing. The basic concept behind nanopores involves using an applied voltage to drive single-stranded DNA molecules through a narrow nanopore, which separates chambers of electrolyte solution. This voltage also drives a flow of electrolyte ions through the pore, measured as an electric current. When molecules pass through the nanopore they modify the flow of ions, and structural information can be extracted by analysis of the duration and magnitude of the resulting current reductions. Nanopore in atomically-thin 2D membranes improve the signal-to-noise ratio for molecular detection and analysis because the resistance to the ionic flow through a pore increases linearly with the pore thickness, so both the magnitudes of the ionic current and the blocked current with a translocating molecule increase with decreasing nanopore height. Specifically, we seek to make solid-state ionic-current based nanopore sequencing possible by combining several components to make a modular multilayer on-chip solid-state ultrathin-pore system that limits the range of motion for DNA in the sensing region of a pore. We do so by creating devices containing a second layer of silicon nitride holes, parallel to primary layer containing the sensing 2D pore that orient DNA within a device to a restricted geometry, yet allow the free motion of ions to maintain a high signal-to-noise ratio. We propose a specific multilayer concept with two independent electrical connections, and corresponding chip device architecture to achieve this goal. In this method, there is a central, highly sensitive 2D pore. A secondary layer is a nanopore array (NPA) sharing the same electrode pair as the sensing 2D pore. These pores are constructed parallel to the “sensing” pore and serve as “feeding” elements to stretch and feed DNA into the sensing pore. We outline the practical implementation of this concept with Si-based technology, including advantages for DNA (and biomolecule) sequencing (analysis) in solution. Our approach eliminates the need for any enzymes and enables DNA molecules to be guided through robust and long-lasting nanopores, facilitated by the custom-designed “array chip”, and at potentially record high sequencing speeds. Illustration 1: Proposed multilayer device concept for this NIH R43 Phase I proposal, relying on minimization of DNA entropic motion: a guiding array and an optimized 2D pore. 1