Chemical modifications to DNA and RNA bases occur in response to endogenous and environmental exposure to reactive species such as oxidizing and alkylating agents as well as during installation of epigenetic markers. Although considerable information about the type and location of epigenetic base changes can be gleaned from bisulfite sequencing, no such methodology is routinely employed for oxidative stress modifications. This project investigates the use of ion channel proteins, both wild-type and engineered, as part of a nanopore platform to detect the presence of base modifications in DNA:DNA duplexes or DNA:RNA duplexes. The hypothesis rests on recent results from these laboratories showing that the latch zone of the alpha-hemolysin ion channel is a sensitive detector of changes in base pairs when double- stranded DNA is electrophoretically driven into the vestibule of the protein cavity. The work proposes that a combination of site-directed mutagenesis and chemical modification of the protein, combined with a biophysical understanding of the protein-nucleic acid-electrolyte interactions, can fine-tune the response of the ion channel for sensing changes in nucleic acid duplexes.
The specific aims are to (1) optimize the latch zone of alpha-hemolysin to sense base modifications in DNA: RNA duplexes, (2) construct DNA probes and examine oxidative damage in the anti-codon region of tRNAs, and (3) explore gamma-hemolysin as a tool to examine DNA damage in translocating double-stranded DNA. A key aspect of the work is to provide a new single-molecule method to examine changes in the bases of transfer RNA that will provide insight into the pathway by which oxidative stress results in tRNA cleavage and inhibition of translation. Given the significant correlations between oxidative stress and disease, and the public focus on micronutrients and antioxidant therapy, technologies that report on modifications to DNA or RNA bases as a function of diet, drugs, and inflammation and disease state are of key importance in modern medicine. Innovative aspects of the project include a novel method for PCR amplification of DNA damage in a way that retains information about the sites of damage, and the use of non-traditional components of hemolysin-type ion channels for sensing of DNA:DNA and DNA:RNA duplexes.
Protein ion channels will be investigated as a means to detect changes in DNA and RNA bases by examining electrical currents produced by single molecules passing through nanopore membranes. The work is focused on oxidative stress-induced damage to double-stranded DNA and to tRNA, both of which are proposed to be important in early stages of disease.
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