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All about oligos impurities and side reactions

Synthetic oligonucleotides have applications in a variety of scientific disciplines, and considerable effort has been put towards their synthetic optimization. However, despite the advancements in oligonucleotide synthesis technology, impurities can still be present in the final product, sometimes presenting unique purification problems due to their chemical similarity to the desired material. These impurities can affect the accuracy and reliability of downstream experiments, and hinder oligonucleotide quantification by optical density, so it is important to be aware of them and take steps to minimize their presence.


A scientist looking at assays results

Coupling reaction failures (called shortmers, n-1) are one of the most commonly seen impurities. During the synthesis process, nucleotide building blocks are added one by one to the growing oligonucleotide chain. If the coupling reaction is incomplete, the 5'-OH from unreacted sequences can couple during the next coupling cycle, leading to truncated sequences. These truncated sequences can interfere with the intended function of the oligonucleotide and produce misleading results in experiments. Due to their very high chemical similarities to the desired product, these shortmers can be particularly challenging to remove using ion-pairing HPLC (IP-HPLC) techniques, and purification using anion exchange HPLC (AX-HPLC) or hydrophilic interactions liquid chromatography (HILIC) methods are recommended in case many shortmers (n-1, n-2, n-3, etc...) are present. The presence of significant levels of shortmers may point out poorly reactive monomers (especially if modified monomers are used) or an ineffective capping step.


Failure sequences that have undergone a capping step, thus containing an acetylated residue at the 5' end, can be difficult to eliminate from the desired material, especially if they are similar in length. To address this issue, a DMT-ON cleavage can be carried out, where the final 5'-DMTr group is not removed before the oligonucleotide cleavage from the resin. This method facilitates the removal of capped sequences as the lack of the terminal DMTr group causes them to elute much faster (via HPLC) compared to the desired product. Additionally, C18-based solid-phase extraction cartridges (SPE) can aid in separating the 5'-DMTr desired product from the capped failure sequences, potentially giving higher recoveries than HPLC-based techniques.


Another common impurity is depurination, which occurs when the glycosidic bond between the nucleobase and the sugar moiety is hydrolyzed, resulting in the loss of the nucleobase, and leaving behind an abasic site. This process is mediated by acid and often occurs during acidic DMTr cleavage. Depurinated oligonucleotides can have reduced hybridization efficiency and stability, affecting the accuracy of experiments such as PCR and sequencing. Depurination can be minimized by using high-quality reagents that are resistant to depurination (dmf-protected guanosine and dibutyl formamidine-protected adenosine), optimizing DMTr-cleavage times to reduce acidic exposure, or using dichloroacetic acid (DCA) instead of TCA.


The formation of guanosine dimers can also be observed in longer sequences. Due to the lower stability of the DMTr group on G compared to other bases, some amount of cleavage can occur during monomer coupling because of the relatively high acidity of tetrazole-based activators. Removal of the 5' protecting group during the coupling cycle leads to the double addition of G residues, yielding sequences that are one nucleoside longer. This side reaction can be minimized by switching the activator to dicyanoimidazole (DCI), which has a lower pKa than tetrazole-based alternatives and limits G deprotections.


A set of HPLC vials containing oligonucleotides samples for analysis

Formation of acrylonitrile (CE) adducts can also be observed in certain cases, resulting from incomplete scavenging of the acrylonitrile-protecting group. In cases where this becomes significant, this side reaction can be eliminated by treating the resin with a tertiary amine (ex. DIPEA) to remove and scavenge acrylonitrile groups before oligonucleotide cleavage.


To minimize the presence of impurities in oligonucleotide synthesis, it is important to use high-quality reagents and equipment, follow optimized protocols, and perform thorough purification steps such as HPLC or PAGE purification. Additionally, regular quality control checks, such as mass spectrometry analysis, can help identify and quantify impurities in the final product. Since some impurities and side products can be challenging to detect by IP-HPLC, it is recommended to perform QC using two different chromatographic methods to ensure all impurities are detected. At Polaris Oligonucleotides, we validate the conformity of all our products by a combination of IP-HPLC-UV and HILIC-MS, to make sure we deliver the highest standards of quality and purity.



The best way to remove impurities that cannot be reduced by synthetic procedures alterations may vary depending on the impurity's nature. HILIC or AX-HPLC approaches are the most effective ways to remove shortmers and G dimers, while IP-HPLC can sometimes be used to remove depurinations and CE adducts.



In conclusion, while oligonucleotide synthesis has become more efficient and reliable over the years, impurities can still be present and impact the accuracy of experiments. By understanding the common impurities in oligonucleotide synthesis and taking steps to minimize their presence, researchers can ensure the reliability and reproducibility of their results.


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