Chemoenzymatic ligation is a promising approach for oligonucleotide and siRNA API manufacture, offering enhanced purity, increased yield, and reduced environmental impact. This innovative technology provides an optimal solution to meet the anticipated surge in demand for siRNA drugs over the coming decade. 🧬𝗪𝗲 𝘂𝘀𝗲 𝘁𝘄𝗼 𝗿𝗼𝗯𝘂𝘀𝘁 𝗺𝗲𝘁𝗵𝗼𝗱𝘀 𝗼𝗳 𝗼𝗹𝗶𝗴𝗼𝗻𝘂𝗰𝗹𝗲𝗼𝘁𝗶𝗱𝗲 𝗹𝗶𝗴𝗮𝘁𝗶𝗼𝗻 1️⃣Firstly, so-called '͟𝘀͟𝘁͟𝗶͟𝗰͟𝗸͟𝘆͟ 𝗲͟𝗻͟𝗱͟'͟ 𝗹͟𝗶͟𝗴͟𝗮͟𝘁͟𝗶͟𝗼͟𝗻͟ involves joining of hybridized oligonucleotide fragments that have complementary single-stranded overhangs (sticky ends) at their termini. Sticky end ligation is highly adaptable and can be performed with different numbers of fragments of various lengths depending on the process and strategy. It is particularly well-suited for the synthesis of siRNA. 👉 For siRNA, Hongene's strategy is to ligate four fragments (2 for each strand), then purify by non-denaturing chromatography purification. This obviates the requirement for separate purification of each siRNA strand and simplifies the manufacturing process. 2️⃣Secondly, oligonucleotides can be synthesized by 𝘀͟𝗽͟𝗹͟𝗶͟𝗻͟𝘁͟𝗲͟𝗱͟ 𝗹͟𝗶͟𝗴͟𝗮͟𝘁͟𝗶͟𝗼͟𝗻͟ where multiple oligonucleotide fragments and corresponding reverse complement splints are hybridized, and then enzymatically ligated. Splinted ligation is modular and with the appropriate design of fragments and templates can be applied to synthesize oligonucleotides including sgRNA and long prime editing guide RNA (pegRNA) with high purity and yield. 👉For sgRNA Hongene's strategy involves the use of DNA splints which are convenient to produce and highly effective. Following ligation of hybridized oligonucleotide fragments, the splints can be removed by chromatography under denaturing conditions or enzymatically under the action of DNase I to furnish the pure sgRNA. Check out our on-demand video to learn more about our experience in using ligation to synthesize siRNA and sgRNA. https://lnkd.in/ehU7T5un #oligonucleotides #RNA #siRNA #sgRNA #RNAtherapeutics #geneediting #chemoenzymaticligation #oligo #drugdevelopment
