Molecular Biology Division, Bhabha Atomic Research Centre, Trombay,Mumbai, India.
Life Sciences, Homi Bhabha National Institute, Anushakti Nagar, Mumbai, India.
Transcription. 2023 Jun-Oct;14(3-5):146-157. doi: 10.1080/21541264.2023.2190296. Epub 2023 Mar 16.
Proximity ligation assay (PLA) is an immunofluorescence assay, which determines in situ interaction of two biomolecules present within 40 nm close proximity. Here, we describe a modification of PLA for visual detection of in situ protein interactions with nascent RNA in a single cell (IPNR-PLA). In IPNR-PLA, nascent RNA is labeled by incorporating 5-fluorouridine (FU), a uridine nucleotide analogue, followed by covalent cross-linking of the interacting partners in proximity to newly synthesized RNA. By using combination of anti-BrdU antibody, which specifically binds to FU, and primary antibody against a protein of interest, the IPNR reaction results in fluorescent puncta as a positive signal, only if the candidate proteins are in proximity to nascent RNA. We have validated this method by demonstrating known CDK9 and elongating RNA pol II interaction with nascent RNA. Finally, we used this method to test for the presence of DNA double strand breaks as well as Poly (ADP-ribose) polymerase 1 (PARP1), an RNA binding protein, in the vicinity of nascent RNA in cancer cells. The capability of performing parallel IF labeling and quantifiable multiparameter measurements within heterogeneous cell populations makes IPNR-PLA very attractive for use in biological studies. Overall, we have developed the IPNR-PLA method for analysis of protein association with nascent RNA with single-cell resolution, which is highly sensitive, quantitative, efficient, and requires little starting experimental material.
邻近连接分析（PLA）是一种免疫荧光分析方法，用于确定 40nm 内两种生物分子的原位相互作用。在这里，我们描述了 PLA 的一种改良方法，用于在单个细胞中可视化检测新生 RNA 与原位蛋白质的相互作用（IPNR-PLA）。在 IPNR-PLA 中，通过掺入 5-氟尿嘧啶核苷（FU），一种尿嘧啶核苷酸类似物，对新生 RNA 进行标记，然后将接近新合成 RNA 的相互作用伴侣进行共价交联。通过使用抗 BrdU 抗体（特异性结合 FU）和针对感兴趣蛋白的初级抗体的组合，只有候选蛋白与新生 RNA 接近时，IPNR 反应才会导致荧光斑点作为阳性信号。我们通过证明 CDK9 和延伸的 RNA pol II 与新生 RNA 的已知相互作用验证了这种方法。最后，我们使用这种方法在癌细胞中检测新生 RNA 附近的双链 DNA 断裂以及多聚（ADP-核糖）聚合酶 1（PARP1），一种 RNA 结合蛋白。在异质细胞群体中进行并行 IF 标记和可量化多参数测量的能力使 IPNR-PLA 非常适合用于生物学研究。总体而言，我们开发了用于分析新生 RNA 与蛋白质相互作用的 IPNR-PLA 方法，具有单细胞分辨率，高度敏感，定量，高效，并且需要很少的初始实验材料。
