QUADRUPLEX PRIMING AMPLIFICATION AND HIV-INTEGRASE APTAMER IN THE PRESENCE OF LEAD IONS

The aim of this research was to implement Quadruplex Priming Amplification (QPA) and to perform a spectroscopic study of an HIV-integrase aptamer in the presence of lead ions.

Quadruplex Priming Amplification (QPA) is a simple amplification assay in which isothermal amplification is performed using only a DNA polymerase, and detection is conducted by the intrinsic fluorescence of the primers. QPA employs specific G-rich sequences (G3T) as primers that, upon polymerase elongation at defined temperatures, spontaneously dissociate from the primer-binding sites (PBS) and fold into a monomolecular quadruplex. The G3T sequence is based on a DNA aptamer designed against HIV-1 integrase. Fluorescent nucleotide analogs, such as 3-methyl isoxanthopterin (3MI), when incorporated into these primers, emit light upon quadruplex formation and permit simple, specific, and sensitive quantification without the need for attached probe molecules.

Previously, QPA assays were developed using truncated targets and potassium cations. Here, we designed QPA assays with a truncated target and lead cations within a temperature range of 50-60°C. Lead cations resulted in significantly faster amplification compared with potassium cations. In addition, we performed a spectrophotometric study of the G3T sequence in the presence of lead cations. Lead cations exhibited highly stable quadruplex formation.

The target molecule can be extended with a complementary sequence derived from a pathogen, enabling pathogen detection. QPA can be applied as a simple and inexpensive diagnostic method for point-of-care (POC) applications, as well as for the development of faster amplification strategies, which will make this method more suitable for molecular diagnostics. Although we use low concentrations, given the toxicity of lead, suitable disposal methods are imperative.