Molecular Reading Head for Single-Molecule DNA sequencin

描述（由申请人提供）：拟议研究的目标是评估一种新的单分子DNA测序技术，该技术有可能在数小时内对基因组尺寸的分子进行测序。DNA附着在由分子环（环糊精）组成的轮烷络合物上，该络合物自旋在丙烯低聚物上。丙烯低聚物的远端附着在固定表面上，环糊精环共价附着在AFM探针上。当原子力显微镜探针从固定表面抽离时，DNA通过环糊精环，一次通过一个碱基。当环通过每个基时分子摩擦的波动被记录为原子力显微镜悬臂的挠度。如果这些数据可以用长DNA分子的碱基序列来解释，那么用这种新技术就可以快速地对单个DNA分子进行测序。

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.