ZNA® Oligonucleotides

Zip nucleic acids (ZNA®s) are oligonucleotides conjugated with cationic spermine units (1). These units increase affinity of the ZNA® oligo to its target by decreasing electrostatic repulsion between negatively charged anionic single strand nucleic acids. This results in improved hybridization, thus enhancing and accelerating target recognition (Figure 1; 1; 2).

The possibility of modulating the global charge of the ZNA® oligonucleotide by the number of cationic spermine moieties attached to the nucleic acid oligomer is key to easily predict the melting temperature of ZNA®-DNA/ZNA®-RNA hybrids. Tm increases linearly with the length of the oligocation (Figure 2; 1).

ZNA®s enable specific and sensitive reactions when used as primers for PCR and Reverse Transcription (Figure 3-5). Moreover, ZNA® probes provide broad flexibility in assay design and represent an effective alternative to Minor Groove Binder (MGB)- and Locked Nucleic Acid (LNA)-containing oligonucleotides (3; 4).

The relative Tm increasing effect of ZNA® or other duplex stabilizing groups is naturally higher on shorter oligonucleotides used as primers or probes. Therefore, metabion offers ZNA® length ranges starting from 8mers for primers and 10mers for dual labeled probes!

The number of cationic spermines is a key factor in the oligo design, as a high number of cationic spermines may result in a global charge shift of the oligonucleotide and consequent solubility issues. metabion offers ZNA-2 and ZNA-3 (cationic) building blocks for (anionic) primers ranging from 8 to 15mers, and dual labeled probes ranging from 10 to 17mers, respectively. Attachment of ZNA-4 and ZNA-5 building blocks to primers is allowed from 16mers (ZNA-4), and 20mers (ZNA-5), respectively. Attachment of ZNA-4 and ZNA-5 building blocks to dual labeled probes is allowed from 18mers (ZNA-4), and 22mers (ZNA-5), respectively.

Additional affinity-to-target enhancement of primers and probes while maintaining specificity can be provided by incorporation of our base analogues

C-5 propynyl-dC (pdC) raising melting temperature by ~2.8 °C per substitution

and/or

C-5 propynyl-dU (pdU) raising melting temperature by ~1.7 °C per substitution.

On top of this, metabion also offers 5´ZNA® modified dual labeled probes, introducing certain 5´reporters (e.g. FAM, Fluo, JOE, ROX, TAMRA, CY3, CY5, CY5.5, …) through “click” chemistry.

These probes are not degraded during PCR. Therefore, if appropriately designed, they allow for high resolution, post-PCR melt curve analysis. Please inquire at info@metabion.com.

ZNA® oligonucleotides have been applied successfully to a variety of applications, including antisense technology and in-situ hybridization. Visit our Portfolio for information about how we can assist you with your experiments!

Figure 1. ZNA probes retain full SNP discrimination ability and specificity at higher Tm (modified from 4).

Figure 2. Number of Z-units increases oligo Tm. The Tm increase per conjugated spermine is only dependent on the length of the oligonucleotide (modified from 1).

Figure 3. Improved signal-to-noise ratio using long probes. (A) Model for the greater quenching fluorescence of ZNA probes: the cationic charges of spermine units (Z) stabilize the probe in a coil conformation, reducing the distance (R) between the fluorophore (F) and the quencher (Q). (B) PCR detection with long ZNA dual-labeled probes. Amplifications of target genomic DNA were detected using ZNA hydrolysis probes (red) and their unmodified DNA counterpart (black dotted) containing 22 (circle) and 33 (triangle) bases. Raw fluorescence data is shown (modified from 4).

Figure 4. Conventional gradient PCR. Target or control genomic DNA was amplified following an annealing temperature gradient procedure with DNA-L1 or ZNA-L1 primer pair containing four spermines or five spermines. Final reactions were analysed on 4% agarose gel stained with ethidium bromide (3).

Figure 5. (A) ZNA primers improve the accuracy of low-abundant gene expression measurement. Quantification of target in three independent RT-qPCR reactions. Bars represent the Cq value for each RT reaction and error bars indicate qPCR duplicate standard deviations. (B) Fast cycling PCR. Mean Cq values and standard deviations from quadruplicate reactions carried out with ZNA (red bars), DNA (black bars) or LNA (grey bars) primers as a function of annealing/extension time (modified from 3).

References and links

1. Noir R., Kotera M., Pons B., Remy JS., Behr JP. Oligonucleotide-oligospermine conjugates (zip nucleic acids): a convenient means of finely tuning hybridization temperatures. J Am Chem Soc. 2008 Oct 8;130(40):13500-5.
2. Voirin E., Behr JP., Kotera M. Versatile synthesis of oligodeoxyribonucleotide-oligospermine conjugates. Nat Protoc. 2007;2(6):1360-7.
3. Moreau V., Voirin E., Paris C, Kotera M., Nothisen M., Rémy JS., Behr JP., Erbacher P., Lenne-Samuel N. Zip Nucleic Acids: new high affinity oligonucleotides as potent primers for PCR and reverse transcription. Nucleic Acids Res. 2009 Oct;37(19):e130.
4. Paris C., Moreau V., Deglane G., Voirin E., Erbacher P., Lenne-Samuel N. Zip nucleic acids are potent hydrolysis probes for quantitative PCR. Nucleic Acids Res. 2010 Apr;38(7):e95.
5. ZNA®-FAQs.
6. ZNA® products are sold for research use only. Please refer to our “Trademarks and License” webpage for more information.