HYBRIDIZATION OF AN OLIGONUCLEOTIDE PROBE WITH DUPLEX DN

寡核苷酸探针与双链 DN 的杂交

• 批准号: 2826039
• 负责人: MAXIM D FRANK-KAMENETSKII
• 金额: $ 37.75万
• 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 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2826039
• 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; transposon /insertion element

The goal of the project consists of developing a new approach for hybridization of an oligonucleotide (ON) to double-stranded DNA (dsDNA). A major tool is the peptide nucleic acid (PNA), in which DNA nucleobases are attached to a polyamide backbone. A special class of PNA molecules, bis-PNA, is known to effectively and sequence-specifically invade into short homopurine tracts of dsDNA locally displacing the complementary strand of DNA and forming the P-loop. A major idea underlying the project is that two bis-PNAs (PNA "openers") bound to two closely located sites on dsDNA open the double helix between them. This makes one of DNA strands locally accessible for hybridization with an ON via Watson-Crick pairing. Such a hybrid, the PD-loop, must form very sequence- specifically because only the DNA site opened by PNA openers is accessible for binding with the complementary probe. To reach the objectives of the project, we will study the efficiency of formation of PD-loops and their stability under various conditions. Various constructions of PD-like loops will be checked and the nature of openers and probes will be varied. Oligodeoxyribonucleotides, which form PD-loops, oligoribonucleotides, which form PR-loops, and PNA oligomers, which form PP-loops, will be tested under a variety of conditions. We will study factors influencing stability and specificity of PD-, PR- and PP-loops. Two major methods will be used in the project. When possible, we will use the gel electrophoretic mobility shift assay. It is based on difference in gel mobility of a naked dsDNA fragment and the same fragment carrying the PD-, PR- or PP-loops. More generally applicable assay will be based on affinity capture of dsDNA via PD-loop formation. In this assay, the probe is biotinylated and iron microbeads covered with streptavidin are used for magnetic separation of the probe complex with dsDNA from control DNA, which does not carry the PD-loop. We will also use "molecular beacons" for monitoring the PD-loop formation. The major result of the project will be the development of two new approaches for manipulating with dsDNA: (i) ON/PNA-assisted affinity capture (OPAC) of dsDNA for isolation of a specific dsDNA fragment from a complex mixture of dsDNA fragments; (ii) in vitro and in situ hybridization of dsDNA with a probe. These techniques are expected to be more convenient and more sequence specific than existing methods based on hybridization with single-stranded (denatured) DNA and single-stranded RNA. The project will open totally new opportunities for development of DNA diagnostics and for isolation of genes in an intact form.

该项目的目标包括开发一种新的寡核苷酸（ON）与双链DNA（dsDNA）杂交的方法。一个主要的工具是肽核酸（PNA），其中DNA核碱基连接到聚酰胺骨架。已知一类特殊的PNA分子，bis-PNA，有效地和序列特异性地侵入dsDNA的短高嘌呤束，局部置换DNA的互补链并形成P-环。该项目的一个主要思想是，两个双PNA（PNA“开放器”）结合到dsDNA上两个位置接近的位点上，打开它们之间的双螺旋。这使得DNA链中的一条通过沃森-克里克配对与ON局部杂交。这样的杂合体，PD环，必须形成非常序列特异性的，因为只有由PNA开放剂打开的DNA位点才可与互补探针结合。为了达到该项目的目标，我们将研究在各种条件下形成PD环的效率及其稳定性。将检查PD样环的各种构造，并且将改变开放器和探针的性质。将在各种条件下检测形成PD环的寡脱氧核糖核苷酸、形成PR环的寡核糖核苷酸和形成PP环的PNA寡聚体。我们将研究影响PD-，PR-和PP-环的稳定性和特异性的因素。该项目将采用两种主要方法。如果可能，我们将使用凝胶电泳迁移率变动分析。其基于裸dsDNA片段和携带PD-、PR-或PP-环的相同片段的凝胶迁移率的差异。更普遍适用的测定将基于经由PD环形成的dsDNA的亲和捕获。在该测定中，探针被生物素化，并且用链霉亲和素覆盖的铁微珠用于磁性分离探针与dsDNA的复合物与不携带PD环的对照DNA。我们还将使用“分子信标”来监测PD环的形成。该项目的主要成果是开发了两种新的dsDNA操作方法：（i）对dsDNA进行ON/PNA辅助亲和捕获（OPAC），用于从dsDNA片段的复杂混合物中分离特定的dsDNA片段;（ii）dsDNA与探针的体外和原位杂交。预计这些技术比现有的基于与单链（变性）DNA和单链RNA杂交的方法更方便和更序列特异性。该项目将为DNA诊断的发展和完整形式的基因分离开辟全新的机会。