Abstract
Antisense oligonucleotides (ASODNs) have been widely used as an important tool for regulating gene expression, and developed into therapeutics. Natural ODNs are susceptible to nuclease degradation, nucleic acid analogues, however, have less side effects, stronger stability and more potent activities. Large-scale de novo synthesis of a certain oligonucleotide has been very difficult and costly. In a previous preliminary study, we developed the polymerase-endonuclease amplification reaction (PEAR) for amplification and large-scale preparation of natural antisense ODNs. Here we extended the method in preparation of a widely used modified oligonucleotide with 5′-O-(1-Thiotriphosphate) modifications. Using electrospray ionization liquid chromatography mass spectrometry (ESI/LC/MS) detection, the purity of the PEAR product was measured as high as 100.0%. Using PEAR a large amount of a specific oligonucleotide can be produced starting from a small amount of synthetic seeds. It is suggested that PEAR can be a useful tool for large-scale production of modified oligonucleotides.
Highlights
Synthetic oligonucleotides (ODNs) have been widely used as an important tool for regulating gene expression, and developed into drugs for gene therapy, especially antisense oligonucleotides (ASODNs) [1] and CpG oligonucleotides (CpG-ODNs) [2]
In 2010, Lanford treated chronically infected chimpanzees with a locked nucleic acid (LNA) modified oligonucleotide (SPC3649) complementary to miRNA miR-122 leads to long-lasting suppression of hepatitis C virus (HCV) viremia, with no evidence of viral resistance or side effects in the treated animals [11]
The ultimate maximum yield of modified polymerase-endonuclease amplification reaction (PEAR) products, ca. 200 ng/mL, is basically equivalent to that of the natural ones, which is not limited by the initial concentration of the target and the template, but by the concentration of substrates
Summary
Synthetic oligonucleotides (ODNs) have been widely used as an important tool for regulating gene expression, and developed into drugs for gene therapy, especially antisense oligonucleotides (ASODNs) [1] and CpG oligonucleotides (CpG-ODNs) [2]. ASODNs are used to inhibit the expression of pathogenic or viral genes by targeting their transcripts, including messenger RNA (mRNA) or microRNA (miRNA). Modarresi and his colleagues reported that inhibition of a natural antisense transcript (NAT), BDNF-AS, by ASODNs or siRNAs can transiently and reversibly upregulate the expression of a specific gene, brain-derived neurotrophic factor (BDNF), leads to increased protein levels and induces neuronal outgrowth and differentiation both in vitro and in vivo [3]. In 2010, Lanford treated chronically infected chimpanzees with a locked nucleic acid (LNA) modified oligonucleotide (SPC3649) complementary to miRNA miR-122 leads to long-lasting suppression of hepatitis C virus (HCV) viremia, with no evidence of viral resistance or side effects in the treated animals [11]
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