HYBRIDIZATION OF AN OLIGONUCLEOTIDE PROBE WITH DUPLEX DN

Boston University, Boston MA

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Abstract

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.