A FRET-based assay for characterization of alternative splicing events using peptide nucleic acid fluorescence in situ hybridization.
A FRET-based assay for characterization of alternative splicing events using peptide nucleic acid fluorescence in situ hybridization.
复制标题
DOI：
10.1093/nar/gkp551
复制
发表时间：
2009-09
影响因子：
14.9
通讯作者：
Artero R
中科院分区：
文献类型：
--
作者：
Blanco AM;Rausell L;Aguado B;Perez-Alonso M;Artero R
我们描述了一种检测RNA选择性剪接变体的定量方法，该方法结合了荧光标记肽核酸（PNA）探针与共聚焦显微镜Förster共振能量转移（FRET）的原位杂交。使用与给定剪接连接两侧序列互补的PNA探针，可以在细胞内通过FRET测量特异性地量化产生这种剪接连接的RNA异构体。为了证明这一概念，我们分析了源于淋巴细胞抗原6 （LY6）复合体，位点G5B (LY6G5B) pre-mRNA的两个备选剪接事件。它们的特征是去除第一个内含子（完全剪接异构体，FSI）或保留该内含子（内含子保留异构体，IRI）。使用标记有供体（Cy3）和受体（Cy5）荧光团的PNA探针对，适用于FRET，侧翼FSI和IRI特异性剪接，特异性检测HeLa细胞中的两种mRNA亚型。我们已经观察到该方法在探针间距为5 - 11nt时有效。这些数据支持这种基于fret的PNA荧光原位杂交（FP-FISH）方法提供了一种概念上的新方法，不仅可以在亚细胞水平上表征剪接变体异构体结构、位置和动力学，还可以潜在地表征各种近距离RNA-RNA相互作用。
We describe a quantitative method for detecting RNA alternative splicing variants that combines in situ hybridization of fluorescently labeled peptide nucleic acid (PNA) probes with confocal microscopy Förster resonance energy transfer (FRET). The use of PNA probes complementary to sequences flanking a given splice junction allows to specifically quantify, within the cell, the RNA isoform generating such splice junction by FRET measure. As a proof of concept we analyzed two alternative splicing events originating from lymphocyte antigen 6 (LY6) complex, locus G5B (LY6G5B) pre-mRNA. These are characterized by the removal of the first intron (Fully Spliced Isoform, FSI) or by retention of such intron (Intron-Retained Isoform, IRI). The use of PNA probe pairs labeled with donor (Cy3) and acceptor (Cy5) fluorophores, suitable to FRET, flanking FSI and IRI specific splice junctions specifically detected both mRNA isoforms in HeLa cells. We have observed that the method works efficiently with probes 5–11 nt apart. The data supports that this FRET-based PNA fluorescence in situ hybridization (FP–FISH) method offers a conceptually new approach for characterizing at the subcellular level not only splice variant isoform structure, location and dynamics but also potentially a wide variety of close range RNA–RNA interactions.
登录
查看更多内容
影响因子：
4.5
作者：
Brodsky, AS;Silver, PA
通讯作者：
Silver, PA
影响因子：
3.2
作者：
BAUMAN, JGJ;WIEGANT, J;VANDUIJN, P
通讯作者：
VANDUIJN, P
影响因子：
5.3
作者：
BOND, VC;WOLD, B
通讯作者：
WOLD, B
影响因子：
2.5
作者：
Megias, Diego;Marrero, Raquel;Montoya, Maria C.
通讯作者：
Montoya, Maria C.
影响因子：
9.3
作者：
Bonifazi, E;Gulotta, F;Novelli, G
通讯作者：
Novelli, G