Zip nucleic acids are potent hydrolysis probes for quantitative PCR

Abstract

Zip nucleic acids (ZNAs) are oligonucleotides conjugated with cationic spermine units that increase affinity for their target. ZNAs were recently shown to enable specific and sensitive reactions when used as primers for polymerase chain reaction (PCR) and reverse-transcription. Here, we report their use as quantitative PCR hydrolysis probes. Ultraviolet duplex melting data demonstrate that attachment of cationic residues to the 3' end of an oligonucleotide does not alter its ability to discriminate nucleotides nor the destabilization pattern relative to mismatch location in the oligonucleotide sequence. The stability increase provided by the cationic charges allows the use of short dual-labeled probes that significantly improve single-nucleotide polymorphism genotyping. Longer ZNA probes were shown to display reduced background fluorescence, therefore, generating greater sensitivity and signal level as compared to standard probes. ZNA probes thus provide broad flexibility in assay design and also represent an effective alternative to minor groove binder- and locked nucleic-acid-containing probes.

Figure 1.

Identical mismatch discrimination of standard DNA and ZNA oligonucleotides for various mismatches located along the duplex. (A) Sequences of target and probe strands used in UV melting experiments. (B) ΔT_m of standard DNA (white triangles) and ZNA oligonucleotide (black circles) as a function of mismatch position.

Figure 2.

PCR detection and SNP discrimination with short ZNA dual-labeled probe, and comparison with other modified probes. (A) Serial dilution amplifications of target genomic DNA (10 ng to 10 pg) were detected using ZNA short hydrolysis probe (red lines) and its unmodified DNA counterpart (black dotted lines). (B) SNP discrimination. Amplification of 10 ng of wild-type and Leiden genomic DNA were detected using the short ZNA probe (red circle) and a longer unmodified DNA probe. The probes were respectively 17 and 22 bases in length. (C) Amplification of wild-type genomic DNA and detection using short unmodified, ZNA-, MGB- and LNA-containing probes. All probes have identical sequence.

Figure 3.

PCR detection with long ZNA dual-labeled probes. Amplifications of 10 ng of target genomic DNA were detected using ZNA hydrolysis probes (red) and their unmodified DNA counterpart (black dotted) containing 22 bases (circle) and 33 (triangle). (A) Normalized data by the instrument’s software. (B) Raw fluorescence data. (C) Model for the greater quenching fluorescence of ZNA probes: the cationic charges of spermine units stabilize the probe in a coil conformation, reducing the distance R between the fluorophore and the quencher.

Figure 4.

The 5′ nuclease assay using low concentration of ZNA and standard primers. Target genomic DNA (10 and 0.1 ng) was amplified with either ZNA primers (A) or standard primers (B). Reactions were performed with 200 nM (thin lines) and 25 nM (dotted lines) of primers, 200 nM of ZNA-22 hydrolysis probe and JumpStart Taq ReadyMix (Sigma–Aldrich).