PROJECT SUMMARY This project aims to improve DNA and RNA sequencing technology by at least an order of magnitude by dramatically improving the ability to control silicon nitride nanopore surface chemistry and to modify silicon nitride nanopore size. While silicon nitride is a conventional material for nanopore sequencing applications, its complex charged native surface chemistry can present a challenging and complicated environment for a charged nucleic acid biopolymer passing through a nanopore not much larger than itself. Highly desirable long read lengths heighten the need for chemical control over the nanopore surface. Coating the nanopore surface with even a single molecular layer will change the nanopore diameter, which thus also provides for molecular-scale tuning of nanopore dimensions. Broadly, chemically tuned nanopore surface chemistry affords control over motion of (native or labelled) nucleic acid polymers through the pore through electrostatics, specific chemical interactions, and electrokinetics (e.g. electroosmosis). It offers the potential for passivation against fouling in complex matrices, thereby supporting more minimal sample processing. It also affords control over interfacial phenomena that can affect nanopore current noise. Thin-film silicon nitride is a widely used nanofabrication material with widespread commercial utility, so that its continued use in a host of nanopore sequencing implementations is warranted in spite of its often challenging surface chemistry. But efforts to control and improve its surface chemistry using silane chemistry have not gained traction in the field, in significant part because the chemistry is inherently challenging to implement, the more so given the variability of the silicon nitride oxide coating. We thus propose to develop a radically different type of surface chemical modification strategy that is simple to implement, produces highly reliable results, and that can be used to install surface coatings with a wide variety of chemical properties and sizes. We propose to test the surface coatings through their effect on the nanopore conductance and current noise, and on the sequence-specific signal characteristics when sensing well-defined sequences of DNA. The project will be implemented by an interdisciplinary team that combines more than 20 years of Principal and Co-Investigator experience in physical organic chemistry and chemical synthesis (MK); materials science (MK&JRD); and nanofabrication (JRD), with a decade of experience in nanopore science begun in nanopore genotyping (JRD), including a specialization in nanopore surface chemistry modification and characterization.