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首页> 外文期刊>Nucleic Acids Research >Determination of interactions between structured nucleic acids by fluorescence resonance energy transfer (FRET): selection of target sites for functional nucleic acids.
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Determination of interactions between structured nucleic acids by fluorescence resonance energy transfer (FRET): selection of target sites for functional nucleic acids.

机译：通过荧光共振能量转移（FRET）确定结构化核酸之间的相互作用：选择功能性核酸的靶位点。

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摘要

We previously developed a method for monitoring the integrity of oligonucleotides in vitro and in vivo by quantitating fluorescence resonance energy transfer (FRET) between two different fluorochromes attached to a single oligonucleotide. As an extension of this analysis, we examined changes in the extent of FRET in the presence or absence of target nucleic acids with a specific sequence and a higher-ordered structure. In this system FRET was maximal when probes were free in solution and a decrease in FRET was evidence of successful hybridization. We used a single-stranded oligodeoxyribonucleotide labeled at its 5'-end and its 3'-end with 6-carboxyfluorescein and 6-carboxytetramethylrhodamine, respectively. Incubation of the probe with a single-stranded complementary oligonucleotide reduced the FRET. Moreover, a small change in FRET was also observed when the probe was incubated with an oligonucleotide in which the target site had been embedded in a stable hairpin structure. The decrease in the extent of FRET depended on the length of the stem region of the hairpin structure and also on the higher-ordered structure of the probe. These results indicate that this spectrofluorometric method and FRET probes can be used to estimate the efficacy of hybridization between a probe and its target site within highly ordered structures. This conclusion based on changes in FRET was confirmed by gel-shift assays.
机译：我们之前开发了一种通过定量连接到单个寡核苷酸的两种不同荧光染料之间的荧光共振能量转移（FRET）来监测体外和体内寡核苷酸完整性的方法。作为此分析的扩展，我们检查了在存在或不存在具有特定序列和更高级结构的靶核酸的情况下，FRET范围的变化。在该系统中，当探针在溶液中游离时，FRET达到最大，而FRET的降低是成功杂交的证据。我们使用了在其5'端和3'端分别标记有6个羧基荧光素和6个羧基四甲基若丹明的单链寡脱氧核糖核苷酸。用单链互补寡核苷酸孵育探针可降低FRET。此外，当探针与寡核苷酸孵育时，FRET的变化也很小，在寡核苷酸中，目标位点已嵌入稳定的发夹结构中。 FRET程度的降低取决于发夹结构茎区域的长度，还取决于探针的高阶结构。这些结果表明，这种荧光分光光度法和FRET探针可用于评估探针与目标分子在高度有序结构中的杂交效率。该基于FRET变化的结论已通过凝胶位移分析得到证实。

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