Custom Oligonucleotide Synthesis
Oligonucleotide synthesis for cutting-edge life science
Although the need of custom oligonucleotide synthesis services rises throughout the field of life sciences and drug discovery, providers of high quality custom nucleotides and oligos are still hard to find. Since we have substantial expertise in custom synthesis services and medicinal chemistry without applying automated synthesizers since 1999, our experienced chemists aim for sophisticated chemistry challenges and innovative nucleotide sequences, e.g. with special labels and modifications. Hence, we do not provide standard oligos, but rather custom oligos synthesis services to meet the individual requirements of our client’s research projects. Those oligonucleotides are synthesized with defined sequences and can be modified with chemical structures to meet your scientific needs and can be used in a great variety of molecular biology applications, which range from sequencing, site-directed mutagenesis, and microarrays to PCR, real-time PCR and various therapeutic applications.
Range of custom oligonucleotide modifications and labels
As modified oligonucleotides (DNA or RNA) are key to understand the stereochemical and mechanistic aspects of most biochemical processes, access to custom oligos is indispensable. To meets those needs, Taros provides customized oligos covering a modified backbone, bases, sugar and internucleotidic linkages. Instead of standard nucleotides we rather strive for synthesis of more challenging ones and offer a great variety of modifications, tags and labels including:
- Unnatural and uncommon nucleoside or nucleotide
- Thiophosphate analogues: chiral thiophosphate moiety
- Isotope labelled nucleoside or nucleotide
- Fluorescent marked nucleoside or nucleotide
- Biotinylated and tagged nucleoside or nucleotide
- Cyclic nucleotides, cGMPs
- Different linkers, spacers and PEGs
- Backbone and Cross Linker Modifications
- Base Modifications and inverted Bases
- Photoactivable Labels
Oligonucleotide synthesis for diverse research applications
Since 1999 we offer custom synthesis services with high success rates so we are capable of optimizing the appropriate oligonucleotide synthesis method for your purposes! Our experienced chemists are well prepared to advise you before synthesis and offer support with the experimental design and application. You benefit from our personal customer service with one continuous project manager, who ensures effective transparent communication on the progress of the project as well as on finding solutions for potential problems. We can help you to select which custom oligo or synthetic nucleotide and assist you in selecting the proper nucleotide specifications (e.g. purity and amount of the nucleotide, modifications, analytical options and the overall delivery formats) to meet your request. After each oligonucleotide synthesis service, we offer a routinely analysis of the product by 1H-NMR, 31P-NMR, LC-MS or HPLC to confirm the exact structure, identity and quality of the compound. As a result, our custom oligos are suitable for applications in molecular biology research and therapeutical applications. Taros uses freeze-drying for the gentle drying and stabilization of high-quality products from oligonucleotide synthesis and therefore our lyophilized products can be stored for much longer at the appropriate temperature. For detailed information on our custom chemical services please consider our Taros FAQ – here we highlight key issues for potential customers.
Which method does Taros use for the preparation of target oligonucleotides?
The most common method applied at Taros for the preparation of target oligonucleotides is Phosphoramidite synthesis, in solution or solid-phase manner. For those who are not familiar with this kind of approach, let us explain you this four-step cycle for nucleotide synthesis:
To couple nucleotides in order to form an desired oligo, many manufacturers rely on the well-known and highly optimized phosphoamidite synthesis approach. Here, a solid synthesis platform (e.g. a resin) is used as a base for synthesis. Once the correct initial nucleotide or modification has been fused to the platform, a four-step cycle, in which one nucleotide is added one by one, starts.
Each cycle consists of a deblocking, coupling, capping and oxidation for each addition of A, C, T or G.
In order to perform a nucleotide addition from 3’→5’, an initial removal of the protecting group dimethoxytrityl (DMT) on the 5’ carbon at the receiving oligo is needed. This is achieved by treating the growing oligo with a deblocking reagent which cleaves the protecting group and gives access to a free 5’ hydroxyl group. This hydroxyl group is capable of reacting with the next nucleotide.
Once the 5’ hydroxyl group is accessible, the oligo is ready to couple to the next nucleotide in the oligo sequence. The desired nucleotide is introduced to the reaction and reacts with a weak acid, resulting in a phosphoramidite intermediate. Once this conversion happened, the nucleotide interaction with the unblocked hydroxyl group at the 5’ end of the growing oligo is enabled, resulting in a covalent attachment of the new nucleotide through a phosphite triester bond.
Although this method has been around for quite some time and a lot of optimization has been performed, a 100% accuracy of coupling is not possible to achieve. Even with very pure reagents and precise chemistry, some 5’ hydroxyl groups will not be coupled in the previous step, resulting in a free 5’ hydroxyl group. If this is not blocked in any way, this very reactive group can couple to a new nucleotide in the next cycle, which leads to a sequence containing a deletion. Subsequently, this would give rise to various sequences containing deletions in different parts of the desired sequence. This can be easily avoided by capping the uncoupled molecule to prevent its extension within the next cycles. By removing those half-built sequences in the purification steps that follow the synthesis, we aim to minimize the inevitable presence of truncated molecules within the final product. This way, the process obtains very high coupling efficiencies with low levels of truncated or failed products.
The newly formed phosphite triester bond of the reactive nucleotide sequence needs to be stabilized in the last step of the cycle. This is achieved by adding an oxidizing mixture to the reaction, which oxidized the phosphite into a phosphate, which in turn results in a stable phosphotriester bond between the two nucleotides. Once this is stabilized, the growing oligo is ready to receive a new nucleotide to its sequence in a new cycle.
This cycle repeats as many times as needed to obtain the desired oligonucleotide sequence in the end. Once the last nucleotide is fused to the sequence, the oligos undergo a process which deblocks the whole molecule by removing the remaining protecting groups which are still attached. It also includes the removal of the DMT group that blocks the last nucleotide, as well as various nucleotide-specific protection groups. Once everything is removed, the oligo is cleaved from the solid synthesis platform and collected. Ultimately, the final and functional single-stranded DNA molecule is obtained and can be further processed to purification.