Poster abstracts

Poster number 35 submitted by Besik Kankia

Real-time optical assays for monitoring nucleic acid strand-exchange, cleavage and elongation reactions

Besik I. Kankia (Department of Chemistry, The Ohio State University, Columbus, OH 43210), Karin Musier-Forsyth (Department of Chemistry, The Ohio State University, Columbus, OH 43210)

Abstract:
Enzymes processing nucleic acids usually are studied by discontinuous gel-electrophoresis assays using radioactive or fluorescence labeled substrate molecules. Besides the fact that the labels can perturb the reaction and pose a health risk to the investigators, the assays usually involve extra experimental steps: quenching the reaction, separation, visualization and quantification of the products. Here we describe real-time and simple optical assays to monitor nucleic acid strand-exchange, DNA/RNA cleavage and elongation of DNA. The methods take advantage of the property of some guanine-reach oligonucleotides to adopt monomolecular quadruplex conformation in the presence of certain cations. The conformation is characterized by significant absorption in long-wavelength range of the ultraviolet region where usually other secondary structures are transparent. The “signal” oligonucleotide is incorporated into reactant duplex which is released into solution upon an enzyme activity. The release is accompanied by fast quadruplex formation and ultraviolet absorption in long-wavelength range.

Keywords: DNA strand-exchange, DNA cleavage, DNA Quadruplex