PROJECT SUMMARY The objective of this proposal is to unlock the full potential of nanopore sequencing. This sequencing method has transformative intrinsic qualities, such as long read length, direct epigenetic modification detection, fast sample-to-answer, portability and low cost- to-entry, but relatively low sequencing accuracy remains a significant drawback. With NHGRI funding, we have built a team that has played a pivotal role in developing nanopore sequencing, first engineering the highly sensitive pore MspA to provide single-nucleotide resolution and then combining it with enzyme control for the first proof-of-concept of nanopore sequencing. Recently, we developed a hybrid- voltage-enzyme control method that provided a substantial increase in nanopore sequencing accuracy. Concurrently, we developed a nanopore single-molecule tool that measures the kinetics of enzymes which move along DNA or RNA at unprecedented detail. Here we build on our previous research and propose well-founded and innovative methods to further increase the accuracy of nanopore sequencing with the ultimate goal of a single-passage base calling accuracy of around 99%. Our specific aims are (1) to use sequence-dependent enzyme kinetics to create a second reader in the nanopore system that runs in tandem to the ion current reader. This reader will provide sequence information that is independent of the ion current reader. We will engineer enzymes with accentuated sequence-dependent kinetics. (2) We will systematically map the sequence-dependent enzyme kinetics and splice this information into novel base calling algorithms. In addition, we will measure the ion current of all four different orientations of the pore-enzyme-DNA complex to maximize the accuracy with this new method. (3) We will engineer better sequence specificity into the ion current reader by creating a robust platform that permits asymmetric assembly of MspA. We will then design MspA with an asymmetric pore constriction to reduce Brownian motion of the DNA and enhance nucleotide recognition. Our team's success to date has enabled us to form partnerships with prominent collaborators and to gain support from many excellent labs in academia and industry, whose expertise assists us in each step forward. We will work with our partner labs to complete the aims outlined in this proposal.