What are the main routes of RNA synthesis?

RNA is transforming from a "messenger molecule" into a "drug" and a "tool." siRNA with silent genes, ASO for regulated splicing, sgRNA for guiding gene editing, and ordinary single-stranded RNA with various chemical modifications. There are three main routes for RNA synthesis:

• Chemical synthesis, i.e., solid-phase synthesis of phosphorous, suitable for oligonucleotides of about 20–100 nt, with flexible modification;

• For in vitro transcription, DNA templates are added to RNA polymerase such as T7, suitable for longer RNAs such as mRNA and sgRNA;

• Enzymatic ligation and enzymatic synthesis, used for long-chain splicing, cyclization, capping, and tail addition.

   

  

What are the advantages and disadvantages of chemical synthesis, IVT, and enzymatic synthesis?

• The advantage of chemical synthesis is that sequences and modifications can be precisely controlled, making it suitable for siRNA, ASOs, and short-chain RNA with end modifications; The disadvantages are limited length, cost increases with length and number of modifications, and low yield of long chains.

• The advantage of IVT is that it can generate relatively long RNA at once, is relatively low-cost, and is suitable for mRNA and sgRNA; The disadvantage is that the product is highly heterogeneous, may retain DNA templates, enzymes, NTPs, and double-stranded RNA, requiring purification, and modifications can only be introduced by modifying NTPs or post-transcriptional enzymatic methods.

• The advantages of enzymatic synthesis are mild conditions and the ability to introduce special structures; The downside is that efficiency, scale, and sequence universality are still evolving.

What role do common modifications play in RNA synthesis?

RNA modifications can be divided into four categories: sugar modification, skeleton modification, base modification, and end/conjugate modification. GentleGen offers nearly a hundred selectable individual products.

1. 2'-OMe, 2'-F, 2'-MOE, LNA, cEt enhance nuclease stability and target affinity;

2. PS enhances stability and protein binding, but excessive amounts may increase toxicity, complement activation, and coagulation effects;

3. Modifications such as m1Ψ and m5C reduce mRNA immunogenicity and improve translation;

4. GalNAc achieves hepatocyte targeting and is key to hepatic delivery of siRNA and ASO;

5. Fluorescence and biotin are used for detection, enrichment, and imaging.

However, excessive modification may reduce affinity for targets or proteins, affecting RISC loading, RNase H cleavage, Cas9 activity, metabolism, and increasing hepatotoxicity.

GentleGen RNA Synthesis: Nearly a hundred precise and controllable modifications

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GentleGen's one-stop oligonucleotide service platform is not simply a combination of various services, but deeply integrates core capabilities such as target evaluation, sequence design, synthesis purification, active screening, and sequence modification & conjugation, truly achieving a "just imagine and get" R&D experience.

For detailed information about related services, please send an email to marketing@gentlegen.com.