HYBRIDIZATION OF AN OLIGONUCLEOTIDE PROBE WITH DUPLEX DN

该项目的目标是开发一种新的方法，将寡核苷酸(ON)与双链DNA(DsDNA)杂交。一个主要的工具是肽核酸(PNA)，其中DNA碱基连接到聚酰胺骨架上。一类特殊的PNA分子，双PNA，被认为能够有效地和序列特异性地侵入dsDNA的短同嘌呤小束，局部取代DNA的互补链并形成P-环。该项目背后的一个主要想法是，两个与dsDNA上两个位置相近的位点结合的双PNA(PNA“启动子”)打开了它们之间的双螺旋。这使得一条DNA链可以在当地与On-Via Watson-Crick配对进行杂交。这样的杂交，PD-环，必须形成非常序列-特别是因为只有由PNA开放器打开的DNA位点才能与互补探针结合。为了达到项目的目标，我们将研究PD环的形成效率及其在不同条件下的稳定性。将检查类PD环路的各种结构，开启器和探头的性质将有所不同。形成PD环的寡核苷酸、形成PR环的寡核苷酸和形成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。该项目将采用两种主要方法。如果可能，我们将使用凝胶电泳迁移率改变分析。它是基于裸露的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。更普遍适用的分析将基于通过PD环形成对dsDNA的亲和捕获。在本实验中，探针是生物素化的，覆盖有链霉亲和素的铁微球用于从不携带PD-环的对照DNA中分离与dsDNA形成的探针复合体。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处理dsDNA的新方法：(I)ON/PNA辅助亲和捕获(OPAC)dsDNA，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。预计这些技术将比现有的基于单链(变性)DNA和单链RNA杂交的方法更方便，更具序列特异性。该项目将为DNA诊断学的发展和以完整形式分离基因开辟全新的机会。

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.