Zip nucleic acids are potent hydrolysis probes for quantitative PCR

Area of Science:

• Molecular Biology
• Nucleic Acid Chemistry

Background:

• Zip nucleic acids (ZNAs) are modified oligonucleotides with enhanced target affinity due to conjugated cationic spermine units.
• Previous studies demonstrated ZNA utility as primers in polymerase chain reaction (PCR) and reverse-transcription reactions.

Purpose of the Study:

• To investigate the application of ZNAs as quantitative PCR (qPCR) hydrolysis probes.
• To evaluate the impact of ZNA modification on probe performance, including nucleotide discrimination, stability, and genotyping accuracy.

Main Methods:

• Utilized ultraviolet (UV) duplex melting analysis to assess ZNA probe stability and nucleotide discrimination.
• Compared the performance of ZNA probes with standard probes in single-nucleotide polymorphism (SNP) genotyping assays.
• Evaluated background fluorescence and signal levels of ZNA probes versus conventional probes.

Main Results:

• ZNA probes maintained nucleotide discrimination abilities and mismatch destabilization patterns similar to standard oligonucleotides.
• The increased stability of ZNA probes enabled the use of shorter probes for improved SNP genotyping.
• Longer ZNA probes exhibited reduced background fluorescence, leading to enhanced sensitivity and signal output compared to standard probes.

Conclusions:

• ZNA probes are effective for quantitative PCR hydrolysis assays, offering improved performance over standard probes.
• ZNA technology provides flexibility in assay design and serves as a viable alternative to other modified nucleic acids like minor groove binders and locked nucleic acids.