Molecular Reading Head for Single-Molecule DNA sequencin

描述（由申请方提供）：拟定研究的目的是评价一种新型单分子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）组成的轮烷复合物上，该分子环自缠绕在丙烯低聚物上。 丙烯低聚物的远端附着在固定表面上，而环糊精环则共价附着在原子力显微镜探针上。 当AFM探针被拉离固定表面时，DNA通过环糊精环，一次一个碱基。 当环通过每个基底时，分子摩擦的波动被记录为AFM悬臂的偏转。 如果这些数据可以用长DNA分子的碱基序列来解释，那么单DNA分子就可以用这种新技术快速测序。 初步研究似乎表明，DNA可以通过环糊精环被拉。 他们还表明，在收缩过程中的悬臂梁偏转取决于DNA序列。 一个意想不到的发现是双链DNA似乎比单链DNA更容易通过环，并且比单链DNA的情况具有更少的随机波动。 我们的第一个目标是进一步测试“环滑动”模型。 环真的滑过双链DNA的一条链，把互补链剥离吗？ DNA和固定表面之间的序列特异性粘附是否有助于序列相关信号？ 如果这些实验发现了我们系统的问题，我们将适当地修改化学物质。 一旦系统的运行得到验证，我们将开展一项理论和实验计划，旨在了解这些初步观察结果，并确定迄今为止开发的技术的局限性。 在这些研究信息的指导下，将设计改进的分子“阅读头”，优化测序参数，并改进硬件，以实现低聚物的可靠测序，这是随后尝试大规模测序的先决条件。

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.