Hairpin DNAzymes: a new toot for efficient cellular gene silencing
Hairpin DNAzymes: a new toot for efficient cellular gene silencing
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DOI：
10.1002/jgm.1061
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发表时间：
2007-08-01
影响因子：
3.5
通讯作者：
Beeson, David
中科院分区：
文献类型：
--
作者：
Abdelgany, Amr;Wood, Matthew;Beeson, David
背景基于RNA的基因沉默是一种潜在的有效的治疗策略。催化的10-23DNAzyme在Watson-Crick的基础上通过互补的序列臂与靶RNA结合，因此可以被靶向有效地切割特定的mRNA物种。然而，对于体内应用来说，稳定DNAzyme以防止核溶解攻击是必要的。可以在结合臂中引入化学修饰来增加稳定性，但这些修饰可能会改变催化活性，在某些情况下会增加细胞毒性。方法我们设计了新颖的10-23 DNAzyme结构，在DNAzyme结合臂的两端都引入了茎环发夹。体外测定了发夹状DNA酶(HpDNAzyme)对编码肌乙酰胆碱受体(AChR)α亚基的P-32标记的cRNA的催化活性。通过将hpDNAzyme与Bal-31、DNase1或HeLa细胞提取液孵育，检测hpDNAzyme对核溶解降解的抗性。结果hpDNAzyme具有明显的抗核降解作用，在细胞培养系统中，hpDNAzyme远比标准的10-23DNAzyme更有效地下调靶基因的蛋白表达。结论hpDNAzyme提供了一种新的分子工具，无需化学修饰即可在细胞内获得高效的基因沉默，可能具有潜在的治疗应用价值。版权所有(C)2007 John Wiley&Sons，Ltd.
Background RNA-based gene silencing is potentially a powerful therapeutic strategy. Catalytic 10-23 DNAzymes bind to target RNA by complimentary sequence arms on a Watson-Crick basis and thus can be targeted to effectively cleave specific mRNA species. However, for in vivo applications it is necessary to stabilise DNAzymes against nucleolytic attack. Chemical modifications can be introduced into the binding arms to increase stability but these may alter catalytic activity and in some cases increase cell toxicity.Methods We designed novel 10-23 DNAzyme structures that incorporate stem-loop hairpins at either end on the DNAzyme binding arms. The catalytic activity of hairpin DNAzymes (hpDNAzyme) were tested in vitro against P-32-labelled cRNA encoding the muscle acetylcholine receptor (AChR) a-subunit. Resistance of hpDNAzymes to nucleolytic degradation was tested by incubation of the hpDNAzymes with Bal-31, DNase1 or HeLa cell extract. Gene silencing by hpDNAzymes was assessed by measuring reduced fluorescence from DsRed2 and EGFP reporters in cell culture systems, and reduced I-125-alpha-bungarotoxin binding in cells transfected with cDNA encoding the AChR.Results We show that hpDNAzymes show remarkable resistance to nucleolytic degradation, and demonstrate that in cell culture systems the hpDNAzymes are far more effective than standard 10-23 DNAzymes in down-regulating protein expression from target mRNA species.Conclusion hpDNAzymes provide new molecular tools that, without chemical modification, give highly efficient gene silencing in cells, and may have potential therapeutic applications. Copyright (c) 2007 John Wiley & Sons, Ltd.