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.
| Smolina, Irina V; Kuhn, Heiko; Lee, Charles et al. (2008) Fluorescence-based detection of short DNA sequences under non-denaturing conditions. Bioorg Med Chem 16:84-93 |
| Kuhn, Heiko (2007) A nonisotopic assay for unambiguous assignment of DNA glycosylase functionality. Anal Biochem 366:105-7 |
| Liang, Xingguo; Kuhn, Heiko; Frank-Kamenetskii, Maxim D (2006) Monitoring single-stranded DNA secondary structure formation by determining the topological state of DNA catenanes. Biophys J 90:2877-89 |
| Smolina, Irina V; Demidov, Vadim V; Soldatenkov, Viatcheslav A et al. (2005) End invasion of peptide nucleic acids (PNAs) with mixed-base composition into linear DNA duplexes. Nucleic Acids Res 33:e146 |
| Protozanova, Ekaterina; Yakovchuk, Peter; Frank-Kamenetskii, Maxim D (2004) Stacked-unstacked equilibrium at the nick site of DNA. J Mol Biol 342:775-85 |
| Abibi, Ayome; Protozanova, Ekaterina; Demidov, Vadim V et al. (2004) Specific versus nonspecific binding of cationic PNAs to duplex DNA. Biophys J 86:3070-8 |
| Kuhn, Heiko; Cherny, Dmitry I; Demidov, Vadim V et al. (2004) Inducing and modulating anisotropic DNA bends by pseudocomplementary peptide nucleic acids. Proc Natl Acad Sci U S A 101:7548-53 |
| Demidov, Vadim V (2003) PNA and LNA throw light on DNA. Trends Biotechnol 21:4-7 |
| Kuhn, Heiko; Hu, Yongbo; Frank-Kamenetskii, Maxim D et al. (2003) Artificial site-specific DNA-nicking system based on common restriction enzyme assisted by PNA openers. Biochemistry 42:4985-92 |
| Protozanova, Ekaterina; Demidov, Vadim V; Nielsen, Peter E et al. (2003) Pseudocomplementary PNAs as selective modifiers of protein activity on duplex DNA: the case of type IIs restriction enzymes. Nucleic Acids Res 31:3929-35 |
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