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Published on: March 5, 2018
Plastic antibody for DNA damage: fluorescent imaging of BPDE-dG adducts in genomic DNA
Junfa Yin1, Zhixin Wang, Maoyong Song
1State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 100085, China.
Abstract:
Exogenous and endogenous genotoxic carcinogens and their in vivo metabolites may result in DNA damage and cause adverse health effects. It is very difficult to specifically detect trace DNA damage in the presence of a large excess of unmodified nucleosides. Here we report a molecularly imprinted polymer (MIP) nanocomposite, namely nanoMIP, which can be used as a "plastic antibody" for specific recognition of benzo[a]pyrene diol epoxide (BPDE)-DNA adduct. Enhanced binding affinity (880 nM) of nanoMIPs was achieved by using two tailor-made functional monomers. The property of binding kinetics was greatly improved in virtue of the well-defined nanostructure, which was fabricated by initiators for continuous activator regeneration-atom transfer radical polymerization (ICAR-ATRP). The BPDE-adducted DNA can be specifically captured by synthetic nanoMIP. By taking advantage of this antibody-mimicking behavior, we further developed a fluorescently imaged particle counting immunoassay (FIPCIA) method for ultrasensitive detection of BPDE-ssDNA adducts using a laser scanning confocal microscope (LSCM). The number of countable fluorescent dots is proportional to the content of BPDE-ssDNA adducts in the DNA sample. The proposed plastic antibody-based FIPCIA method can detect traces of BPDE-DNA adducts as low as 18 pM in human lung carcinoma A549 cells. This highly-sensitive detection of DNA lesions offers a promising alternative to immunogenic antibody-based immunoassays for genomics and DNA modification analysis.
Insights
Researchers developed a novel plastic antibody (nanoMIP) for detecting DNA damage from carcinogens like benzo[a]pyrene diol epoxide (BPDE). This method enables ultrasensitive detection of BPDE-DNA adducts in cells.
Area of Science:
- Biomaterials Science
- Analytical Chemistry
- Molecular Biology
Background:
- Genotoxic carcinogens can cause DNA damage, leading to adverse health effects.
- Detecting trace DNA damage in complex biological samples is challenging.
- Existing antibody-based methods can be immunogenic and less versatile.
Purpose of the Study:
- To develop a "plastic antibody" (nanoMIP) for specific recognition of benzo[a]pyrene diol epoxide (BPDE)-DNA adducts.
- To create a highly sensitive immunoassay for detecting DNA damage.
- To offer an alternative to traditional antibody-based assays for genomic analysis.
Main Methods:
- Fabrication of molecularly imprinted polymer (MIP) nanocomposites (nanoMIPs) using initiators for continuous activator regeneration-atom transfer radical polymerization (ICAR-ATRP).
- Development of a fluorescently imaged particle counting immunoassay (FIPCIA) utilizing nanoMIPs for capturing BPDE-DNA adducts.
- Detection of BPDE-ssDNA adducts using laser scanning confocal microscopy (LSCM).
Main Results:
- NanoMIPs demonstrated enhanced binding affinity (880 nM) for BPDE-DNA adducts.
- The FIPCIA method achieved ultrasensitive detection of BPDE-ssDNA adducts down to 18 pM.
- The number of fluorescent dots correlated directly with the concentration of BPDE-ssDNA adducts.
Conclusions:
- The developed nanoMIP-based FIPCIA is a highly sensitive and specific method for detecting DNA damage.
- This plastic antibody approach provides a promising alternative to immunogenic antibody-based immunoassays.
- The method has potential applications in genomics and DNA modification analysis.
