Detection of acceptor sites for antisense oligonucleotides on native folded RNA by fluorescence spectroscopy
Detection of acceptor sites for antisense oligonucleotides on native folded RNA by fluorescence spectroscopy
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DOI：
10.1016/s0968-0896(03)00227-x
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发表时间：
2003-07-03
影响因子：
3.5
通讯作者：
Murakami, A
中科院分区：
文献类型：
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作者：
Mahara, A;Iwase, R;Murakami, A
反义策略通过抑制翻译步骤在治疗疾病和研究基因功能方面具有很高的潜力。对于该策略来说，在生理条件下检测反义分子在mRNA上的受体位置是必不可少的。我们提出了一种新的分析方法，用于检测反义分子的高灵敏度的受体位点。以含有2‘-O-(1-丙叉甲基)尿苷的2’-O-甲基寡核苷酸(OMUpy)为荧光探针。在单链OMUpy中很少观察到由芘引起的荧光强度。当OMUpy与互补的寡核苷酸杂交时，375 nm处的荧光强度显著增强。通过时间分辨荧光光谱、圆二色吸收光谱和紫外吸收光谱的测量发现，增加的原因是芘的局域化。这些结果表明，OMUpy的荧光强度的变化可以作为监测杂交的有用指标。在本研究中，我们选择了大肠杆菌。以16S-rRNA为模型RNA，根据已报道的16S-rRNA的二级结构，选择7个区域进行OMUpy的探测。OMUpy与16S-rRNA等摩尔混合物的荧光强度随序列的不同而变化。其中，能与16S-rRNA的887-896nT区域杂交的OMUpy-8系统的增长最为显著。这些结果表明，OMUpy-8靶点暴露于调控分子，并表明本文提出的方法对设计反义分子是有用的。(C)2003爱思唯尔科学有限公司。保留所有权利。
Antisense strategy has high potential for curing diseases and studying gene functions by suppressing the translation step. For the strategy, it is essential to detect acceptor sites of antisense molecules on mRNA under physiological conditions. We propose a new analytical method for the detection of acceptor sites of antisense molecules with high sensitivity. 2'-O-Methyloligoribonucleotide containing 2'-O-(1-pyrenylmethyl)uridine (OMUpy) was chosen as the fluorescence probe. The fluorescence intensity due to the pyrene in single-stranded OMUpy was scarcely observed. When OMUpy was hybridized with the complementary oligoRNA, the fluorescence intensity at 375 nm was remarkably increased. It was found that the increase was derived from the localization of the pyrene by the measurements of time-resolved fluorescence spectroscopy, CD and UV absorption spectra. These results suggest that the change of the fluorescence intensity of OMUpy can be a useful index to monitor hybridization. In this study, we chose Escherichia coli. 16S-rRNA as the model RNA and chose seven regions for probing by OMUpy based on the reported secondary structure of 16S-rRNA. The fluorescence intensity of an equimolar mixture of OMUpy with 16S-rRNA varied depending on the sequence. In particular, the increment in the system of OMUpy-8, which can hybridize with region 887-896 nt of 16S-rRNA, was most significant among the systems. These results indicated that the site targeted by OMUpy-8 was exposed to regulatory molecules, and suggest that the method presented here is useful to design antisense molecules. (C) 2003 Elsevier Science Ltd. All rights reserved.