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Updated: Aug 11, 2026
Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons
Published on: August 6, 2012
Direct Quantification of Damaged Nucleotides in Oligonucleotides Using an Aerolysin Single Molecule Interface
Jiajun Wang1,2, Meng-Yin Li1, Jie Yang2
1State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, 210023, Nanjing, China.
Abstract:
DNA lesions such as metholcytosine(mC), 8-OXO-guanine (OG), inosine (I), etc. could cause genetic diseases. Identification of the varieties of lesion bases are usually beyond the capability of conventional DNA sequencing which is mainly designed to discriminate four bases only. Therefore, lesion detection remains a challenge due to massive varieties and less distinguishable readouts for structural variations at the molecular level. Moreover, standard amplification and labeling hardly work in DNA lesion detection. Herein, we designed a single molecule interface from the mutant aerolysin (K238Q), whose sensing region shows high compatibility to capture and then directly convert a minor lesion into distinguishable electrochemical readouts. Compared with previous single molecule sensing interfaces, the temporal resolution of the K238Q aerolysin nanopore is enhanced by two orders, which has the best sensing performance in all reported aerolysin nanopores. In this work, the novel K238Q could discriminate directly at least three types of lesions (mC, OG, I) without labeling and quantify modification sites under the mixed heterocomposition conditions of the oligonucleotide. Such a nanopore electrochemistry approach could be further applied to diagnose genetic diseases at high sensitivity.
Insights
Detecting DNA lesions like methylated cytosine (mC) is challenging. A novel K238Q aerolysin nanopore interface directly detects and distinguishes multiple DNA lesions, enabling sensitive genetic disease diagnosis.
Area of Science:
- Biochemistry
- Nanotechnology
- Molecular Biology
Background:
- Genetic diseases can arise from various DNA lesions, including methylated cytosine (mC), 8-oxo-guanine (OG), and inosine (I).
- Conventional DNA sequencing struggles to identify these diverse lesions due to limitations in base discrimination and lack of suitable amplification/labeling methods.
- Current methods for lesion detection face challenges due to the wide variety of lesions and difficulty in obtaining distinguishable molecular readouts.
Purpose of the Study:
- To develop a novel single-molecule interface for direct and sensitive detection of DNA lesions.
- To overcome the limitations of conventional sequencing and existing detection methods for various DNA modifications.
- To establish a nanopore electrochemistry approach for quantifying lesion sites in mixed DNA compositions.
Main Methods:
- Engineered a mutant aerolysin (K238Q) to create a single-molecule sensing interface.
- Utilized the K238Q aerolysin nanopore for direct capture and electrochemical readout of DNA lesions.
- Enhanced the temporal resolution of the nanopore for improved sensing performance.
Main Results:
- The K238Q aerolysin nanopore demonstrated significantly enhanced temporal resolution, outperforming previously reported aerolysin nanopores.
- Successfully discriminated and quantified at least three types of DNA lesions (mC, OG, I) without the need for labeling.
- Quantified modification sites within mixed heterocomposition oligonucleotides, showcasing its capability in complex samples.
Conclusions:
- The K238Q aerolysin nanopore interface provides a sensitive and direct method for identifying multiple DNA lesions.
- This nanopore electrochemistry approach overcomes key challenges in DNA lesion detection, offering high sensitivity.
- The developed technology holds potential for diagnosing genetic diseases by detecting DNA damage with high precision.
