Light-activated RNA interference using double-stranded siRNA precursors modified using a remarkable regiospecificity of diazo-based photolabile groups.
Light-activated RNA interference using double-stranded siRNA precursors modified using a remarkable regiospecificity of diazo-based photolabile groups.
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DOI：
10.1093/nar/gkp415
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发表时间：
2009-07
影响因子：
14.9
通讯作者：
Friedman SH
中科院分区：
文献类型：
--
作者：
Shah S;Jain PK;Kala A;Karunakaran D;Friedman SH
基于重氮基的耐光性基团的前体已被广泛用于修饰核酸，目的是用光来切换生物过程。这些过程包括转录、翻译和RNA干扰。在这些情况下，耐光性基团通常被描述为修饰RNA和DNA的磷酸骨架。在这项工作中，我们发现这些重氮基试剂实际上与主链磷酸盐反应很差。相反，它们表现出对末端磷酸盐的显著专一性和非常温和的核苷酸碱基修饰。此外，这些末端修饰的光解保护比碱基修饰的位点容易得多。在这项研究中，我们使用RNA双链和模式核苷酸来表征这种区域特异性，并使用LC/MS/MS进行分析。我们还将这种对区域特异性的理解应用于我们的光激活RNA干扰(LARI)技术。我们检测了27聚体siRNA双链前体(‘dsRNA’)，并用光可裂解的二甲氧基硝基苯乙基(DMNPE)对它们进行了修饰。通过在dsRNA中掺入末端磷酸盐，我们能够引导DMNPE在这些末端位置反应。这些修饰的dsRNA双链显示了比我们之前描述的DMNPE修饰的siRNA更好的性能，可以通过光切换的表达范围增加了两倍。
Diazo-based precursors of photolabile groups have been used extensively for modifying nucleic acids, with the intention of toggling biological processes with light. These processes include transcription, translation and RNA interference. In these cases, the photolabile groups have been typically depicted as modifying the phosphate backbone of RNA and DNA. In this work we find that these diazo-based reagents in fact react very poorly with backbone phosphates. Instead, they show a remarkable specificity for terminal phosphates and very modest modification of the nucleobases. Furthermore, the photo deprotection of these terminal modifications is shown to be much more facile than nucleobase modified sites. In this study we have characterized this regiospecificity using RNA duplexes and model nucleotides, analyzed using LC/MS/MS. We have also applied this understanding of the regio-specificity to our technique of light activated RNA interference (LARI). We examined 27-mer double-stranded precursors of siRNA (‘dsRNA’), and have modified them using the photo-cleavable di-methoxy nitro phenyl ethyl group (DMNPE) group. By incorporating terminal phosphates in the dsRNA, we are able to guide DMNPE to react at these terminal locations. These modified dsRNA duplexes show superior performance to our previously described DMNPE-modified siRNA, with the range of expression that can be toggled by light increasing by a factor of two.
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影响因子：
3.4
作者：
Nguyen, Quan N.;Chavli, Rajesh V.;McMaster, Gary K.
通讯作者：
McMaster, Gary K.
影响因子：
4.8
作者：
Monroe, WT;McQuain, MM;Haselton, FR
通讯作者：
Haselton, FR
影响因子：
14.9
作者：
Amarzguioui, M;Holen, T;Prydz, H
通讯作者：
Prydz, H
影响因子：
--
作者：
Blidner, Richard A.;Svoboda, Kurt R.;Monroe, W. Todd
通讯作者：
Monroe, W. Todd
影响因子：
14.9
作者：
Chaulk, SG;MacMillan, AM
通讯作者：
MacMillan, AM