Issue 22, 2020

Rolling circle amplification-driven encoding of different fluorescent molecules for simultaneous detection of multiple DNA repair enzymes at the single-molecule level

Abstract

DNA repair enzymes (e.g., DNA glycosylases) play a critical role in the repair of DNA lesions, and their aberrant levels are associated with various diseases. Herein, we develop a sensitive method for simultaneous detection of multiple DNA repair enzymes based on the integration of single-molecule detection with rolling circle amplification (RCA)-driven encoding of different fluorescent molecules. We use human alkyladenine DNA glycosylase (hAAG) and uracil DNA glycosylase (UDG) as the target analytes. We design a bifunctional double-stranded DNA (dsDNA) substrate with a hypoxanthine base (I) in one strand for hAAG recognition and an uracil (U) base in the other strand for UDG recognition, whose cleavage by APE1 generates two corresponding primers. The resultant two primers can hybridize with their respective circular templates to initiate RCA, resulting in the incorporation of multiple Cy3-dCTP and Cy5-dGTP nucleotides into the amplified products. After magnetic separation and exonuclease cleavage, the Cy3 and Cy5 fluorescent molecules in the amplified products are released into the solution and subsequently quantified by total internal reflection fluorescence (TIRF)-based single-molecule detection, with Cy3 indicating the presence of hAAG and Cy5 indicating the presence of UDG. This strategy greatly increases the number of fluorescent molecules per concatemer through the introduction of RCA-driven encoding of different fluorescent molecules, without the requirement of any specially labeled detection probes for simultaneous detection. Due to the high amplification efficiency of RCA and the high signal-to-ratio of single-molecule detection, this method can achieve a detection limit of 6.10 × 10−9 U mL−1 for hAAG and 1.54 × 10−9 U mL−1 for UDG. It can be further applied for simultaneous detection of multiple DNA glycosylases in cancer cells at the single-cell level and the screening of DNA glycosylase inhibitors, holding great potential in early clinical diagnosis and drug discovery.

Graphical abstract: Rolling circle amplification-driven encoding of different fluorescent molecules for simultaneous detection of multiple DNA repair enzymes at the single-molecule level

Supplementary files

Article information

Article type
Edge Article
Submitted
20 Mar 2020
Accepted
16 May 2020
First published
18 May 2020
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY license

Chem. Sci., 2020,11, 5724-5734

Rolling circle amplification-driven encoding of different fluorescent molecules for simultaneous detection of multiple DNA repair enzymes at the single-molecule level

C. Li, H. Chen, J. Hu and C. Zhang, Chem. Sci., 2020, 11, 5724 DOI: 10.1039/D0SC01652G

This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. You can use material from this article in other publications without requesting further permissions from the RSC, provided that the correct acknowledgement is given.

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