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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.
ACS Central Science
|January 29, 2020
Summary
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.
