Photomodulating RNA cleavage using photolabile circular antisense oligodeoxynucleotides.
Photomodulating RNA cleavage using photolabile circular antisense oligodeoxynucleotides.
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DOI：
10.1093/nar/gkq079
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发表时间：
2010-06
影响因子：
14.9
通讯作者：
Li Z
中科院分区：
文献类型：
--
作者：
Tang X;Su M;Yu L;Lv C;Wang J;Li Z
笼型反义寡脱氧核苷酸（asODNs）是利用光可切割连接剂将线性寡脱氧核苷酸的两端连接而合成的。其中两个（H30和H40）具有发夹状结构，其热稳定性（ΔTm = 17.5°C和11.6°C）与未固定的相比差异很大。另外三个（C20， C30和C40）没有稳定的二级结构，中间有20个脱氧核苷酸与40-mer RNA互补。所有笼型asODN都有限制开放，这提供了对RNA/asODN相互作用的控制。RNase H实验结果表明，在H30、H40、C30和C40的光激活下，40-mer RNA的酶切量可达到2 ~ 3倍，而C20的酶切量几乎不存在；然而，光激活导致RNA消化增加了20倍。凝胶转移实验表明，完全结合0.02 μM - 40-mer RNA需要>0.04 μM H40和0.5 μM H30， C40和C30需要>0.2 μM和0.5 μM才能完全结合0.02 μM - 40-mer RNA。然而，即使是4 μM C20也不能完全结合相同浓度的40-mer RNA。通过简单地调整笼中asODNs的环大小，我们可以成功地光调节它们与mRNA的杂交以及RNase H在光激活下对靶RNA的水解。
Caged antisense oligodeoxynucleotides (asODNs) are synthesized by linking two ends of linear oligodeoxynucleotides using a photocleavable linker. Two of them (H30 and H40) have hairpin-like structures which show a large difference in thermal stability (ΔTm = 17.5°C and 11.6°C) comparing to uncaged ones. The other three (C20, C30 and C40) without stable secondary structures have the middle 20 deoxynucleotides complementary to 40-mer RNA. All caged asODNs have restricted opening which provides control over RNA/asODN interaction. RNase H assay results showed that 40-mer RNA digestion could be photo-modulated 2- to 3-fold upon light-activation with H30, H40, C30 and C40, while with C20, RNA digestion was almost not detectable; however, photo-activation triggered >20-fold increase of RNA digestion. And gel shift assays showed that it needed >0.04 μM H40 and 0.5 μM H30 to completely bind 0.02 μM 40-mer RNA, and for C40 and C30, it needed >0.2 μM and 0.5 μM for 0.02 μM 40-mer RNA binding. However, even 4 μM C20 was not able to fully bind the same concentration of 40-mer RNA. By simple adjustment of ring size of caged asODNs, we could successfully photoregulate their hybridization with mRNA and target RNA hydrolysis by RNase H with light activation.
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