Thermal stability of quadruplex primers for highly versatile isothermal DNA amplification

- Driving force of QPA comes from the thermal stabilities of primer/template and quadruplex.
- Purine bases incorporated at loop positions significantly destabilize the quadruplex.
- 5′-End dinucleotide extension also destabilizes the quadruplex.

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.

Scheme showing linear QPA, which isothermally amplifies the DNA signal and allows real-time monitoring through incorporated fluorescence nucleotides.