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寡核苷酸和其他探针与双链（ds）DNA杂交的创新策略的开发。 主要工具是一类特殊的嘧啶肽核酸（PNA; DNA合成模拟物）-阳离子双-PNA或PNA“开放剂”，已知其有效地侵入dsDNA的短嘌呤束。该项目的一个主要想法是，一对PNA开放器与dsDNA上紧密定位的嘌呤位点结合，打开了其间的双螺旋。这使得DNA靶标局部可接近以通过沃森-克里克配对结合探针。这样的复合物，PD环和相关结构，形成高度序列特异性，因为只有由两个开放器共同打开的dsDNA位点可用于随后结合探针。该项目的目标是制定适用于基因组DNA的稳健的基于PD环的测定。 为了达到项目目标，将消除原始PD环设计中固有的序列限制（PNA开放器只能靶向位置接近的嘌呤位点）。将设计具有较短开放器和嘌呤位点之间较长间隙的扩展PD环。具有嵌入剂和额外正电荷的PNA开放剂将用于其稳定化。Tris-PNA构建体也将被尝试作为对延长的PD-环有利的PNA开放剂。增强的亲和性DNA探针和更稳定的PNA探针也将用于此目的。具有扩展的三链体识别的PNA开放剂将用于具有很少嘧啶的大多数嘌呤位点。一个伪互补PNA修饰，pcPNA，将被用作一个开放，以大大减轻PD环序列的限制。pcPNA开放子与在双链体边缘侵入混合嘌呤-嘧啶dsDNA序列的假互补寡核苷酸的组合将最终导致基本上序列通用的PD环的设计。 除了软化序列限制之外，相对于大量过量的不相关基因组DNA，考虑到少量的DNA靶标，需要DNA诊断的高灵敏度。使用能够更稳定地附着于靶位点的环化PD探针（耳环）进行双链体DNA捕获，将用于富集具有指定PD靶标的DNA分析物。预期环状探针的滚环超扩增（HRCA）提供产生dsDNA产物的必要灵敏度，其中原始PD位点重复超过百万次。最后，因此多重复制的PD位点将被PNA开放剂选择性地暴露，并用分子信标进行荧光检测。将详细说明同时将PNA开放器和分子信标靶向HRCA倍增的dsDNA靶点以实时监测序列不受限制的PD环杂交的策略。基于PD环的人工切口酶系统将被设计为用于定点多重标记megaDNA的替代方法。所有这些策略将在粗提物中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的分离和分析开辟全新的机会。

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.