Phosphorothioate (S-oligos/PTO) is the simplest and most widely used nuclease-resistant chemistry available for antisense applications, often used for both in vivo and in vitro technologies.
The discovery of antisense oligonucleotides, as gene expression level inhibitors, introduced a new approach to the development of drug therapeutics, since antisense drugs are designed to inhibit the production of disease-causing proteins. This offers 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) consists in a sulfur atom replacing a non-bridging oxygen in the oligo phosphate backbone. Chimeric designs can partially or completely overcome toxicity or other artifacts caused by phosphorothioates at high concentrations due to greater nonspecific protein binding than unmodified phosphodiester oligos.
Our chimeric antisense oligonucleotides - Portfolio
- Phosphorothioates (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 purifying all antisense oligos by HPLC followed by a Na+ salt exchange prior to use in cells or live animals to ensure removal of the salts used in the purification. Indeed, this additional SEC purification is a size exclusion chromatography purification that removes salts and other small molecules, whose quantity and/or quality can be toxic for cells. In 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).
We are very happy to offer our customers Phosphorothioates (PTOs), 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.
FAQ – you ask, we answer
#
There are two ways of ordering:
- The preferred way is order transmission through our Web Order Portal for most convenient online shopping (please refer here for further details).
- You can order by sending us an e-mail at oligo@metabion.com with our pre-formatted excel order file as attachment. Download respective Order Form.
When you write your e-mail, please make sure to include the following required information in the excel template:
- Name of the oligo/s.
- Sequence of the oligo/s in 5’-3’ orientation. Please indicate any PTO-bond using a star (*).
- Yield range.
- Purification 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 choose to transmit orders via e-mail using your own format(s), we need to alert you that above mentioned information in bold print 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 e-mails. Please note that only files updated to the latest excel/word version (e.g. .xlsx or .docx) are accepted.
How can you convert files to newer formats?
Open
the file in a recent Office program and save the file in a newer format.
For this go to “File” → “Save as” and choose the newer format from the
“Save as type” dropdown menu.
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).
#
You may want to consider the following:
#
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.
#
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
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.
#
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/spectra 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 printout/PDF documentation is
provided.
MALDI-ToF
Explicitly ordered and performed MALDI-ToF check. Product delivered with
MALDI-ToF spectra. Additional charges apply.
ESI-ToF
Explicitly ordered and performed ESI-ToF check. Product delivered with ESI-ToF spectra. Additional charges apply.
#
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 Size-Exclusion Chromatography (SEC) purification. This is a size exclusion chromatography purification that removes salts and other small molecules, whose quantity and/or quality can be toxic for cells.
#
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.