- What is the concept of ZNA™?
- What synthesis chemistry is used to produce ZNAs™?
- Does my ZNA™ have a phosphate on the 5' end?
- What synthesis scales are offered for ZNA™ oligonucleotides?
- What delivery time can I expect for my ZNA™ orders?
- How is my ZNA™ QCed?
- Are there minimum and/or maximum length limits for ZNA™ oligonucleotides?
- How do I receive my ZNA™ oligonucleotides?
- Solubility of ZNA™ and recommended stock solution concentration
- How stable is my ZNA™, and what storage conditions are recommended?
- What kind of documentation do I get with my ZNAs™?
- Are there any design constraints to be considered for ZNAs™?
- How can the Tm increasing effect of ZNA™ be calculated?
- ZNA™ in comparison with MGB™-conjugated or LNA™-modified oligonucleotides
- What concentration of ZNA™ primers and probes are recommended for PCR applications?
- How can I order metabion ZNA™ oligos?
1. What is the concept of ZNA™?
Oligonucleotides are at the heart of some of the most powerful molecular biology techniques, such as 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.
2. What synthesis chemistry is used to produce ZNAs™?
ZNA™- synthesis follows standard solid phase, machine-compatible phosphoramidite chemistry. Spermine units are chemically introduced during oligonucleotide synthesis using an appropriately protected spermine phosphoramidite.
3. Does my ZNA™ have a phosphate on the 5' end?
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.
4. What synthesis scales are offered for ZNA™ oligonucleotides?
ZNAs™ are delivered in a certain range of yields for increased transparency and easy calculation of the quantity needed and to be expected.
| Scale Guaranteed yield (=), range of yields (><)* in nmol |
|---|
| >/= 5 <10 |
| >/=10 <20 |
| >/=20 <30 |
| >/=30 <50 |
| >/=50 <70 |
| >/=70 please inquire |
*based on oligonucleotides of 8-40 bases in length
5. What delivery time can I expect for my ZNA™ orders?
Prompt processing of any orders is a matter of course at metabion.
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.
In case, an oligo does not pass QC entailing re-synthesis, delivery time will be extended by 1-2 days.
6. How is my ZNA™ QCed?
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.the product will finally be released.
7. Are there minimum and/or maximum length limits for ZNA™ oligonucleotides?
Coupling efficiency during solid phase synthesis is the major limiting factor for 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. Deliberately considering the field of applications for ZNA™, we are routinely offering ZNAs™ in a range of 8 (for primers), and 10 (for probes), respectively, to 40 nucleotides (standard length range).
If your needs are outside this range, don’t hesitate to discuss your projects with our specialists.
8. How do I receive my ZNA™ oligonucleotides?
All ZNA™ oligos will be delivered concentration adjusted (default 100 µM) and dissolved in H2O (pH 7-9).
9. Solubility of ZNA™ and recommended stock solution concentration
Because of their less anionic nature as compared to pure nucleic acids, ZNAs™ 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.
If,- during your experiments -, you have to dry and re-dissolve your ZNA™, TE buffer pH 7.4 is the best recommended solvent. In case of solubilisation difficulties, stepwise addition of 50 mM NH4 OH in 50 µl portions will bring ZNAs™s into solution. Usually, “one drop” (50 µl) shall suffice. Standard stock concentration for ZNAs™ is usually the same as for other PCR primers (100 µM, the delivered concentration). Working solutions (10 µM) or higher dilutions should only be used for a short time and ideally prepared instantaneously prior to application.
10. How stable is my ZNA™, and what storage conditions are recommended?
For ZNAs™ storage at -20 °C in TE buffer pH 7.4 is recommended. For modified – specially fluorescently labelled - oligonucleotides –please avoid or minimize exposure to light because of its 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 fluorescent activity fades away. Therefore, keep highly concentrated aliquots in the freezer, thaw them preferably only once, dilute them just before you use the sample and store the aliquot dark at 4 °C. Under these conditions, the oligos will remain stable for several months, even years. Repeated freeze-thaw cycles must be avoided, as it will denature the oligo itself and affect the integrity of any added label negatively . Avoid the use of distilled water, since pH may be as low as 4-5. Heating ZNA™ in basic buffers induces spermine cleavage and must be avoided.
For general recommendations, please have a look at our Fluorescent Handling Guide.
11. What kind of documentation do I get with my ZNAs™?
On the tube label you will find basic information such as oligo name, name of the person who ordered, 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. Additionally Mass check QC documentation will be provided.
12. Are there any design constraints to be considered for ZNAs™?
Since Z-units do not interfere with hybridization specificity, all general guidelines for designing primers and probes apply. Z-units cannot convert poor oligo design into a well performing oligo! ZNAs™ will improve a given sequence by increased affinity for the complementary sequence mainly because they accelerate hybridization to target through enhanced kinetics.
metabion offers ZNA™ building blocks of 2 - 5 consecutive Z-units for primers and building blocks of 2 – 4 Z-units for probes.
Oligonucleotide sequence and positioning of the cationic units (5’ or 3’) do not affect the general Tm increase induced by the introduction of the respective spermine moiety.
As far as sequence composition is concerned however - make sure that general oligonucleotide design guidelines are paid attention to.
13. How can the Tm increasing effect of ZNA™ be calculated?
The relative Tm increasing effect of ZNA or other duplex stabilizing groups is naturally higher on shorter oligonucleotides used as primers or probes. Hence, we have extended our ZNA length ranges now starting from 8mers for primers and 10mers for dual labeled probes!
The Tm increases significantly and quite linearly with the number of grafted Z units. The approximate Tm of 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
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.
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.
Please note:
There are many different algorithms to calculate the Tm of an oligo. All are just approximations for 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.
14. ZNA™ in comparison with MGB™-conjugated or LNA™-modified oligonucleotides
MGB™ (Minor Groove Binder) and LNA™ 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 there is neither a proper calculation of Tm is possible nor an a priori guarantee can be provided. Their effect is very much dependent on sequence composition and GC content, and each assay has to be optimized. MGB™ is often supported by “Superbases”, which increase affinity.
The very same goes for ZNA, and metabion offers as well 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 and C-5 propynyl-dU (pdU) raising melting temperature by ~1.7 °C per substitution.
Moreover ZNA probes provide broad flexibility in assay design and represent therefore an effective alternative to Minor Groove Binder (MGB)- and Locked Nucleic Acid (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!
15. What concentration of ZNA™ primers and probes are recommended for PCR applications?
ZNAs™ display high sensitivity in PCR applications, because of enhanced kinetics due to facilitated « target scanning » as a consequence of reduced charge repulsion between the single stranded nucleic acids. This is the reason, why ZNA™ primers and probes should be used in lower concentrations than other DNA-oligos. Typical concentrations are 100-200 nM for primers and 200 nM for probes. Even less than 50 nM have been proven to show positive results without losing sensitivity. This may be specially important and favorable for multiplex assays, which - by nature - require a « heavy primer load ».
16. How can I order metabion ZNA™ oligos?
By sending us an e-mail with our excel order file as attachment. Please download this file here and send it to zna@metabion.com.
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.
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 to postmaster@metabion.com.