siRNA, as a type of RNA interference (RNAi) technology, has demonstrated significant value and potential in the fields of gene silencing and targeted therapy. During the development of siRNA therapeutics, bivalent siRNA has garnered increasing attention due to its unique structural design and functional advantages. However, its special bivalent structure also poses several challenges for the traditional SPOS (Solid-Phase Oligonucleotide Synthesis) method, such as lower purity and yield of bivalent siRNA, and difficulties in scaling up production.
In recent years, Hongene has deeply explored and developed enzymatic ligation synthesis technology, significantly improving the synthesis purity and yield of various complex small nucleic acids, while achieving production scale-up and cost reduction. This article will be divided into two parts. The first part will introduce Hongene's case study on bivalent siRNA synthesis, aiming to demonstrate how enzymatic ligation technology overcomes the purity bottleneck of the SPOS method, facilitating the preparation of next-generation RNAi therapeutics!
01 Synthesis Challenges of Bivalent exNA siRNA
Figure 1, A illustrates the subject of this case study, the bivalent exNA siRNA. This bivalent design consists of two siRNA sense strands linked at their 3'-ends by a TEG (tetraethylene glycol) linker, which enhances its in vivo stability, tissue-targeting capability, and duration of efficacy. Furthermore, the 3'-ends of both antisense strands each contain two exNA modifications. Unlike standard phosphoramidite monomers, the exNA monomer incorporates an extra carbon between the 5'-carbon and the 5'-OH (Figure 1, B). This extension helps the siRNA resist exonuclease degradation.
Although the bivalent structure and exNA modifications offer the aforementioned advantages, they also present the following challenges for the SPOS method. Firstly, the linker connects two sets of 16-nt sense strands in opposite orientations, meaning one set runs 5'-to-3' while the other runs 3'-to-5'. Synthesizing the sense strand via SPOS requires the initial use of reverse phosphoramidites, followed later by standard phosphoramidites. This specific SPOS approach reduces product purity and yield. Secondly, the exNA modifications introduced on the antisense strands, along with PS (phosphorothioate) modifications introduced at all termini, further increase the difficulty of purity control.
02 Hongene's Enzymatic Ligation Technology Empowers and Solves Synthesis Challenges
Figure 2 illustrates Hongene's high-purity enzymatic ligation method for bivalent siRNA. This method requires SPOS synthesis only of fragments of the sense and antisense strands (not the full-length strands). Due to the significant reduction in synthesis length, the purity and yield of these fragments are higher than those of full-length strands. Finally, a ligase connects the individual fragments to form the complete bivalent siRNA.
The specific Ligation synthesis process includes the following steps:
- First, use SPOS to synthesize four fragments separately (Figure 2, A), where fragments 1 and 3 are sense strand fragments, and fragments 2 and 4 are antisense strand fragments.
- Second, perform a one-pot enzymatic ligation of the four fragments. First, the four fragments hybridize with each other to form sticky ends. These sticky ends then pair to create ligation sites for the enzyme (Figure 2B).
- Third, the ligase connects the sites, forming the complete bivalent siRNA (Figure 2C).
In this case, Hongene adopted a purified-to-purified (P2P) scheme. This means SPOS-synthesized fragments are purified before being used for enzymatic ligation, and the bivalent siRNA obtained from ligation undergoes purification again. As shown in Table 1, all four fragments achieved purities of >93% after purification, thereby ensuring high purity of the ligation product.
As shown by the IPRP-HPLC-MS identification results in Figure 3, even the crude product of the bivalent exNA siRNA obtained via enzymatic ligation reached a high purity of 91.9%. After purification, the final product purity was as high as 96.7%, indicating that Hongene's enzymatic ligation technology achieves a very high fragment conversion rate. Furthermore, the final purification step easily removed all unreacted fragments, and no new structurally related impurities were observed during the ligation process.
03 The "Winning" Keys of Enzymatic Ligation Technology
In this case, the enzymatic ligation synthesis of bivalent exNA siRNA demonstrated the following characteristics and advantages:
- Higher purity of bivalent exNA siRNA. Compared to using SPOS alone (80-85%), enzymatic ligation synthesis achieved final product purity above 95%.
- Higher yield. Although the P2P scheme achieved only a 19% yield, adopting a crude-to-purified (C2P) scheme can double the final yield without significantly sacrificing purity (>90%).
- Lower cost. The crude-to-crude (C2C) scheme under research by Hongene can eliminate column purification, further reducing production costs. Additionally, enzymatic ligation significantly reduces the consumption of reagents required for SPOS.
- Scalable production. Hongene has already achieved kilogram-scale process amplification for Ligation technology, which is compatible with single-use reactor systems.
- High suitability for siRNA. Typically, enzymatic ligation synthesis requires preparing additional DNA templates to assist fragment pairing. However, the sense and antisense strands of double-stranded siRNA serve as templates for each other, thus eliminating the need for DNA templates and DNase digestion steps, as well as the annealing step in siRNA synthesis.
04 Enzymatic Ligation Technology Leads the Iteration of Small Nucleic Acid Synthesis
With the continuous advancement and expanding application scope of small nucleic acid technology, structurally complex small nucleic acid drugs like bivalent siRNA are poised for breakthroughs in multiple fields. Enzymatic ligation technology, with its unique jigsaw-puzzle-like synthesis approach, will become a new-generation synthesis platform meeting the dual demands of industrial-scale production and clinical quality.
Hongene continues to deepen its expertise in enzymatic ligation technology. Beyond establishing the synthesis process for bivalent siRNA, it has also proposed a two-step enzymatic ligation method applied to sgRNA synthesis (details in the next part). In the near future, we believe enzymatic ligation technology will become an indispensable part of the CMC system for small nucleic acid drugs, aiding novel small nucleic acid therapeutics on their path to industrialization and broader accessibility!
Currently, the enzyme-based synthesis process technology independently developed by Hongene has taken the lead globally in achieving kilogram-scale industrial production. Even more significantly, the world's first siRNA drug utilizing an enzymatic process supported by Hongene has been approved for clinical trials in China!