Nucleic acid molecules (DNA-and RNA) can be driven into and through a nanoscopic pore (nanopore) such as a-hemolysin by an applied electric field. Because of steric restrictions, only one molecule of single stranded nucleic acid at a time can occupy the pore of the channel, and each strand passes through in strict linear sequence from one end of the nucleic acid to the other. While in the pore, the molecule blocks ionic current that otherwise flows unimpeded, and modulations of this ionic current blockade provide information about structure and composition of the nucleic acid. If modulations of the blockade by individual nucleotides can be resolved, a suitable nanopore has the potential to sequence single molecules of DNA at much faster rates than current methods. The research proposed here incorporates three specific aims related to nanopore analysis of nucleic acids. In the first, we propose to use secondary structure as a way to slow the translocation process, so that a given base will spend defined amounts of time in the limiting aperture of the pore. This tests the hypothesis that translocation rates are related to the amount of secondary structure that must be overcome as a molecule traverses the pore.
The second aim will develop specialized computational methods to analyze signals from individual molecules or populations of molecules and then use multidimensional wavelet analysis to isolate signal from noise. This approach will help identify signal modulations related to single nucleotides in nucleic acid molecules tranversing the pore.
The third aim i s to design and fabricate a robust stable nanopore as an alternative to the present fluid lipid bilayer with embedded a-hemolysin. The hypothesis to be tested is that ionic current blockades are produced by the fractional volume occupied in the pore by specific components of the translocating nucleic acid strand. The success of our current research, together with the milestones proposed here, will markedly advance our progress toward the overall goal of providing an ultrarapid method for determining concentration, length, secondary structure and sequence information in nucleic acids.
| Akeson, M; Branton, D; Kasianowicz, J J et al. (1999) Microsecond time-scale discrimination among polycytidylic acid, polyadenylic acid, and polyuridylic acid as homopolymers or as segments within single RNA molecules. Biophys J 77:3227-33 |
