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Large scale oligonucleotide synthesis case sharing


The success of mRNA vaccine in 2020 makes nucleic acid medicine a hot track in the pharmaceutical industry. It also makes small nucleic acid drugs, which belong to nucleic acid drugs, attract wide attention. In fact, as early as 1998, there were small nucleic acid drugs on the market. Up to now, a total of 15 small nucleic acid drugs have been approved by FDA (2 ASO drugs and 1 aptamer drug have been delisted due to low sales and other reasons). According to statistics, the global market size of small nucleic acid drugs has increased from US $10 million to US $3.25 billion from 2016 to 2021, with a compound annual growth rate of 217.8%. Showing great growth potential.

Although both belong to nucleic acid drugs, the production processes of mRNA and small nucleic acid drugs are quite different. MRNA usually has a large molecular weight, up to thousands of nt. At present, it is mostly prepared by in vitro transcription (IVT) using linear plasmids as templates. Small nucleic acid drugs mainly include antisense oligonucleotide (ASO), small interfering RNA (siRNA), microRNA (miRNA), small activating RNA (saRNA), messenger RNA (mRNA) and RNA aptamer (Aptamer), which are generally less than 100nt, usually 20-30nt. At present, solid phase synthesis is still the main preparation process.

Oligonucleotide synthesis is a multi-step continuous reaction. A cycle mainly includes four steps: Detritification, Coupling, Oxidation, O or S, Capping. One nucleotide monomer is added to each cycle. It is required that the side reactions in the oligonucleotide synthesis reaction should be as few as possible, otherwise the yield of the target product may be very low and the purification will be very difficult. Therefore, the oligonucleotide synthesis efficiency and cycle number (monomer number) of each cycle directly determine the purity and yield of the final product.


Fig. 1 Oligonucleotide synthesis process - showing a cycle

For example, when the efficiency of each step is 99.0% for the same synthesis of 30 base oligonucleotides, the final calculated yield of oligonucleotide synthesis is 74.72%; If the single step yield is reduced to 98.0%, the final yield will be reduced to 55.66%. When the single step synthesis efficiency is 99.0%, the oligonucleotide yield of 10 monomers can reach 91.35%, while the oligonucleotide yield of 100 monomers can only obtain 36.97%. Therefore, the solid phase synthesis process is generally less than 100 monomers, and the synthesis of oligonucleotides with more than 60 monomers is more challenging.


Fig. 2 Calculation of single step coupling efficiency and final yield of oligonucleotides

The key points of solid phase synthesis of oligonucleotides, including the quality of reagents and monomers used, and the synthesis process parameters, are also closely related to the oligonucleotide synthesis equipment used. At the early stage of the development of nucleic acid drugs, Insys keenly found the demand for large-scale nucleic acid synthesizers in relevant markets. Based on its own patented plunger pump technology and research on oligonucleotide synthesis process, it launched the Unique AutoOligo nucleic acid synthesizer series in September 2021. At present, the products have covered AutoOligo25, AutoOligo100, AutoOligo150, and the pilot scale AutoOligo600. Oligonucleotide synthesis amount 10μmol-50mmol

Insys nucleic acid synthesizer products are widely used in the synthesis of DNA, RNA and various modified oligonucleotides after being marketed, and successfully used in the synthesis of 85 base oligonucleotides.

Case sharing of Insys nucleic acid synthesizer:

Case 1: Synthesis of 85 base super long tRNA

The idea of using inhibitory tRNA to treat human diseases can be traced back to about 40 years ago. However, because other therapeutic strategies (such as gene replacement) produced better results in mice at that time, the progress of tRNA therapy research was stagnant. Until 2014, a research team led by Carla Oliveira, a Portuguese cancer geneticist, found that inhibitory tRNA had the potential to treat or prevent hereditary cancer syndrome caused by nonsense mutation. With the rise of nucleic acid drugs, tRNA has also become one of the hot areas of research and investment

Sequence: RNA 85 base

Target molecular weight: 23,890

Synthesis instrument: AutoOligo100 nucleic acid synthesis instrument

Synthetic column: 6.3ml synthetic column

Carrier information: CPG, 1.8g

Synthesis scale: 57 μ mol

Purity: 98% after one-step C18 reverse phase purification


Fig. 3 Mass spectrum of case 1 after one-step purification


Fig. 4 HPLC chromatogram of case 1 after one-step purification

Case 2: 21 base methoxyfluoro RNA synthesis, high synthesis efficiency and high purity

Sequence: RNA 21 base methoxyfluoro

Target molecular weight: 6596.3

Synthesis instrument: AutoOligo600 nucleic acid synthesis instrument

Synthetic column: FineLINE 35 24ml

Carrier information: Nitto Phase ®  HL UnyLinkerTM 250

Synthesis scale: 0.660mmol

Single coupling efficiency: 99.5%

Synthetic purity: 91.268%


Fig. 5 Synthetic chromatogram of Case 2


Fig. 6 Case 2 Synthetic Mass Spectrometry


Figure 7 HPLC Detection Map of Case 2

Case 3: 30 base total thioDNA synthesis

Sequence: DNA 30 base total thio

Target molecular weight: 9670

Synthesis instrument: AutoOligo100 nucleic acid synthesis instrument

Synthetic column: 6.3ml synthetic column

Carrier information: Nitto Phase ®  HL UnyLinkerTM 250

Synthesis scale: 0.175mmol

Single coupling efficiency: 99.4%

Purity: 85.372%


Fig. 8 Synthetic chromatogram of Case 3


Figure 9 Mass Spectrum of Case 3


Figure 10 3HPLC Detection Spectrum of Case 3

Introductions to Insys Nucleic Acid Synthesizer:

Unique AutoOligo Large Scale Nucleic Acid Synthesizer


Unique AutoOligo is a flexible and intuitive fully automatic oligonucleotide synthesizer, which can be used for 10 μ The rapid synthesis of mol - 50mmol nucleic acid sample is suitable for clinical research, medical development and the synthesis of molecular diagnostic probes.

Unique AutoOligo nucleic acid synthesizer has four models:

Unique AutoOligo25, synthesis amount 10 μmol  ~ 100 μmol

Unique AutoOligo100, synthetic volume 50 μmol  ~  9mmol

Unique AutoOligo150, synthesis capacity 150 μmol  ~ 12mmol

Unique AutoOligo600, synthesis volume 1mmol-50mmol.

Unique AutoPure Protein | Nucleic Acid Purification System


Unique AutoPure is a flexible and intuitive protein purification chromatography system, which is used to purify microgram to gram level proteins, polypeptides, nucleic acids, polysaccharides and other target substances.

Unique CDSystem is a powerful, advanced and highly stable chromatographic data management system, integrating functions such as instrument control, method management, data analysis, standard curve, report editing, user authority management, audit tracking, digital signature, network data, etc.