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JACS:南京大学张辰宇教授课题组发表微小核糖核酸化学生物学研究进展

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摘要 : 近日,国际期刊《J. Am. Chem. Soc》杂志上在线发表了南京大学生命科学学院张辰宇教授研究组的一篇研究论文,论文报道了在微小核糖核酸化学生物学研究中的重要进展。

近日,国际期刊《J. Am. Chem. Soc》杂志上在线发表了南京大学生命科学学院张辰宇教授研究组的一篇研究论文,论文报道了在微小核糖核酸化学生物学研究中的重要进展。研究在通过实验方法鉴定micrornA靶基因方面实现了重要突破,为利用化学生物学手段研究细胞内microRNA提供了新的工具及思路。李金波副研究员和2015级博士研究生黄磊为共同第一作者,张辰宇教授与张艳教授为共同通讯作者。

微小核糖核酸(microRNA)是细胞内源的非编码rna,与靶基因结合后可调控基因表达。对microRNA靶基因的解析有助于了解microRNA在细胞内的生物过程,为针对microRNA的疾病诊断以及治疗提供新的理论基础或方法。目前microRNA靶基因的鉴定主要依赖于生物信息学预测的方法。已知在3’末端修饰生物素的microRNA在细胞内会丧失其调控基因表达的功能,因此用化学生物学方法鉴定microRNA在细胞内的靶基因一直是一个难题。针对这一挑战,张艳课题组与张辰宇教授研究团队合作开展了深入的学科交叉研究,提出了以可发生光点击化学反应的四氮唑基团为化学标签对microRNA进行化学修饰(图1),这一修饰相比于生物素修饰可避免microRNA细胞内功能的丧失。待可发生光点击化学反应的microRNA与细胞内靶基因结合后,通过一步法的光点击化学反应在microRNA与其结合的靶基因上修饰生物素基团,进而利用链霉素的亲和作用成功实现了对细胞内microRNA靶基因的富集、分离与鉴定

JACS:南京大学张辰宇教授课题组发表微小核糖核酸化学生物学研究进展
图1、基于“光点击化学”的微小核糖核酸细胞内靶基因鉴定方法

JACS:南京大学张辰宇教授课题组发表微小核糖核酸化学生物学研究进展
图2、课题组围绕细胞内microRNA开展的化学生物学研究

原文链接:

Photoclickable MicroRNA for the IntraCellular Target Identification of MicroRNAs

原文摘要:

MicroRNAs (miRNAs) are important gene regulators that bind with target genes and repress target gene expression at the post-transcriptional level. The identification of target genes associated with miRNAs inside different cells is a major challenge in miRNA chemical biology due to the lack of functional miRNAs bearing appropriate tags. Here we report photoclickable miRNAs as appropriately pretagged miRNAs that keep the intracellular function of miRNAs and allow the addition of molecular handles through photoclick reaction. The photoclickable miRNAs upon transfection inside cells were able to form functional complexes with target genes and repress target gene expression. Target genes associated with the photoclickable miRNAs in the complexes were then tagged with the molecular handle through photoclick reaction for pull-down and identification. Using photoclickable miR-106a, miR-27, and miR-122, we first verified that their intracellular function was comparable to that of intact miRNAs, which showed obvious advantage over corresponding biotinylated miRNAs. After attaching the biotin handle to the associated complexes containing the photoclickable miRNAs through the tetrazole-ene photoclick reaction, target genes previously bound with these miRNAs inside cells were successfully pulled town and analyzed. The application of this strategy was demonstrated by the identification of several new target genes of miR-122, followed by revealing a novel regulatory pathway in HepG2 cells with regard to the role of PEG10 in miR-122-promoted cell apoptosis.

DOI:10.1021/jacs.6b08521

作者:张辰宇 点击:

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