Zip nucleic acids (ZNA®s) are oligonucleotides conjugated with cationic spermine units. This asset decreases the electrostatic repulsion between single-stranded nucleic acids (i.e. between primer/probe and target DNA during annealing), therefore increasing the affinity of ZNA® primers and probes to their targets.
The global charge of a ZNA® oligonucleotide-oligocation can be modulated by specifying the number of cationic spermine moieties attached to the nucleic acid oligomer. The Tm of ZNA®increases linearly with the length of the oligocation. Therefore, it is possible to easily predict the melting temperature (Tm) of ZNA®-DNA or ZNA®-RNA hybrids.
Despite sharing similar properties to LNA and MGB in terms of higher target affinity (increased Tm), ZNA® has unique features, such as higher Tm for short probes, which enhances discrimination (SNP genotyping, miRNA detection) and can be used to reduce the cycling time or improve the accuracy of quantification of low abundant transcripts, as well as better quenching for long probes (low fluorescence background and higher signal-to-noise ratio).
ZNA® oligos advantages at a glance
...featuring
- Enhanced/accelerated target recognition
- Increased sensitivity
- High specificity
- Increased Tm for short probes
- Improved quenching for long probes
- Increased flexibility in terms of
- Efficiency at low oligo concentration
- Efficiency at high annealing temperatures
- Mg2+ concentration adjustment
- Design/sequence composition constraints
- Probes for post PCR melting curve analysis
- Enhanced primer/probe sensitivity and specificity through incorporation of base analoga (pdC, pdU)
...providing
- Efficient mismatch/SNP/allelic discrimination
- Earlier Ct values
- Improved quantification accuracy of low abundancy transcripts
- Improved RNA to cDNA conversion
- Higher signal level
- Targeting highly conserved, specific (including A/T-rich) sequences
- Universal cycling conditions due to 'Tm leveling' effects (stabilization of A/T-rich duplexes), hence supporting multiplex assays
- Broad flexibility in assay design and represent therefore an excellent alternative to MGBs and LNAs
...hence qualifying for
a wide range of Nucleic Acid-based applications like
- PCR / real-time PCR/RT-PCR
- Microarrays/Capture probing
- Northern Blot / Dot Blot
- (Fluorescent) In-Situ Hybridization (ISH)
- Blocking of alleles (clamp PCR)
- Next generation sequencing
- Gene silencing, such as DNA antisense and siRNA/RNAi
- Nucleic acid cell delivery, without the use of transfection reagents.
In PCR applications, ZNA®s show high sensitivity as a result of
enhanced target recognition and "scanning" kinetics. In multiplex
assays, which use multiple primer sets, this can be a great advantage.
Our ZNA primers and probes – Portfolio
- Primers as short as 10mers and dual labelled probes with a minimum length of 8 nucleotides/bases, because the relative Tm increase effect of ZNA (or other duplex stabilizing groups) is naturally greater on the shorter oligonucleotides used as primers or probes;
- The possibility to add ZNA®-building blocks, both at the 3’ and at the 5’-end of your DNA sequence;
- Standard length range between 10 and 40 nucleotides for primers and probes. For longer sequences, please inquire;
- For primers, 4 standard ZNA® building block modifications: ZNA-2, ZNA-3, ZNA-4, and ZNA-5!
- ZNA-2 for oligos ≥ 8mers
- ZNA-3 for oligos ≥ 12mers
- ZNA-4 for oligos ≥ 16mers
- ZNA-5 for oligos ≥ 20mers
- For probes, 3 standard ZNA-Quencher (Q*ZNA-X) building block modifications for 3´-labeling (For 5´-ZNA-Reporter labeling, please inquire!); the respective choice of 3´-building blocks will be made automatically by metabion according to the following scheme:
- 3' Q*ZNA-2 for probes ≥ 8mers <14
- 3' Q*ZNA-3 for probes ≥ 14mers <18
- 3' Q*ZNA-4 for probes ≥ 18mers ≤40
- ZNA, alike MGB, is often supported by “Superbases”, which increase affinity. metabion offers Tm modifying bases to complement the use of ZNA primers and probes: i.e., C-5 propynyl-dC (pdC) and C-5 propynyl-dU (pdU), which will further raise the Tm to ~2.8°C and ~1.7°C per substitution, respectively;
- HPLC and documented Mass-Check QC included;
- 5´ZNA modified dual labelled probes, introducing 5´reporters like FAM, Fluo, JOE, ROX, TAMRA, etc (see table below).
With appropriate design, ZNA® probes can be used for high resolution post-PCR melting curve analysis (HRM). Please inquire at info@metabion.com and we will prepare a design for you!
All ZNA® oligos will be delivered dissolved in purified distilled water (pH 7-9), with a concentration of 100 µM (for special requests, please inquire).
*Please note that absorption and emission wavelengths are influenced by the solvent (pH, salt conditions, etc.), oligo sequence and other factors.
- Ban C., Ramakrishnan B., Sundaralingam M. Crystal structure of the highly distorted chimeric decamer r(C)d(CGGCGCCG)r(G).spermine complex-spermine binding to phosphate only and minor groove tertiary base-pairing. Nucleic Acids Res. 1994 Dec 11;22(24):5466-76.
- Trevisan S., Nonis A., Begheldo M., Manoli A., Palme K., Caporale G., Ruperti B., Quaggiotti S. Expression and tissue-specific localization of nitrate-responsive miRNAs in roots of maize seedlings. Plant Cell Environ. 2012 Jun;35(6):1137-55.
- Gagnon KT., Watts JK., Pendergraff HM., Montaillier C., Thai D., Potier P., Corey DR. Antisense and antigene inhibition of gene expression by cell-permeable oligonucleotide-oligospermine conjugates. J Am Chem Soc. 2011 Jun 8;133(22):8404-7.
- Evers MM., Toonen LJ., van Roon-Mom WM. Antisense oligonucleotides in therapy for neurodegenerative disorders. Adv Drug Deliv Rev. 2015 Jun 29;87:90-103.
- Paris C., Moreau V., Deglane G., Karim L., Couturier B., Bonnet ME., Kedinger V., Messmer M., Bolcato-Bellemin AL., Behr JP., Erbacher P., Lenne-Samuel N. Conjugating phosphospermines to siRNAs for improved stability in serum, intracellular delivery and RNAi-mediated gene silencing. Mol Pharm. 2012 Dec 3;9(12):3464-75.
FAQ – you ask, we answer
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By sending us an e-mail with our pre-formatted excel order file as attachment. Please download this file here and send it to zna@metabion.com.
Please provide us with your e-mail address for ensuring an automatic electronic order confirmation right upon synthesis start. In the unlikely case, that you do not receive a confirmation within a few hours from sending your order, please inform us immediately.
Jump on our Web Order Portal, look for the product category "DNA Primer" or “ZNA Probes” under “Custom Synthesis Services”, open the respective order form and enter your oligo. Options provided are self-explanatory. The system shall guide you through the ordering process.
If you want to connect your eProcurement System with our Web Order Portal (e.g. OCI - Open Catalog Interface), please simply contact our Customer Service (customerservice@metabion.com).
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The relative Tm increase induced by ZNA or other duplex stabilizing groups is naturally larger on shorter oligonucleotides used as primers or probes. Hence, we offer ZNA primers as short as 10mers and dual-labelled probes as short as 8mers!
The Tm increases significantly and quite linearly with the number of grafted ZNA® spermine units. The approximate Tm of a ZNA® can be calculated applying the following equation:
Tm (ZNA®) = Tm (DNA) + 36z/(N-3.2) (1)
z: number of cationic units
N: number of nucleotides
Example:
Sequence 5´ATATATAT 3´ 8mer Tm(DNA) = 16 °C
plus
ZNA-2 building block
Tm(ZNA) = 16 + 36*2/(8-3.2) = 31 °C
ZNA-2 modification almost doubles Tm; Tm increase of approx 15°C!
Paying respect to the global charge of the ZNA-oligonucleotide-oligocation conjugates raising solubility issues, we additionally offer 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.
Considering the ZNA solubility issues and the global charge of a ZNA-oligonucleotide conjugate, a ratio of 1 spermine each 4 nucleotides is appropriate. Accordingly, we offer ZNA-4 building blocks for an unmodified 16mer, for example. In case of dual labeled probes, ZNA-4 is most suitable for a 18mer.
The Tm can be additionally increased, while maintaining specificity, by incorporation of our base analogues:
- C-5 propynyl-dC (pdC) raising melting temperature by ~2.8 °C per substitution
- C-5 propynyl-dU (pdU) raising melting temperature by ~1.7 °C per substitution.
Please note:
There are many different algorithms to calculate the Tm of an oligo. All are just approximations of the actual Tm of a specific oligo under specific conditions (salt concentrations, pH, temperature, sequence composition, oligo length and other biophysical/ biochemical parameters and reaction conditions). Optimization is always recommended.
References
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Since Z-units do not interfere with hybridization specificity, all general guidelines for designing primers and probes apply.
Remember that ZNA-units cannot convert poor oligo design into a well performing oligo! ZNA® will improve a given sequence by increasing its affinity to the complementary target sequence mainly due to faster hybridization to the target.
metabion offers ZNA® building blocks of 2 - 5 consecutive ZNA-units spermine units for primers and of 2 – 4 spermine units for probes.
The increase in Tm induced by the introduction of spermine moieties is independent both from the oligonucleotide sequence and from the position of the cationic units (5’ or 3’).
To check your sequence, you can read our FAQ "Are there guidelines that should be taken into account when designing oligonucleotides?".
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All ZNA® oligos will be delivered at a set concentration of 100 µM, dissolved in H2O (pH 7–9). For special requests, please inquire.
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MGB (Minor Groove Binder) and LNA (Locked nucleic acids) bases are known to alter the Tm of a oligo sequence. ZNA® shows similar properties concerning increased affinity to their target (Tm increase), especially when it comes to short sequence probes. Additionally, ZNA® has unique features like the enhanced/accelerated target recognition, which can be used to shorten cycling times or for improved quantification accuracy of low abundant transcripts.
You will find approximations about the Tm increasing effect of MGB and LNA, but an exact calculation of the final Tm is not possible, and cannot be provided a priori. The effect of Tm increase is highly dependent on sequence composition and GC content; therefore, each assay has to be optimized.
MGB is often supported by “Superbases”, which increase affinity.
The same applies to ZNA, and metabion offers Tm modifying bases to complement the use of ZNA primers and probes:
- C-5 propynyl-dC (pdC) raising melting temperature by ~2.8 °C per substitution
- C-5 propynyl-dU (pdU) raising melting temperature by ~1.7 °C per substitution.
In summary, ZNA probes provide broad flexibility in assay design and represent therefore an effective alternative to MGB- and LNA -containing oligonucleotides.
Of course ZNA® will not be the solution for all your needs and applications, nor do MGB or LNA. It is a wonderful and promising technology, which has to be explored. Be a part of it!
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Due to their lower anionic nature (lower negative charge), compared to pure nucleic acids, ZNA® may be less soluble than other DNA/RNA-oligos in PCR grade water. The solubility of ZNA®s depends mainly on the ratio between nucleotides (anionic)/ spermines (cationic), pH and salt concentration. To avoid solubility issues, ZNA® oligos are delivered already dissolved by default..
If during your experiments you have to dry and re-dissolve your ZNA®, we recommend TE buffer pH 7.4. In case of solubility problems, we recommend adding 50 µl of 50 mM NH4 OH stepwise. Normally, the first 50 µl aliquot is already sufficient to bring a ZNA® oligo into solution. Standard stock concentration for ZNA®s is usually the same as for other PCR primers (100 µM is the delivered concentration). Working solutions of 10 µM should only be used for a short time and ideally prepared instantaneously prior to application.
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ZNA®s are delivered in yield ranges for increased transparency and easy calculation of the quantity needed and to be expected. Final yield range is the actual amount that we guarantee to deliver. For example, for a yield range of 5–10 nmol, we guarantee to deliver the final product in a yield between 5 and 10 nmols. See the table below:
*based on oligonucleotides of 8-40 bases in length.
Please note that OD260 values are a measure of total nucleotides´ optical density. Hence, neither purity nor amount of ordered substance are transparently reflected. For simplification and exemplification reasons look at the following:
1 OD of the 20mer 5´CAT CGT ATT CGA TGC TAC GT 3´
translates into approximately 5 nmol.
1 OD of the 40mer 5´CAT CGT ATT CGA TGC TAC GT CAT CGT ATT CGA TGC TAC GT 3´
translates into approximately 2.5 nmol.
Therefore, a 1 OD guaranteed amount of delivered product can vary significantly, while metabion´s commitment to delivered yields in nmol does not allow for ambiguity in terms of what you expect and pay for.
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Coupling efficiency during solid phase synthesis is the major factor that limits length and/or quality of the stepwise built molecule. Moreover, any modifications/labels added to or incorporated into the nucleotide chain affect the integrity of the oligomer. Sequence composition and synthesis scales are contributing factors as well. We routinely offer ZNA®s of 40 nucleotides in length. Minimum standard length is 8 nucleotides (for probes), and 10 nucleotides (for primers).
If your needs are outside this range, don’t hesitate to discuss your projects with our specialists.
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All ZNA® oligos are routinely analyzed by checking and verifying the molecular weight by Mass Check analysis (ESI- or MALDI-ToF) for actual molecular weight determination and comparing the result with to the theoretical and to be expected mass. If practice meets theory applying our strict quality criteria are the basis for final product release.
Mass Check analysis (ESI- or MALDI-ToF) is routinely performed on all ZNA®s. If the theoretical expected mass matches the molecular weight determined by the mass check, the product is released.
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Turnaround time of ZNA® primers (synthesis, purification and QC by Mass-Check) is about 4–5 working days.
Turnaround time of ZNA® probes (synthesis, purification and QC by Mass-Check) is about 5–6 working days.
Shipping time within Europe normally doesn't exceed 1 day. For countries outside Europe, please inquire.
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We recommend to store ZNA®s at –20 °C in TE buffer (pH 7.4). For modified – especially fluorescently labelled-oligonucleotides – please avoid or minimize exposure to light, to avoid any bleaching effect. Also we recommend storing dye-labelled oligos highly concentrated, unless you use the working dilutions within 24 hours. The higher the dilution factor, the faster the fluorescent activity fades away. Therefore, keep highly concentrated aliquots in the freezer, thaw them preferably only once, dilute them just before use. Store the thawn aliquot in the dark. Under these conditions, the ZNA oligos will remain stable for several months. Repeated freeze-thaw cycles must be avoided, as this will denature the oligo and compromise the integrity of any added label. Avoid the use of non-sterile distilled water, since its pH may be as low as 4–5. Heating ZNA® in basic buffers induces spermine cleavage and must be avoided.