FAQs ZNA primers and probes
Oligonucleotides are at the heart of some of the most powerful molecular biology techniques like PCR, DNA chips and in situ hybridization. Moreover, lots of hope has been pinned for oligonucleotides as a generic, yet very selective, class of drugs if they were able to cross cell membranes. Many chemical modifications have been developed, including thiophosphate, peptide nucleic acid, locked nucleic acid and morpholino oligonucleotides, to improve their properties. Owing to the polyanionic nature of oligonucleotides/nucleic acids, conjugation to a polycationic moiety is the rational approach to decrease charge repulsion and thus improve hybridization properties. Moreover, polycations are known to carry oligonucleotides into cells, hence their conjugates are expected to behave similarly. Various synthetic approaches for introducing cations into oligonucleotides have been described, including phosphate backbone replacement, nucleotide modifications or end-conjugation, for which enhanced hybridization, strand invasion and eventually cell penetration were observed.
Among cation-modified oligonucleotides, those leaving the oligonucleotide moiety intact retain mismatch discrimination and remain substrates of nucleic acid–processing enzymes. Moreover, molecular biology applications require rapid on-demand synthesis of oligonucleotide sequences, and this requirement was not fulfilled by previous synthetic approaches.
By conjugation of spermine derivatives as cationic moieties (Z-units) to – by nature – polyanionic oligonucleotides, a versatile concept to increase affinity of oligonucleotides for their target by decreasing electrostatic repulsions has been developed and proven.
ZNA oligos result from the conjugation of a polycationic moiety to an oligonucleotide. This decreases the charge repulsion between the oligonucleotide and its target sequence, both naturally anionic.
ZNA®- synthesis follows standard solid phase, machine-compatible phosphoramidite chemistry. Spermine units are chemically introduced during oligonucleotide synthesis using an appropriately protected spermine phosphoramidite.
Oligos are made using a DNA synthesizer, which is basically a computer-controlled reagent delivery system. The first base is attached to a solid support, usually a glass or polystyrene bead, which is designed to anchor the growing DNA chain in the reaction column. DNA synthesis consists of a series of chemical reactions.
|I||Deblocking||The first base, attached to the solid support via a chemical linker, is deprotected by removing the protecting group (trityl-group). This produces a free 5´ OH group to react with the next base.|
|II||Coupling||The next base is activated and couples to the 5’-OH-group of the last base of the chain.|
|III||Capping||Any of the first bases that failed to react are capped. These failed bases will play no further part in the synthesis cycle.|
|IV||Oxidation||The bond between the first base and the successfully coupled second base is oxidized to stabilize the growing chain.|
|V=I||Deblocking||The 5´ trityl-group is removed from the base which has been added.|
Each cycle of reactions results in the addition of a single DNA base. A chain of DNA bases can be built by repeating the synthesis cycles until the desired length is achieved.
Unless requested, oligos are synthesized without either 3´or 5´ phosphate. The same goes for ZNA® oligos. The 5´ and/ or 3’-phosphate is available as a modification at additional charge.
ZNAs® 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 5-10nmol, we guarantee to deliver the final product in a yield between 5 and 10 nmols. See the table below:
Guaranteed Yield Range* in nmol
|≥ 5 <10|
|≥ 10 <20|
|≥ 20 <30|
|≥ 30 <50|
|≥ 50 <70|
|≥ 70 please inquire|
*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.
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.
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, and. 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 ZNAs. If the theoretical, expected mass matches the molecular weight determined by the mass check, the product is released.
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 ZNAs® 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.
All ZNA® oligos will be delivered at a set concentration of 100 µM, dissolved in H2O (pH 7-9). For special requests, please inquire.
Because of their less anionic nature as compared to pure nucleic acids, ZNA® may be less soluble than other DNA/RNA-oligos in PCR grade water. ZNA® solubility depends mainly on the ratio of nucleotides (anionic)/ spermines (cationic) (N/S), pH and salt concentration. To avoid solubility issues, ZNA® oligos will be delivered already dissolved by default.
Due to their lower negative charge, compared to regular nucleic acids, ZNA may be less soluble in PCR grade water. The solubility of ZNAs depends mainly on the ratio between nucleotides (anionic)/ spermines (cationic), pH and salt concentration. To avoid solubility issues, ZNA® oligos are delivered already dissolved.
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 ZNAs® 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.
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 towed aliquot at 4 °C in the dark. Under these conditions, the ZNA oligos will remain stable for several months, even years. 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.
On the tube label you will find basic information such as oligo name, name of the person who made the order, oligo sequence including modifications, oligo ID, delivered quantity of DNA and molecular weight. In addition, you will receive a technical data sheet including more detailed information on physico-chemical properties of the oligo like base composition, GC-content, synthesis scale, purification grade, quality control information, etc. You will also receive a printout of the Mass-Check, free of charge.
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 FAQ "Are there guidelines that should be taken into account when designing oligonucleotides?"
Yes, they are as follows:
|Sequence Length - metabion can routinely synthesize DNA oligonucleotides from 5 to 220 bases (see above). Most sequences range from 18 to 30 bases with the average of 24 bases. Remember that the longer the oligonucleotide, the lower the percentage of full length product in the crude synthesis. This results in lower yields after purification.|
|Sequence Composition - Make sure your sequence is free of hairpins and self-complementary regions. Also, more than six of the same consecutive bases (i.e. GGGGGGG) can be problematic and reduce final yields.|
|Modification Placement - Whenever possible, place modifications at the 5' end. Automated DNA synthesis occurs in the 3' to 5' direction. Each nucleotide addition is less than 100% efficient, resulting in a small proportion of the oligonucleotide being truncated and capped at each position. Placing the modification at the 5' end ensures that only the full length oligo is modified. Furthermore, because most modifications are more hydrophobic than unmodified oligonucleotides, the full-length modified oligo binds more tightly to the reverse phase media during HPLC purification. This enhances the separation between the full-length, modified oligonucleotide sequences and the truncated, unmodified oligo sequences.|
|Synthesis Scale - The term "synthesis scale" refers to the amount of derivatized solid support used. The final quantity of product delivered will depend on sequence length, sequence secondary structure, type of modification used, position of modification, number of modifications per oligonucleotide and purification methods used. For further information click here.|
|Purification Method - Choose a purification method on the basis of the level of purity required for your specific application.|
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) (Noir et al., JACS 2008)
z: number of cationic units
N: number of nucleotides
Sequence 5´ATATATAT 3´ 8mer Tm(DNA) = 16 °C
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.
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.
MGB (Minor Groove Binder) PNA (peptide nucleic acids ) 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-, PNA- 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!
ZNA® display high sensitivity in PCR applications, due to enhanced kinetics in target recognition and “scanning”. This is the reason why ZNA® primers and probes should be used in lower concentration, compared to other DNA-oligos. Typical concentrations are 100-200 nM for primers and 200 nM for probes. A concentration even lower than 50 nM have been proven successful in PCR, without lost of sensitivity. This may represent a great advantage in multiplex assays, which use multiple sets of primers.
Please provide us with your email 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.
Secure order facility: If you want to place your order in a secure way, you can encrypt your e-mail or Excel-File with PGP (Pretty Good Privacy). Please request our public key by sending an email email@example.com.
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 (firstname.lastname@example.org).