Single-Molecule DNA Sequencing with Engineered Nanopores

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DESCRIPTION (provided by applicant): In nanopore strand sequencing, a single strand of DNA moves through a narrow pore and the bases are identified as they pass a reading head. Here, we focus on the remaining tasks required to put into practice strand sequencing with the a-hemolysin (aHL) protein nanopore. Nanopore sequencing is a rapid real-time technology; it does not require the time-consuming cyclic addition of reagents. After implementing a chip with 106 pores, we expect nanopore sequencing to achieve a 15-minute genome by 2014 with a very short sample preparation time. In addition, nanopore sequencing will be able to identify modified bases and to sequence RNA directly. Over the past four years, we have made significant progress; we have shown that all four nucleobases can be identified within intact DNA strands and demonstrated real-time single- nucleotide strand translocation driven by DNA polymerase. We are now in a position to integrate these findings, and with a nanopore array, achieve ultrarapid sequencing. In the next funding period, we will: 1. Refine base recognition by using aHL nanopores, engineered by conventional mutagenesis, unnatural amino acid mutagenesis and targeted chemical modification, to produce DNA reading heads fit for real-time sequencing. 2. Achieve control of strand translocation for non-enzymatic DNA sequencing. The speed of DNA movement will be slowed by the use of rotaxanes, made from small molecules or engineered protein rings, so that bases can be detected by available recording techniques. 3. In a parallel effort, control DNA movement enzymatically by using DNA polymerase. The polymerase will also be employed in two novel sequencing modes, based on nanopore detection of conformational changes associated with nucleobase incorporation. 4. Develop chips containing up to 106 aHL nanopores. First, the prototype of an optically-detected 106-chip will be developed. Second, aHL pores will be placed in arrays of apertures that have been bored into a silicon nitride film with an electron beam, thereby avoiding the use of lipid bilayers altogether. In year 4, these crucial aspects of nanopore sequencing will be integrated into an ultrarapid sequencing device.