Hybridization of Oligonucleotide Probes with Duplex DNA

描述(由申请人提供)：该项目继续并显著扩展了将寡核苷酸和其他探针杂交到双链DNA的创新策略的开发。一种主要的工具是一种特殊类型的嘧啶多肽核酸(PNA；一种DNA合成模拟物)-阳离子双PNA或PNA‘开启物’，已知能有效地入侵dsDNA的短嘌呤区域。该项目背后的一个主要想法是，一对PNA开启子结合到dsDNA上紧密定位的嘌呤位点，打开介于两者之间的双螺旋。这使得DNA目标可以通过沃森-克里克配对在本地访问以结合探针。这种复合体，即Pd-环和相关结构，形成了高度的序列--特别是因为只有由两个启动子协同打开的dsDNA位点可用于随后与探针的结合。该项目的目标是制定适用于基因组DNA的稳健的基于PD-loop的分析方法。 为了实现项目目标，将消除原始PD环设计中固有的序列限制(只有位置较近的嘌呤位点才能成为PNA开启剂的靶点)。将设计具有较短的开启器和较长的嘌呤位置之间的间隙的扩展的PD环。将使用带有嵌入剂和额外正电荷的PNA开启器来稳定它们。Tris-PNA结构也将作为有利于扩展PD-环的PNA开启剂进行试验。增强亲和力的DNA探针和更稳定的PNA探针也将用于这一目的。具有扩展的三链识别的PNA开启剂将用于主要含有少量嘧啶的嘌呤部位。伪互补PNA修饰物PCPNA将被用作启动子，以基本上解除PD-环序列限制。PcPNA启动子与侵入位于双链边缘的混合的嘌呤-嘧啶dsDNA序列的假互补寡核苷酸的组合最终将导致基本序列通用的PD-环的设计。 除了软化序列限制外，还需要高灵敏度的DNA诊断，因为相对于大量无关的基因组DNA，只有少量的DNA靶标。带有环状钯探针(耳环)的双链DNA捕获能够更稳定地附着到目标位置，将用于富集具有指定钯目标的DNA分析物。环状探针的滚环超扩增(HRCA)有望提供所需的灵敏度，从而产生原始PD位点重复超过百万次的dsDNA产物。最后，这样复制的多个Pd位点将被PNA开放剂选择性地曝光，并用分子信标进行荧光检测。将阐述同时将PNA开放剂和分子信标定向到HRCA倍增的dsDNA靶标以实时监测序列非限制性PD环杂交的策略。基于PD环的人工镍酶系统将被设计为一种用于巨型数据库DNA的位点定向多重标记的替代方法。所有这些策略都将在粗提液中dsDNA标记的多重检测中进行测试。该项目的实施将为病原体的DNA诊断以及完整形式的基因分离和分析带来全新的机会。

DESCRIPTION (provided by applicant):The project continues and significantly extends the development of an innovative strategy for hybridization of oligonucleotides and other probes to double-stranded (ds)DNA. A primary tool is a special class of pyrimidine peptide nucleic acid (PNA; a DNA synthetic mimic) - cationic bis-PNAs or PNA 'openers', known to effectively invade short purine tracts of dsDNA. A major idea underlying the project is that a pair of PNA openers bound to closely located purine sites on dsDNA opens the double helix in between. This makes the DNA target locally accessible for binding the probe via Watson-Crick pairing. Such a complex, the PD-loop and related structures, forms highly sequence-specifically because only the dsDNA site opened in concert by two openers is available for subsequent binding the probe. The goal of the project is to elaborate robust PD-loop based assays applicable to genomic DNA. To reach the project objectives, the sequence limitations intrinsic in the original PD-loop design (only closely located purine sites could be targeted by PNA openers) will be eliminated. Extended PD-loops with shorter openers and longer gaps between purine sites will be designed. PNA openers with intercalators and extra positive charges will be used for their stabilization. Tris-PNA constructs will also be tried as PNA openers advantageous for extended PD-loops. Enhanced-affinity DNA probes and more stable PNA probes will be involved for this purpose, too. PNA openers with the extended triplex recognition will be used for mostly purine sites with few pyrimidines. A pseudocomplementary PNA modification, pcPNA, will be employed as an opener to substantially relieve the PD-loop sequence limitations. A combination of pcPNA openers with pseudocomplementary oligonucleotides invading the mixed purine-pyrimidine dsDNA sequence at the edge of the duplex will finally result in the design of essentially sequence-universal PD-loops. Besides softening the sequence limitations, high sensitivity of DNA diagnostics is required given a small amount of the DNA target relatively to a huge excess of unrelated genomic DNA. Duplex DNA capture with circularized PD-probes (earrings) enabling more stable attachment to target site will be used for enrichment of DNA analytes with the designated PD-target. The rolling-circle hyperamplification (HRCA) of circular probes is expected to provide requisite sensitivity yielding the dsDNA product in which the original PD-site repeats more than million times. Finally, thus multiply copied PD-sites will be selectively exposed by PNA openers and fluorescently detected with molecular beacons. The strategies with simultaneous targeting the PNA openers and molecular beacons to the HRCA-multiplied dsDNA targets for the real-time monitoring of sequence-unrestricted PD-loop hybridization will be elaborated. PD-loop based artificial nickase systems will be designed as an alternative method for site-directed multiple labeling of megabase DNAs. All these strategies will be tested in multiplex detection of dsDNA markers in crude extracts. The implementation of the project will open totally new opportunities for DNA diagnostics of pathogens and for isolation and analysis of genes in an intact form.