Antisense/PTOs/2‘ OMe RNA
Antisense oligonucleotides represent an excellent tool to inhibit gene expression levels both in vitro and in vivo. Their discovery introduced a new approach to the development of drug therapeutics, since antisense drugs are designed to inhibit the production of disease-causing proteins. This offer the potential to develop highly selective and less toxic drugs.
Modifying antisense oligonucleotides, either as synthetic DNA or RNA, is key to prevent their nuclease-based degradation in cells and to increase their affinity to specific target mRNAs. Phosphorothioate (S-oligos/PTO) is the simplest and most widely used nuclease-resistant chemistry available for antisense applications. It consists in a sulfur atom replacing a non-bridging oxygen in the oligo phosphate backbone.
At high concentrations, phosphorothioates can cause toxicity or other artifacts, due to their greater non-specific protein binding, compared to unmodified phosphodiester oligos. These problems can be solved, in part or completely, by using chimeric designs.
Metabion offers:
- Phosphorothioate (PTOs)/phosphodiester chimera, which typically have a central core of unmodified DNA and one to four S-modified internucleoside linkages at both 5’ and 3’ end. At least 3 PTO bonds at the 5’ and at the 3’-end of an oligonucleotide are generally recommended to prevent degradation by exonucleases. Adding PTO linkages in the central core of your oligonucleotide (thus making a full-PTO oligonucleotide) will additionally prevent endonucleases-related degradation. However, this might also lead to cytotoxicity effects.
- DNA/ 2'-O-Methyl RNA chimera. The presence of 2'-O-Methyl RNA increases both the affinity of the antisense oligo to the target mRNA (by increasing the Tm), and its nuclease resistance. However, 2'-O-Methyl RNA bases do not activate RNase H cleavage, which can compromise the action of the antisense oligo. Therefore, preferred designs incorporate 2'-O-modified RNA in chimeric antisense oligos that retain an RNase H activating domain of DNA (or phosphorothioate DNA).
- Replacement of dC with 5-Methyl-dC, which is particularly useful in the context of CpG motives, since 5-Methyl dC reduces adverse immune responses in vivo, along with moderately increasing the Tm.
An alternative to DNA antisense is RNA interference (RNAi). To order siRNA click here.
metabion recommends that all antisense oligos receive HPLC purification and that oligos undergo a Na+ salt exchange before use in cells or live animals to ensure that salts used in purification are removed. This way, metabion's antisense oligos are ready-to-use for in vivo applications.
metabion’s antisense oligonucleotides have been successfully applied for the splice correction of a deep intronic mutation (1)
(1) Bonifert T., Gonzalez Menendez I., Battke F., Theurer Y., Synofzik M., Schöls L., and Wissinger B.. Antisense Oligonucleotide Mediated Splice Correction of a Deep Intronic Mutation in OPA1. Molecular Therapy Nucleic Acids. 5, 2016, Vol. e390.
We are very happy to offer our customers Phosphorothioates (PTO), 2'O-Methyl RNA, and 5-Me-dC as modifications to DNA oligonucleotides. For synthesis scale and final yield, please refer to the table below. For larger synthesis scales, please inquire.
| Scale (µmol) | Final Yield (OD) |
|---|---|
| 0,2 | 5 |
| 1 | 15 |
| 10 | 100 |
In order to minimize cytotoxicity issues, we recommend using our SEC Cartridge purification service, which is to be intended as an add-on service.
In principle, antisense oligonucleotides can be synthesized as unmodified phosphodiester oligos. However, these are likely to be rapidly degraded in cells by nucleases. Therefore, antisense oligonucleotides are usually synthesized as chimeric oligonucleotides. Metabion offers:
- Phosphorothioate (PTOs)/phosphodiester chimera, which typically have a central core of unmodified DNA and one to four S-modified internucleoside linkages at both 5’ and 3’ end. At least 3 PTO bonds at the 5’ and at the 3’-end of an oligonucleotide are generally recommended to prevent degradation by exonucleases. Adding PTO linkages in the central core of your oligonucleotide (thus making a full-PTO oligonucleotide) will additionally prevent endonucleases-related degradation. However, this might also lead to cytotoxicity effects.
- DNA/ 2'-O-Methyl RNA bases
- Replacement of dC with 5-Methyl-dC, especially in the context of a CpG motif.
Phosphodiester and phosphorothioate 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.
Coupling efficiency is a way of measuring how efficiently the DNA synthesizer is adding new bases to the growing DNA chain. If every available base on the DNA chain reacted successfully with the new base, the coupling efficiency would be 100%. Few chemical reactions are 100% efficient. The industry standard for coupling efficiency during DNA synthesis is around 98,5%, with maximum coupling efficiency obtainable being around 99%. This means that at every coupling step approximately at least 1% of the available bases fail to react with the new base being added. Coupling efficiency is significantly influenced by the quality of raw material (amidites and solutions), instruments and synthesis protocols used.
Please note that Metabion regularly overperforms the industry standards written above, reaching a coupling efficiency of up to 99,7%, even for long unmodified oligonucleotides.
Moreover, Metabion's QC (Quality Control) system ensures that every new batch of chemicals passes strict quality controls. Our machines are serviced by a well organised maintenance program and synthesis cycles are perfectly adjusted to the type of ordered oligo.
Coupling efficiency is important because the effects are cumulative during DNA synthesis. The Table below shows the effect of a 1% difference in coupling efficiency and how this influences the amount of full-length product, following the synthesis of oligos of different length. Considering a relatively short oligo of 20 bases, a 1% difference in coupling efficiency can result in a 15% difference, in terms of full-length final product.
| Effect of coupling efficiency on % full-length product following DNA synthesis | ||||
|---|---|---|---|---|
| Oligo length | Coupling effency of | N. of bases added |
||
| 98% | 99% | 99,5% | ||
| % full length product (crude) | ||||
| 2 | 98% | 99% | 100% | 1 |
| 10 | 83% | 91% | 96% | 9 |
| 20 | 68% | 83% | 91% | 19 |
| 30 | 56% | 75% | 86% | 29 |
| 40 | 45% | 68% | 82% | 39 |
| 50 | 37% | 61% | 78% | 49 |
| 80 | 20% | 45% | 67% | 79 |
| 100 | 14% | 37% | 61% | 99 |
| 120 | 9% | 30% | 55% | 119 |
| 150 | 5% | 22% | 47% | 149 |
| 180 | 3% | 17% | 41% | 179 |
| 200 | 2% | 14% | 37% | 199 |
| 220 | 1% | 11% | 33% | 219 |
The table also shows that the longer an oligo, the lower the yield of full length product that can be expected, due to limitations set by chemistry. Assuming a coupling efficiency of 99% for every single base addition (industry standard is 98.5 % in average), the raw product of a 95-mer synthesis would consist of only 38.5 % full length oligonucleotide. Separating full length and failure sequences from each other by HPLC purification results in additional loss, so that low yields are a normal matter of fact.
Note that metabion regularly exceeds 99 % of coupling efficiency, reaching a coupling efficiency of up to 99,7%, even for long unmodified oligonucleotides.
Every DNA base (in terms of DNA synthesis chemistry, we are speaking of phosphoramidite monomers and amidites) added during DNA synthesis has a dimethoxy-trityl (trityl) protecting group attached to the 5´-hydroxyl position. This acid labile trityl-group is bound to the 5’-end of each support-bound monomer and protects the corresponding base from undergoing unwanted chemical reactions during the synthesis cycle. The trityl-group is removed in the first step of each synthesis cycle, immediately before a new base is added, until the elongation of the nucleotide chain is complete. The final trityl-group is removed before delivery (Unless otherwise requested).
The trityl-group is colorless when attached to a DNA base but it gives a characteristic orange color once removed. The intensity of this color can be measured by UV spectrophotometry and it is directly related to the number of trityl molecules present. Following the first coupling step, the amount of trityl released during deblocking is directly proportional to the amount of full-length oligo synthesized in the previous cycle. When the trityl is cleaved during the deblocking step, the resulting trityl cation is orange in color. The intensity of this color can be measure by UV spectrophotometry. By comparing the intensities of the trityl cation produced after the first and last coupling steps, one can calculate the average successful base coupling per cycle and hence the coupling efficiency.
DNA synthesis is a complicated process, which has improved significantly over the last years. Despite these improvements, all manufacturers have an inherent failure rate. We are constantly developing our processes and systems to minimize these losses; however, it is inevitable that we will occasionally have to re-synthesize some oligos. Please note that metabion performs strict quality controls on each and every oligo synthesized. If an oligo does not pass our quality tests, it will be resynthesized.
The expected average in-house turnover time for antisense/PTOs is 3-4 working days. Please note that we perform strict quality controls on each and every oligo. In case one or more oligos do not pass our quality control, they will need to be resynthesized. This may, of course, result in a delay.
Given the use of antisense oligos in cells and in vivo, high purity is essential.
High pressure liquid chromatography (HPLC) purification is standard (no additional charge) for antisense/ PTOs. For applications in cells and in vivo, we recommend an additional SEC. This is a size exclusion chromatography purification that removes salts and other small molecules, whose quantity and/ or quality can be toxic for cells.
Unless requested, antisense/ PTO are synthesized with neither 3´nor 5´ phosphate. Both, the 5´and the 3´-end will carry a Hydroxyl-group. The 5´ and/or 3’-phosphate is available as a modification at additional charge. (please inquire)
There is a normal degree of variation in the appearance of the supplied dry pellets. Variation in appearance per se does not indicate a quality defect. In general, appearance of unmodified and dye-labeled antisense/ PTO pellets may vary from powdery to hyaloid. The color of unmodified antisense/ PTO pellets may range from transparent over off-white and yellowish to tan. The pellets of labeled antisense/ PTO are colored according to the dye attached.
Purified sterile water, TE or any biological buffers (i.e. with physiological pH) are acceptable as diluents. The recommended diluent volume is 100 µl - 1 ml, the concentration depending on the application to be used and the yield of the resulting product. For application in cells, we do recommend to use sterile water/buffer. The use of a sterile diluent will also increase the stability of your antisense/PTO. For more information, please read FAQ "How stable is my antisense/PTO once I have resuspended it?".
To gain a maximum shelf life for oligonucleotides, samples should generally be stored dehydrated at ≤ -15°C in absence of light. Under the mentioned conditions, samples are stable for at least 6 months. In case of a longer storage period, oligos should be pretested for molecular integrity prior to experimental use. If your dsDNA is resuspended in a sterile diluent, it will be stable at 20°C for several days to weeks, at 4°C for about a month. If stored frozen at –20°C or –70°C, it will remain stable for several months. Repeated freeze-thaw cycles should be avoided, as this will denature the dsDNA. Moreover, the oligo stability in solution depends on the pH. Dissolving an oligo into acidic solutions may result in its degradation. Therefore, avoid the use of non-sterile distilled water as a diluent, since solution pH may be as low as 4-5.
In addition to what above advised, we recommend that you minimize the exposure of modified antisense/PTOs– especially if fluorescently labelled - to light, to avoid any bleaching effect.
Moreover, we recommend storing dye-labelled antisense/ PTOs highly concentrated and not in working dilutions, if you are not planning to use it within 24 hours. The higher the dilution factor, the faster the fluorescent activity fades away. Therefore, try to store highly concentrated aliquots frozen, thaw them only once, dilute them just before you use the probe and store the aliquots at 4°C in the dark.
You may want to consider the following:
| Sequence Length The usual size of an antisense is around 20 bases, which assures good stability and target specificity. |
Sequence Composition
|
| Modification Placement Whenever possible, place modifications at the 5' end. Automated DNA synthesis occurs in the 3' to 5' direction. Each nucleotide addition is 98-99% efficient resulting in 1-2% 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. |
| Purification Method Metabion purifies each and every antisense oligo with HPLC. However, we strongly suggest to additionally purify your antisense/ PTOs with a SEC, to minimize cytotoxicity for in vivo use. |
Metabion is dedicated to reliably deliver high quality products. While every production step is performed in light of achieving best quality, the product is released only if it passes our final inspection. Mass Spectrometry has become the state-of-the-art technology for verifying the integrity of oligonucleotides, and metabion has been the first custom oligo house who introduced routine mass checks into its operations. Each and every oligo is characterized by either MALDI- or ESI-ToF and stringent release criteria are applied.
Mass Spectrometry allows for the most sensitive detection of low-level by-products/impurities such as
- n-1/n-x oligos
- Depurination
- Incomplete Deprotection
- Acrylonitrile adducts
- High Salt Content Identification
Moreover, it is the fastest and most efficient way to identify potential product mix-ups.
We run two different types of Mass Spectrometry (MS) instruments in order to cope best with quality and quantity/throughput issues determined by the specifications of the respective oligo/analyte. While each instrument type precisely characterizes oligonucleotides in terms of composition through direct molecular weight measurement, their field of application is diligently adjusted to suitability considerations.
MALDI-ToF instruments typically have a higher throughput, while the limits of using this technique become manifest, if it comes to analyzing long oligonucleotides, or oligos carrying certain photo-labile modifications (e.g.common quenchers like BHQ®s, Dabcyl used in DLPs).
ESI-ToF is less efficient in terms of throughput but perfectly compensates for resolution issues with long oligos as well as for a potential detrimental laser impact on labile/photosensitive modifications – thus being a "natural" complement to MALDI-ToF analysis.
| Comparison MALDI-ToF and ESI-ToF | ||
|---|---|---|
| Qualification Criteria | MALDI-ToF | ESI-ToF |
| < 60 nts | + | + |
| > 60 nts | - | ++ |
| Photosensitive Modified Oligos | - | + |
| Wobble Oligos | - | + |
| Throughput | ++ | + |
| n-1/n-x Detection | + | + |
| Incomplete Deprotection | + | + |
| Depurination | + | + |
| Mass Accuracy | ++ | ++ |
Synthetic oligonucleotide purification is particularly challenging because of the small differences in size, charge and hydrophobicity between the full-length product and impurities, which often co-elute.
For improved analysis of complex samples like long and/or multiple labeled oligos, metabion offers liquid chromatography (LC) coupled with electrospray ionization mass spectrometry (ESI-MS). The mass spectrometer is connected to a high pressure liquid chromatography (HPLC) system, which allows premium analyte characterization via chromatographical separation, followed by respective molecular weight determination. With this system, the mass of oligonucleotides between 2 and 220 bases can be analysed with high accuracy, resolution and sensitivity. Our expert production team will take care of the method (MALDI or ESI ToF) that best applies to your sample.
Preparative High Pressure Liquid Chromatography (HPLC) deals with isolating the separated components of a sample, and can be done on small-, mid- and large scale operations. In other words, the objective of a preparative HPLC is isolating and purifying a product. Practically, the sample goes from the detector into a fraction collector or it is collected manually.
Analytical HPLC refers to the processes of separating and identifying the components of a sample. It is usually a small-scale process, whose objective is the qualitative and quantitative determination of a compound. The sample goes from the detector into waste.
metabion offers analytical HPLC as an additional (optional) quality control method, complementing our Mass-Check QC, which is performed by default on all our oligos.
For product/quality documentation please see FAQ: What kind of documentation do I get with my RNA oligos?
There are two ways of ordering:
- The preferred way is order transmission through our Web Order Portal for most convenient online shopping.
- You can order by sending us an e-mail with our pre-formatted excel order file as attachment.
DNA Oligo Order WOP download xlsx »
When you write your email, please make sure to address the following questions in the excel template:
• Name of the antisense/PTO?
• Sequence of the antisense/ PTO in 5’-3’ orientation? Please indicate any PTO-bond using a star (*)
• Synthesis scale
• Cartridge Purification, if required
• Delivery form? (dry / in water/ buffer + concentration)
• Modifications? (2'-O-Methyl RNA and 5-Methyl-dC can be selected here)
If you are a new customer, please additionally provide us with
• Your shipping and billing address
• Any other information like Purchase Order number, VAT number (VAT only for customers resident in the EU) etc
In case you opt to transmit orders via email using your own format(s), we need to alert you that above mentioned information is obligatory for processing your order. Due to extra efforts necessary for individual order format transfer into our system, order processing will take longer as compared to preferred web orders and pre-formatted emails.
The label on the antisense/ PTO tube shows basic information like oligo name, name of person who ordered, antisense oligo sequence including modifications, oligo ID, amount of DNA (OD260 and nmol), Tm, and molecular weight.
In addition, you will receive a synthesis report containing more detailed information on the physical-chemical properties of the oligo, such as base composition, base count, purification grade, amount of DNA in nmol, Tm and molecular weight. Antisense DNA/ PTOs are HPLC purified by default and you will also get a printout of the preparative chromatogram.
While each and every oligo produced and delivered is characterized by either MALDI- or ESI-ToF before release, Mass-Check documentation/traces will only be provided if requested at time of order placement. Additional charges apply.
The following terminology is used for differentiating between offered QC options including respective documentation coverage in our order forms and on supporting documents delivered with the products:
Mass Check
Standard quality control performed on each and every oligo. Either MALDI- or ESI-ToF, subject to the "nature of the oligo", and metabion internal procedures. This service is free of charge and no printed/pdf documentation is provided.
MALDI-ToF
Explicitly ordered and performed MALDI-ToF check. Product delivered with MALDI-ToF traces. Additional charges apply.
ESI-ToF
Explicitly ordered and performed ESI-ToF check. Product delivered with ESI-ToF traces. Additional charges apply.
Order Forms
| DNA Oligo Order | download xlsx |
| Also available in our web order portal (WOP) | |
Data Sheets
| Product Data Sheet DNA-Oligos | download PDF |
| Material Safety Data Sheet DNA-Oligos | download PDF |
Credit Card Payment Form
| Credit Card Payment Form | download PDF |