Real time detection of DNA RNA hybridization in living cells
Real time detection of DNA RNA hybridization in living cells
复制标题
DOI：
10.1073/pnas.95.20.11538
复制
发表时间：
1998-09-29
影响因子：
11.1
通讯作者：
Gewitz, AM
中科院分区：
文献类型：
--
作者：
Sokol, DL;Zhang, XL;Gewitz, AM
事实证明，在活细胞中展示反义寡脱氧核苷酸与其 mRNA 靶标之间的杂交极其困难。为了解决反义研究中的这一基本问题，我们合成了“分子信标”(MB) 报告寡脱氧核苷酸，其 5' 和 3' 末端具有匹配的荧光供体和受体发色团。在缺乏互补核酸链的情况下，MB 保持茎环构象，其中荧光共振能量转移阻止信号发射。在与互补序列杂交时，茎环结构打开，增加了供体和受体部分之间的物理距离，从而减少荧光共振能量转移，并允许在信标被适当波长的光激发时发射可检测的信号。溶液杂交研究表明，与对照 MB 相比，在存在互补链的情况下，靶向 MB 的荧光强度可增加多达 60 倍。通过使用配备UV氟化物透镜的荧光显微镜，发现显微注射到细胞中的预先形成的MB/靶序列双链体的检测限大于或等于1 x 10(-1)ag的MB，或类似于10个mRNA分子。基于这种精细的灵敏度，通过将针对vav原癌基因的MB或对照MB显微注射到活细胞中，尝试实时检测活细胞中的MB/靶mRNA杂交。 K562人白血病细胞。 15 分钟内，共聚焦显微镜显示注射了靶向 MB（而非对照 MB）的细胞中出现荧光。这些研究表明寡核苷酸/mRNA 相互作用的实时可视化和定位现在是可能的。 MB 可用于研究活细胞中的 RNA 加工、运输和折叠，我们假设 MB 也可用于在生理条件下寻找可靶向的 mRNA 序列。
Demonstrating hybridization between an antisense oligodeoxynucleotide and its mRNA target has proven to be extremely difficult in living cells. To address this fundamental problem in antisense research, we synthesized "molecular beacon" (MB) reporter oligodeoxynucleotides with matched fluorescent donor and acceptor chromophores on their 5' and 3' ends. In the absence of a complementary nucleic acid strand, the MB remains in a stem-loop conformation where fluorescence resonance energy transfer prevents signal emission. On hybridization with a complementary sequence, the stem-loop structure opens increasing the physical distance between the donor and acceptor moieties thereby reducing fluorescence resonance energy transfer and allowing a detectable signal to be emitted when the beacon is excited by light of the appropriate wavelength. Solution hybridization studies revealed that in the presence of a complementary strand targeted MB could yield up to a 60-fold increase in fluorescence intensity in comparison to control MB. By using a fluorescence microscope fitted with UV fluoride lenses, the detection limit of preformed MB/target sequence duplexes microinjected into cells was found to be greater than or equal to 1 x 10(-1) ag of MB, or similar to 10 molecules of mRNA, On the basis of this exquisite sensitivity, real-time detection of MB/target mRNA hybridization in living cells was attempted by microinjecting MB targeted to the vav protooncogene, or control MB, into K562 human leukemia cells. Within 15 min, confocal microscopy revealed fluorescence in cells injected with targeted, but not control, MB. These studies suggest that realtime visualization and localization of oligonucleotide/mRNA interactions is now possible. MB could find utility in studying RNA processing, trafficking, and folding in living cells, We hypothesize that MB may also prove useful for finding targetable mRNA sequence under physiologic conditions.