Polymerase-Endonuclease Amplification Reaction (PEAR)

PLoS ONE: new technologies for the enzymatic production of high-purity phosphorothioate oligonucleotides

The principle of PEAR (Flash animation)

In July 4, in the latest issue of PLoS ONE, a new method is reported for the enzymatic mass production of high purity gene drugs — phosphorothioate oligonucleotides. This technology was developed by researchers from Ocean University of China, College of Life Sciences. This research will contribute to the development of oligonucleotide drugs.

Currently, oligonucleotides are manufactured by chemical synthesis, but the chemical synthesis of oligonucleotides involves a multi-step reaction, errors are easy to occur, the purity of the product is low, and purification is difficult. The cost of large-scale synthetic oligonucleotides is very high, greatly limits the oligonucleotide drug research and applications. The corresponding author, Dr. Xiaolong Wang from Ocean University of China explains: “We have developed a thermocyclic reaction for the amplification of oligonucleotides, the Polymerase-endonuclease amplification reaction (PEAR), in which the number of oligonucleotide molecules increases exponentially, and achieves the self-replication of oligonucleotides, like a virus, greatly improve the purity of oligonucleotide products.”

Another author of the paper, Dr. Gang Chen said: “This new oligonucleotide production method have many advantages, it can solve many problems in current oligonucleotide manufacturing industry. PEAR product can be used as ‘seeds’ for the next round of amplification directly without any treatment, without adding new primer and templates, the product oligonucleotides remains high purity after multiple round of amplification. Compared with chemical synthesis, the technology does not require expensive equipment, greatly reduces the cost of production, and the process can be easily scaled up for mass production of modified oligonucleotides. ”

PEAR reaction relies on a unique “slipping and cleaving mechanism”, thanks to the use of thermostable DNA polymerase and thermostable restriction endonuclease, only the target oligonucleotides achieves exponential amplification, while non-targets sequences cannot be amplified owing to the lack of sequence repeats and restriction sites. At present, using PEAR the research group has successfully prepared oligonucleotides modified with thio-, fluoro- and methyl- groups. Compared with the chemical synthesis, PEAR product almost has no failure sequence. Using electrospray ionization liquid chromatography mass spectrometry (ESI/LC/MS) detection, the proportion of the target sequence in the PEAR product content was measured as >99.9%.

Now we have designed and manufactured a large-scale thermocycler, in which the individual volume of PEAR reaction is scaled up to 10×10 mL, and the yield is ca. 0.98 mg per reaction.  We can produce ~1.0 gram of double-stranded oligonucleotides in 100 reactions. So the PEAR technology is ready for true large-scale production of theraputical oligonucleotide.




Background Information: Oligonucleotide is a basic research tool in the field of biomedical and life sciences. The recent developments in oligonucleotide drugs have made some major breakthroughs, and the market of oligonucleotide drug has a very promising future. Antisense oligonucleotides have been developed as targeted gene therapeutics for anti-viral, anti-tumor and treatment of hereditary diseases. CpG oligonucleotide can activate the non-specific immune response, and can be used as immunostimulants and vaccine adjuvants. In January 2013, a major achievements of the ISIS Corporation and Genzyme cooperation, the antisense oligonucleotide drugs KYNAMRO ™ (Mipomersen) has been approved by FDA, and are currently available in the market. Mipomersen is a lipid-lowering oligonucleotide drug for the treatment of familial hypercholesterolemia (HoFH) through antisense inhibition of the mRNA of apolipoprotein B-100.

In modern molecular biology laboratories, nucleic acids are commonly amplified by polymerase chain reaction (PCR) technology , but PCR is not suitable for the large scale preparation of oligonucleotides: PCR cannot directly amplify oligonucleotides, because they are too short (often only 18-30 nucleotides in length). In addition, the yield of PCR product is limited by the input primer concentrations.

At present there have been a number of new nucleic acid amplification methods, but only a few method can be used for the amplification of small oligonucleotides. In 2003, in the PNAS magazine it is reported an exponential amplification reaction (EXPAR) for the amplification of oligonucleotides. In June 2, 2013 in the Nature Methods journal it is reported the mass production of DNA molecules using rolling circle replication or bacterial replication process. However, these methods have not been developed in producing modified oligonucleotides that are useful for drug production. Since natural oligonucleotides are easily degradable in vivo, and have a number of side effects, so as in pharmaceutical oligonucleotides, in order to enhance the in vivo stability, the phosphodiester bond must be modified with a phosphorothioate.

Link to the original article:

(1)  Xuxiang Wang, Jianye Zhang, Yingjia Li, Gang Chen, Xiaolong Wang. Enzymatic Synthesis of Modified Oligonucleotides by PEAR Using Phusion and KOD DNA Polymerases. Nucleic Acid Therapeutics. February 2015, 25(1): 27-34. PubMed PMID: 25517220. doi:10.1089/nat.2014.0513

(2) Biao Li, Shihua Dong, Jiajun Wu, Jianye Zhang, Gang Chen, Quanjiang Dong, Xinhong Zhu, Xiaolong Wang* (2013) Preparation of 5′-O-(1-Thiotriphosphate)-Modified Oligonucleotides Using Polymerase-Endonuclease Amplification Reaction (PEAR). PLoS ONE 8(7): e67558. doi:10.1371/journal.pone.0067558.

(3)  Xiaolong WANG*, Deming GOU, Shuang-yong XU. (2010) Polymerase- Endonuclease Amplification Reaction (PEAR) for Large-Scale Enzymatic Production of Antisense Oligonucleotides. PLoS ONE, 5(1): e8430. doi:10.1371/journal.pone.0008430.