Polymerase-endonuclease amplification reaction (PEAR) for large-scale enzymatic production of antisense oligonucleotides.

Xiaolong Wang,Shuang-Yong Xu,Deming Gou

doi:10.1371/journal.pone.0008430

Abstract

Antisense oligonucleotides targeting microRNAs or their mRNA targets prove to be powerful tools for molecular biology research and may eventually emerge as new therapeutic agents. Synthetic oligonucleotides are often contaminated with highly homologous failure sequences. Synthesis of a certain oligonucleotide is difficult to scale up because it requires expensive equipment, hazardous chemicals and a tedious purification process. Here we report a novel thermocyclic reaction, polymerase-endonuclease amplification reaction (PEAR), for the amplification of oligonucleotides. A target oligonucleotide and a tandem repeated antisense probe are subjected to repeated cycles of denaturing, annealing, elongation and cleaving, in which thermostable DNA polymerase elongation and strand slipping generate duplex tandem repeats, and thermostable endonuclease (PspGI) cleavage releases monomeric duplex oligonucleotides. Each round of PEAR achieves over 100-fold amplification. The product can be used in one more round of PEAR directly, and the process can be further repeated. In addition to avoiding dangerous materials and improved product purity, this reaction is easy to scale up and amenable to full automation. PEAR has the potential to be a useful tool for large-scale production of antisense oligonucleotide drugs.

Highlights

MicroRNAs are a family of short noncoding regulatory RNA molecules
Implementation of polymerase-endonuclease amplification reaction (PEAR) A synthetic oligonucleotide and an antisense probe derived from human microRNA miR-375 were used to validate the proposed reaction mechanism
Antisense oligonucleotides to target miRNAs or mRNA are expected to be capable of curing a wide variety of human diseases, including cancer, obesity, cardiovascular and metabolic diseases [3,4] and viral infections [19]

Summary

Introduction

MicroRNAs (miRNAs) are a family of short noncoding regulatory RNA molecules. The miRNA pathway serves as an important post-transcriptional regulation mechanism [1]. Synthetic antisense oligonucleotides targeting miRNAs or their mRNA targets are proving to be powerful tools for molecular biology research [2] and may eventually find application as new therapeutic agents [3,4]. Large quantities (from multi-grams to kilograms) of a specific oligonucleotide have to be produced for commercial production of antisense oligonucleotide drugs. The development of an economical and safe method for industrial production of short oligonucleotides has become necessary. Micrograms to kilograms of a specific oligonucleotide can be produced in a few hours. Synthetic oligonucleotides are often contaminated with a significant fraction of truncated failure sequences, makes the product purification process difficult. The oligonucleotide synthesis process requires expensive equipments, and costly and hazardous chemicals. An organic solvent (dichloromethane or toluene) must be used to dissolve the deblocking reagent, which arises the problem of disposing chemical wastes

Methods

Results

Conclusion