相关实验视频

Updated: Jul 24, 2025

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

Published on: December 5, 2016

单基分辨率检测RNA中的N6-甲基氨酸,通过氨酸脱胺测序

Wen-Xuan Shao^1,2, Yi-Hao Min¹, Wei Chen³

¹College of Chemistry and Molecular Sciences, Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan 430071, China.

Analytical chemistry

|July 4, 2023

概括

一种新的氨酸脱胺测序 (AD-seq) 方法允许简单,单基分辨率检测N6-甲基氨酸 (m6A) RNA修饰. 这种技术使用一种经过修改的酶来区分腺和m6A,有助于理解m6A.

科学领域:

分子生物学分子生物学
在RNA生物学,RNA生物学.
生物化学生物化学

背景情况:

N6-甲基氨酸 (m6A) 是一种普遍存在的RNA修饰,对各种生物过程至关重要.
在单基分辨率下准确检测单个m6A位点对于理解其功能至关重要.
现有的m6A检测方法往往缺乏简单性或单基分辨率.

研究的目的:

开发一种简单且具有成本效益的技术,以单基分辨率检测RNA中的m6A.
为解读RNA中m6A修饰的作用提供一个有价值的工具.

主要方法:

开发了使用进化的tRNA腺脱胺酶 (TadA) 变体 (TadA8e) 或二分体 (TadA-TadA8e) 的腺脱胺测序 (AD-seq).
通过TadA8e/TadA-TadA8e,通过TadA8e/TadA-TadA8e将腺素选择性去胺化,而m6A仍然未被胺化.
差异测序读取区分腺 (阅读为瓜诺) 和m6A (阅读为腺).

主要成果:

AD-seq成功地在RNA中实现了m6A的单基分辨率检测.
该技术通过在埃舍里希亚大肠杆菌23SrRNA中识别单个m6A位点来证明其能力.
m6A修饰被证明可以抵抗TadA8e/TadA-TadA8e的去胺化,这是由于甲基组的固态阻碍.

结论:

AD-seq提供了一种简单,具有成本效益和准确的方法,以单基分辨率检测m6A.
这种技术为研究RNA修饰及其功能影响的研究人员提供了一个强大的新工具.
AD-seq促进了对m6A在各种生理和病理背景中扮演的角色的更深入的理解.

更多相关视频

Improving Small RNA-seq: Less Bias and Better Detection of 2'-O-Methyl RNAs

Improving Small RNA-seq: Less Bias and Better Detection of 2'-O-Methyl RNAs

Published on: September 16, 2019

DNAzyme-dependent Analysis of rRNA 2’-O-Methylation

DNAzyme-dependent Analysis of rRNA 2’-O-Methylation

Published on: September 16, 2019

相关概念视频

RNA Editing

RNA Editing

RNA editing is a post-transcriptional modification where a precursor mRNA (pre-mRNA) nucleotide sequence is changed by base insertion, deletion, or modification. The extent of RNA editing varies from a few hundred bases, in mitochondrial DNA of trypanosomes, to a just single base, in nuclear genes of mammals. Even a single base change in the pre-mRNA can convert a codon for one amino acid into the codon for another amino acid or a stop codon. This type of re-coding can significantly affect the...

RNA-seq

RNA-seq

RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases.
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while...

Nonsense-mediated mRNA Decay

Nonsense-mediated mRNA Decay

The Upf proteins that carry out nonsense-mediated decay (NMD) are found in all eukaryotic organisms, including humans. Each protein has an individual role, but they need to work in collaboration. Upf1 is an ATP-dependent RNA helicase that unwinds the RNA helix. Because Upf1 can unwind any RNA, Upf2 and Upf3 are required to help Upf1 discriminate between nonsense and normal mRNAs.
Usually, Upf3 binds to an Exon Junction Complex (EJC) at mRNA splice sites. If a ribosome fully translates the mRNA,...

RNA Stability

RNA Stability

Intact DNA strands can be found in fossils, while scientists sometimes struggle to keep RNA intact under laboratory conditions. The structural variations between RNA and DNA underlie the differences in their stability and longevity. Because DNA is double-stranded, it is inherently more stable. The single-stranded structure of RNA is less stable but also more flexible and can form weak internal bonds. Additionally, most RNAs in the cell are relatively short, while DNA can be up to 250 million...