洛克菲勒大学Samie R. Jaffrey课题组近日取得一项新成果。他们的最新研究揭示了利用自催化转录物在细胞中高效表达环状RNA适体。相关论文发表在2019年6月出版的《Nature Biotechnology》杂志上。

该研究团队描述了龙卷风(耐用超表达优化龙卷风 RNA)表达系统能够实现RNA快速环化，使RNA寡核苷酸适配子具有高稳定性和高表达水平。龙卷风表达的转录物包含的目的RNA侧翼含有两个Twister核酶。核糖酶迅速进行催化裂解,使末端被普遍存在的内源性RNA连接酶RtcB结扎。使用这种方法,影响力很小的蛋白结合适配子成为细胞信号的有效抑制剂。另外，当表达为线性RNA时，以低水平表达的S-腺苷甲硫氨酸（SAM）的基于RNA的荧光代谢物生物传感器，达到足以在表达为环状RNA时检测细胞内SAM动力学的水平。龙卷风表达系统显著提高哺乳动物细胞基于RNA的应用。

据悉，RNA适体和基于RNA适体的装置可以进行遗传编码并在细胞中表达以探测和操纵细胞功能。然而,他们哺乳动物细胞的应用受限于低表达和快速降解。

附：英文原文

Title: Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts

Author: Jacob L. Litke, Samie R. Jaffrey

Issue&Volume: Volume 37 Issue 6, June 2019

Abstract: RNA aptamers and RNA aptamer-based devices can be genetically encoded and expressed in cells to probe and manipulate cellular function. However, their usefulness in the mammalian cell is limited by low expression and rapid degradation. Here we describe the Tornado (Twister-optimized RNA for durable overexpression) expression system for achieving rapid RNA circularization, resulting in RNA aptamers with high stability and expression levels. Tornado-expressed transcripts contain an RNA of interest flanked by Twister ribozymes. The ribozymes rapidly undergo autocatalytic cleavage, leaving termini that are ligated by the ubiquitous endogenous RNA ligase RtcB. Using this approach, protein-binding aptamers that otherwise have minimal effects in cells become potent inhibitors of cellular signaling. Additionally, an RNA-based fluorescent metabolite biosensor for S-adenosyl methionine (SAM) that is expressed at low levels when expressed as a linear RNA achieves levels sufficient for detection of intracellular SAM dynamics when expressed as a circular RNA. The Tornado expression system thus markedly enhances the utility of RNA-based approaches in the mammalian cell.

DOI: 10.1038/s41587-019-0090-6

Source:https://www.nature.com/articles/s41587-019-0090-6