Designing effective siRNA involves several important factors to ensure high silencing efficiency, specificity, and minimal off-target effects. Below are the key considerations when designing siRNA: 

1. Target Sequence Selection 

   ●    Select an Efficient Target Region: 

Choose a target sequence that is highly conserved among species and is ideally located in the 3' untranslated region (UTR) or coding region to maximize gene silencing. 

   ●    Avoid Off-Target Sequences: 

Ensure that the siRNA sequence does not significantly match other genes in the genome to reduce off-target effects. Tools like BLAST can be used to assess off-target potential. 

   ●    Preferably Avoid Sequence with Secondary Structures: 

Select target sequences that do not form strong secondary structures (such as hairpins), as these can hinder the binding of the siRNA to the mRNA. 

2. siRNA Duplex Design 

   ●    Guide and Passenger Strand Balance: 

The guide strand is the one that binds to the target mRNA, while the passenger strand is degraded. Ensure that the guide strand is selected from the most potent position, usually the middle of the target sequence. 

          ●           The guide strand should have a 5' phosphate, and the passenger strand should have a 3' overhang for efficient loading into the RNA-induced silencing complex (RISC). 

   ●    Strand Stability: 

Ensure a proper balance in the melting temperature (Tm) between the two strands. The 5' end of the guide strand should ideally be more stable than the passenger strand to promote unwinding of the duplex and incorporation of the guide strand into RISC. 

3. siRNA Length 

   ●    Typically 19-23 Nucleotides: 

siRNA is typically designed to be 19-23 base pairs long. This length is optimal for efficient cleavage by the RNA-induced silencing complex (RISC) and minimizes the risk of immune activation. 

4. Chemical Modifications 

   ●    Phosphorothioate Backbone Modification:

Introduce phosphorothioate modifications at the 3' and/or 5' ends of the siRNA to increase stability against nucleases in serum and cellular environments. 

   ●    2'-O-Methyl and 2'-Fluoro Modifications: 

Use these modifications to protect the siRNA from degradation and reduce activation of innate immune responses by Toll-like receptors (TLRs). 

   ●    Cholesterol Conjugation (for in vivo): 

Conjugate cholesterol to the siRNA (typically at the 3' end) to enhance cellular uptake, particularly for liver-targeted siRNAs. 

5. GC Content 

   ●    GC Content of 30-50%: 

Ideally, the GC content of the siRNA should be balanced to ensure optimal stability and efficient hybridization with the target mRNA. Avoid sequences that are too rich in GC, as they may form secondary structures. 

6. Avoid Sequence Homology with Other Genes 

   ●    Minimize Homology with Off-Target Genes: 

Ensure that the siRNA sequence does not have significant homology with unintended genes to minimize off-target silencing effects. This can be assessed through bioinformatics tools to check for off-target sequences. 

7. Avoid Immunostimulatory Sequences 

   ●    Avoid PAMP Motifs: 

Avoid sequences that might trigger immune responses (e.g., patterns recognized by Toll-like receptors or other immune sensors). This can be especially critical for in vivo applications where systemic immune activation could limit efficacy. 

8. 5’ and 3’ End Modifications 

   ●    3' Overhang: 

For efficient incorporation into the RISC complex, siRNAs typically have a 3' overhang of 2–3 nucleotides. This facilitates the unwinding of the duplex and guide strand loading. 

   ●    5' Phosphate Group: 

The guide strand should have a 5' phosphate, which is essential for RISC binding and mRNA cleavage. 

9. Secondary Structure Considerations 

   ●    Minimize Hairpin or G-Quadruplex Formation: 

Avoid sequences that form strong secondary structures, such as hairpins, as these can inhibit efficient siRNA-mRNA hybridization and reduce silencing efficacy. 

10. Target Gene Validation 

   ●    Validate Target mRNA Structure: 

Before siRNA design, ensure that the target mRNA is accessible and that there are no structural elements (like G-quadruplexes) that would prevent siRNA binding. 

Efficient siRNA design involves a careful balance between target specificity, stability, and minimizing immune responses or off-target effects. Key considerations include selecting optimal target sequences, modifying the siRNA backbone for stability, ensuring proper strand orientation and length, and using chemical modifications to improve pharmacokinetics, particularly for in vivo applications. 

GenCefe Biotech provides high-quality siRNA, miRNA, sgRNA, and custom RNA synthesis services. We can design and synthesize RNA Oligos of different lengths, different forms, and with various modifications according to customer’s needs, and also synthesize RNA sequences designed by customers. The RNA products we deliver are all purified by HPLC. ISO 9001-certified facilities and comprehensive quality control reports ensure the delivery of high-quality products.