HYBRIDIZATION OF AN OLIGONUCLEOTIDE PROBE WITH DUPLEX DN

该项目的目标包括开发一种新的方法来杂交寡核苷酸（ON）到双链DNA （dsDNA）。一个主要的工具是肽核酸（PNA），其中DNA核碱基附着在聚酰胺主链上。一类特殊的PNA分子，双PNA，已知可以有效地和序列特异性地侵入短同嘌呤束的dsDNA，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，两个双pnas （PNA“开启者”）结合在dsDNA上两个紧密相连的位点上，打开它们之间的双螺旋结构。这使得其中一条DNA链可以通过沃森-克里克配对与ON进行局部杂交。这种杂合体，即pd环，必须形成非常有序的结构，特别是因为只有被PNA开启器打开的DNA位点才能与互补探针结合。为了达到该项目的目标，我们将研究pd环的形成效率及其在各种条件下的稳定性。各种结构的pd样回路将被检查和性质的开刀和探针将是不同的。形成pd环的寡脱氧核糖核苷酸，形成pr环的寡核糖核苷酸，以及形成pp环的PNA寡聚物，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。本项目将采用两种主要方法。在可能的情况下，我们将使用凝胶电泳迁移率转移试验。这是基于裸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。更普遍适用的分析将基于通过pd环形成的dsDNA的亲和力捕获。在这个实验中，探针被生物素化，覆盖有链霉亲和素的铁微珠被用于与不携带pd环的对照DNA的dsDNA的探针复合物的磁分离。我们还将使用“分子信标”来监测PD-loop的形成。该项目的主要成果将是开发两种新的dsDNA操作方法：(i) ON/ pnas辅助dsDNA亲和捕获（OPAC），从复杂的dsDNA片段混合物中分离出特定的dsDNA片段；（ii）用探针对dsDNA进行体外和原位杂交。这些技术有望比现有的基于单链（变性）DNA和单链RNA杂交的方法更方便和更具序列特异性。该项目将为发展DNA诊断和分离完整形式的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.