Hybridization of Oligonucleotide Probes with Duplex DNA

描述（由申请人提供）：该项目继续并显着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“开启剂”，已知可有效侵入 dsDNA 的短嘌呤束。该项目的一个主要想法是，一对 PNA 开启子与 dsDNA 上位置紧密的嘌呤位点结合，打开其间的双螺旋。这使得 DNA 靶标可在本地访问，以便通过 Watson-Crick 配对结合探针。这种复合物、PD环和相关结构形成高度序列特异性，因为只有由两个开启子协同打开的dsDNA位点可用于随后结合探针。该项目的目标是精心设计适用于基因组 DNA 的稳健的基于 PD 环的检测方法。 为了实现项目目标，原始 PD 环设计中固有的序列限制（PNA 开启子只能靶向位置接近的嘌呤位点）将被消除。将设计具有更短的开启子和更长的嘌呤位点之间的间隙的扩展PD环。带有嵌入剂和额外正电荷的 PNA 开启剂将用于稳定它们。 Tris-PNA 构建体也将被尝试作为有利于扩展 PD 环的 PNA 开启剂。增强亲和力的 DNA 探针和更稳定的 PNA 探针也将用于此目的。具有扩展三链体识别功能的 PNA 开启子将用于大部分嘌呤位点和少量嘧啶。伪互补 PNA 修饰 pcPNA 将用作开启剂，以显着缓解 PD 环序列限制。 pcPNA 开启子与侵入双链体边缘的混合嘌呤-嘧啶 dsDNA 序列的伪互补寡核苷酸的组合最终将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，考虑到相对于大量过量的不相关基因组 DNA 而言，少量的 DNA 靶标还需要高灵敏度的 DNA 诊断。使用环状 PD 探针（耳环）进行双链 DNA 捕获，能够更稳定地附着到目标位点，将用于富集指定 PD 目标的 DNA 分析物。圆形探针的滚环超扩增 (HRCA) 预计将提供必要的灵敏度，产生双链 DNA 产物，其中原始 PD 位点重复超过百万次。最后，多重复制的 PD 位点将被 PNA 开启剂选择性暴露，并用分子信标进行荧光检测。将详细阐述同时将 PNA 开启子和分子信标靶向 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 环的人工切口酶系统将被设计为兆碱基 DNA 定点多重标记的替代方法。所有这些策略都将在粗提物中双链 DNA 标记的多重检测中进行测试。该项目的实施将为病原体 DNA 诊断以及完整基因的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.