ANALYTICAL CHEMISTRY-INDUSTRIAL

Abstract
Abstract Image


RNA modification, such as N1-methyladenosine (m1A), affects the secondary structure of RNA and its ability to recognize specific reader proteins. Methods for detecting site-specific m1A are in demand. We report here a ligation-assisted differentiation approach for quantitative detection of m1A in mRNA with single-base resolution. The methyl group in m1A disrupts the Watson–Crick base pairing with uridine, resulting in a lower ligation efficiency of certain ligases and lower amounts of ligation products. Detection of the ligation products using quantitative real-time PCR provided site-specific evaluation of m1A. We first screened appropriate ligase and found that T3 DNA ligase offered the best discrimination between m1A and adenosine. We successfully detected and quantified m1A at position 1674 of bromodomain containing 2 (BRD2) mRNA from HEK293T cells. In lung carcinoma tissues, the level of m1A at position 1674 of BRD2 mRNA was significantly decreased compared to the tumor-adjacent normal tissues, suggesting that site-specific m1A may be involved in carcinogenesis.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.9b04454.
RNA extraction; expression and purification of recombinant ALKBH3 protein; sequences of oligonucleotides; genders and ages of the lung cancer patients; sequence of BRD2 mRNA from human; quantified band intensity ratios using different RNA templates of RNA1-m1A and RNA1-A by different ligases; measured Ct values of different percentages of RNA1-A and RNA1-m1A in the mixture and data processing for the linear regression of the curves in Figure 4; quantification of m1A at position 1674 in BRD2 mRNA of lung carcinoma tissues and matched tumor-adjacent normal tissues; quantitative evaluation of the level of m1A at the small portion (PDF)
pdf
Terms & Conditions
Electronic Supporting Information files are available without a subscription to ACS Web Editions. The American Chemical Society holds a copyright ownership interest in any copyrightable Supporting Information. Files available from the ACS website may be downloaded for personal use only. Users are not otherwise permitted to reproduce, republish, redistribute, or sell any Supporting Information from the ACS website, either in whole or in part, in either machine-readable form or any other form without permission from the American Chemical Society. For permission to reproduce, republish and redistribute this material, requesters must process their own requests via the RightsLink permission system. Information about how to use the RightsLink permission system can be found at http://pubs.acs.org/page/copyright/permissions.html.

Cited By



This article has not yet been cited by other publications.

x
x

Comments

Popular Posts