Modulating Nucleotide Size in DNA for Detection by Nanopore

描述（由申请人提供）：该提案的目标是设计和合成修饰的核苷酸，以增加其尺寸差异，用于通过纳米孔进行单分子DNA分析。我们将致力于以下几个方面来研究这种方法的可行性：（1）利用固相合成法制备由携带不同大小修饰基团的核苷酸组成的单链DNA，并使用纳米孔测试这些修饰的DNA，以评估从DNA中的每个核苷酸产生不同阻断信号所需的参数;（2）利用目标1中获得的知识，设计并合成带有不同大小基团的修饰核苷酸，用于在聚合酶反应中合成修饰的DNA。然后使用纳米孔检测单链DNA以寻找条件来指导核苷酸的设计和修饰以实现不同的阻断信号;（3）设计和合成携带小官能团的核苷酸作为用于DNA聚合酶反应的钩，以产生钩标记的DNA产物。由于钩的小尺寸，这些核苷酸预期是常用的DNA聚合酶的良好底物，以产生携带钩的DNA产物。然后，携带钩的单链DNA产物将被分离，并选择性地与几个不同的大官能团反应，以增加DNA中核苷酸之间的大小差异。然后，具有修饰的核苷酸的该DNA链将被纳米孔明显地检测以产生序列数据。这里开发的分子工具将有助于实现单碱基分辨率的纳米孔单分子测序的长期目标。

DESCRIPTION (provided by applicant): The goal of the proposal is to design and synthesize modified nucleotides to increase their size difference for single molecule DNA analysis by nanopores. We will pursue the following aims to study the feasibility of this approach: (1) Use solid phase synthesis to prepare single stranded DNA consisting of nucleotides carrying different sized modification groups and test these modified DNAs using nanopores to evaluate the parameters that are required to generate distinct blockade signals from each nucleotide in the DNA; (2) With knowledge gained in aim 1, design and synthesize modified nucleotides carrying different size groups for synthesis of modified DNAs in polymerase reaction. The single stranded DNA will then be detected using nanopores to search for condition to guide the design and modification of the nucleotides to achieve distinct blockade signals; (3) Design and synthesize nucleotides carrying small functional groups as hooks for DNA polymerase reaction to generate hook-labeled DNA products. Due to the small size of the hook, these nucleotides are expected to be good substrates for commonly used DNA polymerase to produce DNA products carrying the hook. The single stranded DNA products carrying the hook will then be isolated and selectively reacted with several different large functional groups to increase the size difference among the nucleotides in DNA. This DNA strand with the modified nucleotides will then be detected distinctly by nanopores to produce sequence data. The molecular tools developed here will facilitate achieving the long-term goal of single molecule sequencing by nanopores at single base resolution.