title: Thermal stability of quadruplex primers for highly versatile isothermal DNA amplification.
abstract: Quadruplex priming amplification (QPA) allows isothermal amplification of nucleic acids with improved yield and simplified detection. This assay is based on a DNA quadruplex, GGGTGGGTGGGTGGG (G3T), which in the presence of specific cations possesses unusually high thermal stability. QPA employs truncated G3T sequences as primers, which upon polymerase elongation, self-dissociate from the binding site and allow the next round of priming without thermal unfolding of amplicons. The rate of amplification strongly depends on the thermal stability of the primer/primer binding site (PBS) complex and to date QPA has been demonstrated to work over a narrow temperature range. To expand the capabilities of QPA, in the present study, we studied the fold and thermodynamic properties of the wild-type G3T and variants containing sequence modifications or extensions at the 5'-end. Circular dichroism studies demonstrate that the substitution of thymidines by other nucleotides or GC addition at the 5'-end does not change the parallel fold of G3T. Thermal unfolding experiments revealed that purine bases incorporated at loop positions and 5'-end dinucleotide extension significantly destabilize the quadruplex, while loop pyrimidines have almost no effect. Overall, the results of these studies suggest that linear isothermal QPA can be performed over a wide temperature range to accommodate both thermophilic and mesophilic DNA polymerases.
authors: Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA.
authors: Jordan
authors: J
authors: Mathias
authors: Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA.
authors: Robert
authors: R
authors: Okyere
authors: Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA.
authors: Levan
authors: L
authors: Lomidze
authors: Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA.
authors: David
authors: D
authors: Gvarjaladze
authors: Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA.
authors: Karin
authors: K
authors: Musier-Forsyth
authors: Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA. Electronic address: kankia.1@osu.edu.
authors: Besik
authors: B
authors: Kankia
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000235:genetics*; D017931:DNA Primers / Q000737:chemistry* / Q000235:genetics; D054856:G-Quadruplexes; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009690:Nucleic Acid Conformation; D013696:Temperature; D013816:Thermodynamics
chemical_list: D017931:DNA Primers; D004247:DNA
keywords: DNA quadruplexes; DNA thermodynamics; Quadruplex priming amplification; Thermal unfolding