Characterization of Interstrand DNA-DNA Cross-Links Using the α-Hemolysin Protein Nanopore. ACS Nano 2015 Dec 22;9(12):11812-9
Date
11/14/2015Pubmed ID
26563913Pubmed Central ID
PMC4826734DOI
10.1021/acsnano.5b03923Scopus ID
2-s2.0-84952360580 (requires institutional sign-in at Scopus site) 34 CitationsAbstract
Nanopore-based sensors have been studied extensively as potential tools for DNA sequencing, characterization of epigenetic modifications such as 5-methylcytosine, and detection of microRNA biomarkers. In the studies described here, the α-hemolysin protein nanopore embedded in a lipid bilayer was used for the detection and characterization of interstrand cross-links in duplex DNA. Interstrand cross-links are important lesions in medicinal chemistry and toxicology because they prevent the strand separation that is required for read-out of genetic information from DNA in cells. In addition, interstrand cross-links are used for the stabilization of duplex DNA in structural biology and materials science. Cross-linked DNA fragments produced unmistakable current signatures in the nanopore experiment. Some cross-linked substrates gave irreversible current blocks of >10 min, while others produced long current blocks (10-100 s) before the double-stranded DNA cross-link translocated through the α-hemolysin channel in a voltage-driven manner. The duration of the current block for the different cross-linked substrates examined here may be dictated by the stability of the duplex region left in the vestibule of the nanopore following partial unzipping of the cross-linked DNA. Construction of calibration curves measuring the frequency of cross-link blocking events (1/τon) as a function of cross-link concentration enabled quantitative determination of the amounts of cross-linked DNA present in samples. The unique current signatures generated by cross-linked DNA in the α-HL nanopore may enable the detection and characterization of DNA cross-links that are important in toxicology, medicine, and materials science.
Author List
Zhang X, Price NE, Fang X, Yang Z, Gu LQ, Gates KSAuthor
Xi Fang Assistant Professor in the Data Science Institute department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
Cross-Linking ReagentsDNA
DNA Damage
Hemolysin Proteins
Nanopores
Nucleic Acid Conformation
