DESCRIPTION (provided by applicant): The goal of the proposed study is to evaluate a novel single-molecule DNA sequencing technology that has the potential to sequence a molecule of genomic dimension in hours. The DNA is attached to a rotaxane complex consisting of a molecular ring (cyclodextrin) that self-threads onto a propylene oligomer. The far end of the propylene oligomer is attached to a fixed surface, and the cyclodextrin ring is covalently attached to an AFM probe. As the AFM probe is pulled away from the fixed surface, the DNA passes through the cyclodextrin ring, one base at a time. Fluctuations in molecular friction as the ring passes each base are recorded as deflections of the AFM cantilever. If these data can be interpreted in terms of the base sequence of long DNA molecules, then single DNA molecules can be sequenced rapidly with this new technology. Preliminary studies appear to show that the DNA can be pulled through a cyclodextrin ring. They also indicate that the cantilever deflection during retraction depends on the DNA sequence. An unanticipated discovery is that double stranded DNA appears to pass the ring more easily than single stranded DNA, and does so with less random fluctuation than is the case for single stranded DNA. Our first goal is to put the 'ring sliding' model to further test. Does the ring really slide over one strand of double-stranded DNA, peeling the complementary strand off? Does sequence-specifc adhesion between the DNA and the fixed surface contribute to the sequence-related signal? If these experiments unearth a problem with our system, we will modify the chemistry appropriately. Once the operation of the system is verified, we will carry out a program of theory and experiment aimed at understanding these initial observations and establishing the limitations of the technology as developed thus far. Guided with information from these studies, improved molecular 'reading heads' will be designed, sequencing parameters will be optimized and hardware will be improved with the goal of reliable sequencing of oligomers, a pre-requisite for subsequent attempts at large-scale sequencing.