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

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 oligo...

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Autores principales:	Xiaolong Wang, Deming Gou, Shuang-yong Xu
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2010
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/384c7d9c97bf43b6bb50c1092c81b81b
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spelling	oai:doaj.org-article:384c7d9c97bf43b6bb50c1092c81b81b2021-11-25T06:26:58ZPolymerase-endonuclease amplification reaction (PEAR) for large-scale enzymatic production of antisense oligonucleotides.1932-620310.1371/journal.pone.0008430https://doaj.org/article/384c7d9c97bf43b6bb50c1092c81b81b2010-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/20062528/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203Antisense 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.Xiaolong WangDeming GouShuang-yong XuPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 5, Iss 1, p e8430 (2010)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Xiaolong Wang Deming Gou Shuang-yong Xu Polymerase-endonuclease amplification reaction (PEAR) for large-scale enzymatic production of antisense oligonucleotides.
description	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.
format	article
author	Xiaolong Wang Deming Gou Shuang-yong Xu
author_facet	Xiaolong Wang Deming Gou Shuang-yong Xu
author_sort	Xiaolong Wang
title	Polymerase-endonuclease amplification reaction (PEAR) for large-scale enzymatic production of antisense oligonucleotides.
title_short	Polymerase-endonuclease amplification reaction (PEAR) for large-scale enzymatic production of antisense oligonucleotides.
title_full	Polymerase-endonuclease amplification reaction (PEAR) for large-scale enzymatic production of antisense oligonucleotides.
title_fullStr	Polymerase-endonuclease amplification reaction (PEAR) for large-scale enzymatic production of antisense oligonucleotides.
title_full_unstemmed	Polymerase-endonuclease amplification reaction (PEAR) for large-scale enzymatic production of antisense oligonucleotides.
title_sort	polymerase-endonuclease amplification reaction (pear) for large-scale enzymatic production of antisense oligonucleotides.
publisher	Public Library of Science (PLoS)
publishDate	2010
url	https://doaj.org/article/384c7d9c97bf43b6bb50c1092c81b81b
work_keys_str_mv	AT xiaolongwang polymeraseendonucleaseamplificationreactionpearforlargescaleenzymaticproductionofantisenseoligonucleotides AT deminggou polymeraseendonucleaseamplificationreactionpearforlargescaleenzymaticproductionofantisenseoligonucleotides AT shuangyongxu polymeraseendonucleaseamplificationreactionpearforlargescaleenzymaticproductionofantisenseoligonucleotides
_version_	1718413673788080128

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

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