HYBRIDIZATION OF AN OLIGONUCLEOTIDE PROBE WITH DUPLEX DN

寡核苷酸探针与双链 DN 的杂交

• 批准号: 6386436
• 负责人: MAXIM D FRANK-KAMENETSKII
• 金额: $ 37.98万
• 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/6386436
• 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寡核苷酸 (ON) 与双链 DNA (dsDNA) 杂交的新方法。一个主要工具是肽核酸 (PNA)，其中 DNA 核碱基连接到聚酰胺主链上。一类特殊的 PNA 分子，bis-PNA，已知能够有效地、序列特异性地侵入 dsDNA 的短同型嘌呤束，局部置换 DNA 的互补链并形成 P 环。该项目的一个主要想法是，两个双-PNA（PNA“开启子”）与 dsDNA 上两个位置紧密的位点结合，打开它们之间的双螺旋。这使得其中一条 DNA 链可以在本地通过 Watson-Crick 配对与 ON 杂交。这种杂合体，即 PD 环，必须形成非常序列化的序列，因为只有由 PNA 开启子打开的 DNA 位点才能与互补探针结合。为了实现该项目的目标，我们将研究PD环的形成效率及其在各种条件下的稳定性。将检查类似 PD 环的各种结构，并且开启剂和探针的性质将有所不同。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寡聚体将在各种条件下进行测试。我们将研究影响 PD-、PR- 和 PP-环稳定性和特异性的因素。该项目将使用两种主要方法。如果可能，我们将使用凝胶电泳迁移率变动分析。它基于裸露 dsDNA 片段和携带 PD-、PR- 或 PP-环的同一片段的凝胶迁移率差异。更普遍适用的测定将基于通过 PD 环形成对 dsDNA 的亲和捕获。在此测定中，探针经过生物素化，并使用链霉亲和素覆盖的铁微珠将探针与 dsDNA 的复合物与对照 DNA（不携带 PD 环）进行磁性分离。我们还将使用“分子信标”来监测 PD 环的形成。该项目的主要成果将是开发两种操纵双链DNA的新方法：（i）双链DNA的ON/PNA辅助亲和捕获（OPAC），用于从双链DNA片段的复杂混合物中分离特定的双链DNA片段； (ii) 双链DNA与探针的体外和原位杂交。这些技术预计比基于单链（变性）DNA 和单链 RNA 杂交的现有方法更方便、更具有序列特异性。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。