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Polymerase-Endonuclease Amplification Reaction (PEAR) for Production of Oligonucleotides And Its Application in Gene Therapy
Xiaolong WangOcean University of [email protected]
青岛市立医院 2010 年 11 月 5 日
Many diseases are caused by genes
Gene therapy a possible solution
Repairing a damaged good gene
Inhibiting a bad or overexpressed gene
I. Gene knockoutII. Gene Knockdown
1. mRNA1) RNAi2) miRNA3) Antisense oligonucleotides
2. miRNA1) Antisense oligonucleotides2) miRNA sponge
RNAi is effective for mRNA knockdown
A. 将双链 RNA 导入细胞。B, C. 双链 RNA 进入细胞后，在RNA 酶家族中的一个成员 Dicer的作用下，被裂解成 21 到 23 个核苷酸的片段，称为 siRNA 。D. siRNA 双链结合一个核酶复合物从而形成 RNA 诱导沉默复合物（ RNA-induced silencing complex, or RISC ）。E F. RISC 复合物同与 siRNA 互补的 mRNA 结合，使 mRNA 被RNA 酶降解。
How siRNA works?
miRNA is a endogenous small RNA
How miRNA works?
* Antisense oligonucleotide (ASO)
Antisense miRNA oligonucleotide (AMO)
J Weiler et al Anti-miRNA oligonucleotidesGene Therapy (2006) 13, 496–502
Modified ASO
Chemically modified oligonucleotide analogs that have been used as anti-miRNA inhibitors (AMOs).
Locked Nucleic Acid (LNA)
Wahlestedt C et al. PNAS 2000;97:5633-5638
Stability in rat blood serum (A) and RNase H activation (B) of DOR-AS-1 oligonucleotides.
Wahlestedt C et al. PNAS 2000;97:5633-5638
RNase H cleavage of a DOR-2 RNA target sequence with DNA and DNA/LNA copolymers.
Wahlestedt C et al. PNAS 2000;97:5633-5638
SCIENCE VOL 327 8 JANUARY 2010
Chemical Synthetic modified Antisense Oligonucleotides is extremely expensive!
Fomivirsen (Vitravene) — the first and only antisense antiviral drug approved by FDA
Fomivirsen (ISIS 2922)
$63.87 USD
A possible way out:
Polymerase-Endonuclease Amplification Reaction (PEAR) for Enzymatic Production of Antisense Oligonucleotides
X’ R’ X’
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Elongation
dNTPsTaq polymerase
Cleaving
Probe Target
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tura
tio
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PspGI
DenaturationAnnealing
X
Annealing
Denaturation
Denaturation
Annealing(slipping)
Elongation
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Annealing
Annealing
Target:PspGI:
Polymerase:
+++++----HLH--HH--++-+-+-+-
M123456789bp
100
40
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M1234567
Input target concentration (nM) 1 10-1 10-2 10-3 10-4 10-5 10-6
bp
10020
10
Polymerase-Endonuclease Amplification Reaction (PEAR) for Enzymatic Production of Antisense Oligonucleotides
(1). PEAR is a minimal DNA replication system, to study the origin and evolution of repetitive DNA in genome, as well as the origin and evolution of genetic material and life.
(2). The repeat PEAR product DNA can be transferred into cells or organisms to study 1) Function of repeat DNA sequences.2) Repeat Gene Sponges 3) Repeat Gene Missile4) Repeat Gene probe
Other potential applications of PEAR
X’ R’ X’
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X R X R X
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X X
X’ R’ X’
X R X
X’
X’
X’ R’ X’
X R X
X’ R’ X’ R’ X’
X’ R’ X’
X R X
Elongation
dNTPsTaq polymerase
Cleaving
Probe Target
De
na
tura
tio
n
PspGI
DenaturationAnnealing
X
Annealing
Denaturation
Denaturation
Annealing(slipping)
Elongation
Cle
av
ing
+
Annealing
Annealing
Acknowledgements
苟德明 博士、教授 深圳大学特聘教授Assistant Professor, University of Illinois at Chicago
Dr. Shuang-yong Xu, Senior ScientistRestriction EnzymesNew England Biolabs
期限 名称 来源2011~2013 聚合酶 -内切酶扩增反应制备反义寡核苷
酸 (81072567)国家自然科学基金
2011~2013 PEAR制备反义寡核苷酸药物(ZR2010HM056)
省自然科学基金
