Hybridization of Oligonucleotide Probes with Duplex DNA

寡核苷酸探针与双链 DNA 的杂交

• 批准号: 6799279
• 负责人: MAXIM D FRANK-KAMENETSKII
• 金额: $ 40.38万
• 依托单位: BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6799279
• 关键词: DNA; binding sites; gel mobility shift assay; in situ hybridization; nucleic acid hybridization; nucleic acid probes; nucleic acid quantitation /detection; nucleic acid structure; oligonucleotides; pyrimidines; synthetic peptide; transposon /insertion element

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.

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双链DNA探针杂交创新策略的发展。主要工具是一类特殊的嘧啶肽核酸（PNA；一种DNA合成模拟物）-阳离子双pnas或PNA“开启器”，已知可有效侵入dsDNA的短嘌呤束。该项目背后的一个主要想法是，一对PNA开子与dsDNA上紧密定位的嘌呤位点结合，打开了两者之间的双螺旋结构。这使得DNA靶标可以通过沃森-克里克配对在局部结合探针。这种复合物，pd环和相关结构，形成高度序列特异性，因为只有由两个开子一起打开的dsDNA位点可用于后续结合探针。该项目的目标是精心设计适用于基因组DNA的基于PD-loop的稳健分析。