Antisense Oligonucleotide (ASO) Design and Synthesis Service
Introduction
ASO is a single-stranded oligonucleotide molecule (18~30nt), which can be a heterozygous single strand of DNA, RNA or DNA/RNA. After entering cells, ASO can lead to mRNA degradation under the action of RNase H1 by complementing the target sequence, thus inhibiting protein expression; or through steric effect to achieve the selective splicing of pre-mRNA, regulate the translation of genes, to achieve the purpose of disease treatment.
To assist customers quickly and efficiently enter the primary screening phase with ASO candidate sequences, Creative Biolabs offers specialized custom ASO drug sequence design and synthesis services. Upon receiving the transcription sequence/ID number of the target sequence, species information (human, murine, rattus, non-human primates (NHP), etc.), and the target modification type, Creative Biolabs can provide different design schemes and a variety of control options such as positive control, negative control, scramble control, etc.
Table.1 Information provided by the customer
| Information | Specific content |
|---|---|
| Species | Human, Murine, Rattus, NHP |
| Target Gene | Transcript sequence/Sequence ID |
| Modification | 2'-MOE, PS, etc. |
| Control | Positive control, Negative control, Scramble control |
Highlights of ASO Design
- Provide professional ASO sequence design, comprehensively consider ASO length, GC content, thermodynamic characteristics, specificity, chemical modification and other characteristics;
- Customers can choose different modified bases, purification methods, and synthesis scale to meet the specific experimental needs;
- Supports high-throughput design, ASOs can be designed for multiple transcripts, cross-species design;
- Design according to various mechanisms of action (based on steric hindrance formation mechanism or mRNA degradation mechanism);
- Improved intracellular stability and reduced toxicity through nuclease-resistant modification and flexible chimeric design.
When designing ASO sequence, we mainly refer to the following design steps:
Fig.1 ASO sequence design and screening.
The selection of a screening ASO sequence is a balance of several factors, with different priorities depending on the intended application of the ASO. Screening is mainly to remove nucleic acid sequences that may cause inflammation or other adverse reactions, such as CpG. If cross-species ASOs sequences are intentionally selected, the selection factors depend on the degree of sequence homology and the importance of cross-species homology to the project.
Fig.2 Basic process of ASO selection.
Chemical Modification
Due to the low stability of oligonucleotides outside the cell, easy degradation by nuclease, and difficulty in entering the cell to function, it is essential to ensure that an ASO possess a stable structure and optimal delivery properties during research and development. Creative Biolabs can provide custom designed ASOs with modified phosphate skeleton, base, and sugar ring, so as to improve their stability, binding potency and affinity, and to reduce off-target effect, dose and frequency of administration. At present, the commonly used modification on the market is 5-10-5 2 '-MOE gapmer substitution modification, which supports more than 200 modifications such as LNA, thioate, methylation, 2' -O-Me, etc. Customers can submit specific requirements according to the anticipated effects of the ASO drugs.
Tab.2 Features of modified
| Type | Sites | Nuclease resistance | Affinity | RNase H | Other Features |
|---|---|---|---|---|---|
| PO ASO | —— | × | |||
| PS ASO | Phosphate Skeleton | ↓ | √ | Shorter half life | |
| 2'-MOE | Sugar | √ | ↓ | √ | Gapmer |
| 2'-F | Sugar | √ | × | Uniform modification | |
| MOP* | Phosphate Skeleton | High stability under alkaline conditions | |||
| LNA | Sugar | √ | √ | √ | Gapmer |
| Cholesterol/Fatty acids | Conjugation | Better targeting and uptake |
Fig.3 Different forms of gap substitution.
*MOP: Site-specific replacement of PS linkages at position 2 and 3 from the 5'-side of the DNA gap with MP or MOP linkages can enhance therapeutic index of toxic ASOs. The bigger steric bulk of the MOP group increased chemical stability under basic conditions1.
Fig.4 Several modified structure diagrams.
Synthesis of ASO
ASO is generally synthesized by chemical synthesis methods. The resultant ASO need to be purified and identified to ensure their synthetic quality and purity. In addition, PCR technology and PEAR technology can effectively reduce the production of impurities.
Fig.5 ASO synthesisDistributed under public domain, from Wiki, without modification.
Highlights of Our ASO Synthesis Service
- High purity;
- Stability;
- Strong consistency of purity from batch to batch;
- Good consistency between quantitative batches.
Creative Biolabs have extensive experience in the design and synthesis of oligonucleotides, and with our advanced production facilities and design expertise, we have helped our customers complete many customized orders. Oligonucleotides are rigorously tested by our internal bioanalysis laboratory before delivery, and are characterized by high purity, high safety and high efficiency. If you would like to know more about the design and synthesis of oligonucleotides, please contact us.
Reference
- Crooke, Stanley T., et al. "Antisense technology: A review." Journal of Biological Chemistry 296 (2021).
