Detection of human immunodeficiency virus RNAs in living cells using Spinach RNA aptamers.
Detection of human immunodeficiency virus RNAs in living cells using Spinach RNA aptamers.
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DOI：
10.1016/j.virusres.2016.11.031
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发表时间：
2017-01-15
期刊：
影响因子：
5
通讯作者：
Margolis DM
中科院分区：
文献类型：
--
作者：
Burch BD;Garrido C;Margolis DM
目前许多用于测量细胞中HIV RNA产生的技术都存在局限性，包括高背景信号或可能破坏细胞环境。荧光团结合RNA适体有可能在对细胞扰动最小的情况下直接在活细胞中观察RNA。我们将一种编码荧光团结合RNA适体（称为菠菜适体）的序列插入HIV基因组中，使得菠菜适体和HIV中的预测RNA二级结构都得以保留。通过测试嵌合HIV - 菠菜RNA增强一种特异性结合菠菜适体的条件性荧光团（DFHBI）荧光的能力，在体外对其进行了功能验证。利用荧光显微镜和PCR验证了HIV - 菠菜RNA在人类细胞中的表达。分别通过qPCR和荧光测定法测量了HIV - 1 gag RNA的产生和荧光。HIV - 菠菜RNA在体外可通过荧光测定法检测到，并且在人类细胞系和原代细胞中都能转录，通过PCR可检测到剪接和未剪接的种类。尽管信号可重复性较弱，但在活细胞中通过荧光显微镜可以看到HIV - 菠菜RNA。表达HIV - 菠菜RNA的细胞能够产生可通过荧光测定法检测到的病毒粒子，但无法检测单个病毒颗粒。总之，我们研究了一种利用菠菜RNA适体在活细胞中检测HIV RNA的新方法。尽管目前的适体/荧光团组合存在局限性，但这是该技术在传染病中的首次应用，为未来研究改进HIV表达的方法奠定了基础。
Many techniques currently used to measure HIV RNA production in cells suffer from limitations that include high background signal or the potential to destroy cellular context. Fluorophore-binding RNA aptamers offer the potential for visualizing RNAs directly in living cells with minimal cellular perturbation. We inserted a sequence encoding a fluorophore-binding RNA aptamer, known as Spinach, into the HIV genome such that predicted RNA secondary structures in both Spinach and HIV were preserved. Chimeric HIV-Spinach RNAs were functionally validated in vitro by testing their ability to enhance the fluorescence of a conditional fluorophore (DFHBI), which specifically binds Spinach. Fluorescence microscopy and PCR were used to verify expression of HIV-Spinach RNAs in human cells. HIV-1 gag RNA production and fluorescence were measured by qPCR and fluorometry, respectively. HIV-Spinach RNAs were fluorometrically detectable in vitro and were transcribed in human cell lines and primary cells, with both spliced and unspliced species detected by PCR. HIV-Spinach RNAs were visible by fluorescence microscopy in living cells, although signal was reproducibly weak. Cells expressing HIV-Spinach RNAs were capable of producing fluorometrically detectable virions, although detection of single viral particles was not possible. In summary, we have investigated a novel method for detecting HIV RNAs in living cells using the Spinach RNA aptamer. Despite the limitations of the present aptamer/fluorophore combination, this is the first application of this technology to an infectious disease and provides a foundation for future research into improved methods for studying HIV expression.
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DOI：
10.1126/science.1207339
发表时间：
2011-07-29
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Paige JS;Wu KY;Jaffrey SR
通讯作者：
Jaffrey SR
影响因子：
--
作者：
Filonov GS;Jaffrey SR
通讯作者：
Jaffrey SR
影响因子：
14.9
作者：
Shu D;Khisamutdinov EF;Zhang L;Guo P
通讯作者：
Guo P
影响因子：
3.7
作者：
Kiselinova M;Pasternak AO;De Spiegelaere W;Vogelaers D;Berkhout B;Vandekerckhove L
通讯作者：
Vandekerckhove L
影响因子：
20.3
作者：
Bosque, Alberto;Planelles, Vicente
通讯作者：
Planelles, Vicente