Antisense oligonucleotides (ASOs) are short, synthetic strands of nucleic acid designed to modulate gene expression by recruiting binding to complementary RNA sequences. A special type of ASO, the gapmer, features a central DNA „gap“ for RNase H activation, flanked by modified nucleotides such as LNAs or 2′-O-methyl groups to enhance binding strength and stability, while taking full advantage of the degradation resistance provided by phosphorthioate linkages.

We offer high fidelity synthesis services for modified gamper combined with optimal purification methods to ensure the precision and efficacy of antisense oligos for your specific applications.

Length

We are equipped to provide custom gapmer oligo synthesis ranging from 15 to 25 nucleotides, along with all the widely used chemical modification options to enhance stability and specificity for RNA interference applications.

Scales and Yields

The yield of therapeutic oligos depends on the purification method, scale of synthesis, and complexity of modifications. To ensure the highest quality oligonucleotides, we recommend using HPLC for purification.

Please inquire if you have any specific requests on scale or yield.

Modifications

Ella Biotech provides a variety of oligo modification options for ASOs, enabling enhanced stability, specificity, and cellular uptake. Most of our standard modifications are also available for ASOs.

Please refer to the Modification Options For Gapmer page for a list of the most commonly used modifications.

For specialized requirements, please contact us to discuss customized modifications that can optimize gapmer performance in your research.

Backbone Modifications And Directionality

Backbone Modifications

A typical gapmer design includes 3-5 chemically modified nucleotides at each end flanking a central region of 6-10 unmodified DNA nucleotides that support RNase H1 activity. This design provides a balance of stability, potency, and ability to recruit RNase H1 for target RNA cleavage.

The following backbone and sugar modifications are available for custom gapmer ASOs:

Directionality

Regular phosphoramidite based oligo synthesis is done in 3′ -> 5′ direction. Change of synthesis direction offers additional functionality and enables synthetic pathways to modifications otherwise inaccessible. 

Please inquire if you need a modification that is not part of our standard offerings.

Purification

ASO can be ordered with any of our available purification options. Please note that higher purification levels improve quality but result in lower yields, especially for longer oligos. For optimal performance, we recommend HPLC purification, which effectively removes sulfurization by-products and ensures high purity oligonucleotides.

For additional details, please refer to Purification Method in the Resources section.
If you require alternative purification options, please contact us.

Delivery Form and Packaging

We offer oligonucleotides in a variety of packaging formats, including single tubes, multi-well plates, as well as bulk options, in a range of concentrations, solutions, and aliquot options. Custom vial labeling is also available upon request.

* Possible concentration may depend on scale/yield, as low yields at high concentrations result in extremely small volumes that are very difficult to handle, e.g. 5 nmol at 5 mM would result in a volume of 1 µL.

Quality and Quality Control (QC)

To enhance nuclease resistance and improve pharmacokinetics, phosphorothioate (PS) linkages are commonly incorporated in gapmer design. However, these modifications introduce a chiral center at each modified phosphate, leading to a high number of possible diastereomers. For an 18-base gapmer, this results in 17 chiral centers and 131,072 potential diastereomers, complicating purification and quality control.

One of the main challenges in gapmer purification is the variability in how diastereomers interact with RP-HPLC columns. This can lead to peak splitting and broadening, potentially overlapping with n-1/n+1 species, making it difficult to achieve high-resolution purification. In addition, distinguishing the true product from minor impurities using traditional analytical methods is both time consuming and costly.

To overcome these challenges, we do thorough QC via ESI-MS instead of analytical HPLC. Unlike RP-HPLC, ESI-MS provides direct confirmation of the molecular weight and purity of gapmer oligos without interference from chiral variations. This approach allows for convenient impurity assessment and provides clear insight into the nature of any impurities, enables us to provide high-quality gapmers with reliable performance in gene silencing applications.


Additional Services

Production Time and Shipment

Gapmer oligo production time varies with modification complexity, yield requirements, and order volume. Oligos with complex modifications take longer, and higher yields or larger orders may also extend production time.

The typical production time for Gapmer oligo synthesis is 10 days (ranging from 7 to 15 working days) for each 96-well plate synthesis. Similarly, the production time for a 50 mg synthesis is also within the same time frame, averaging 10 days.

We use FedEx for both domestic and international shipments. For temperature-sensitive shipments, we offer either dry ice or blue ice (gel packs).