SBIR-STTR Award

The objective of the research is to develop highly sensitive, quantitative and facile assays for measuring DNA damage induced by reactive oxygen species (ROS). ROS have been implicated in aging, cancer and several neurodegenerative diseases. Initially three specific ROS-induced DNA lesions will be of particular interest, namely, the formamido remnant of pyrimidine bases, the thymine glycol lesion and the 6-hydroxy-5, 6-dihydrothymine lesion. These lesions will be measured in the form of modified dinucleosides obtained from enzymatic digests of DNA. The method of detection will be by 32P-post labeling. The chief technological component of the undertaking will be the synthesis of modified dimers for use as carriers and internal standards. These carriers are necessary for selectivity and unequivocal identification of the lesions of interest. The internal standards are required for quantification of the lesions. These materials will be offered in kits that should make the detection of ROS-induced DNA damage convenient, sensitive and straightforward. It is anticipated that the availability of these kits will facilitate research on the relationship between ROS and disease.

This project is a collaboration between ZeptoMetrix Corporation and Roswell Park Cancer Institute. The objective is to develop sensitive, quantitative and facile assays for measuring DNA damage caused by reactive oxygen species (ROS). ROS have been implicated in aging, cancer and several neurological diseases. Seven ROS-induced DNA lesions will be assayed, namely: the formamido remnant of pyrimidine bases, the 8-oxo-7, 9-dihydroguanine (8-oxo-guanine) lesion, the thymine glycol lesion, the 6-hydroxy-5,6-dihydrothymine lesion, the abasic lesion and two double base lesions. 32P-postlabeling or mass spectroscopic methods of detection are employed with the assays. ZeptoMetrix will make products commercially available that facilitate DNA measurements by either method. Products will include reference compounds, carriers and internal standards for assaying the aforementioned lesions. Carriers for 32P-postlabeling provide unequivocal identification of the DNA lesion of interest. Internal standards for MS measurements will be isotopically enriched to a higher mass than the DNA lesion to be quantitated. ZeptoMetrix anticipates that eventually the largest application of these products will be for evaluating the repair capabilities of cells and tissues. Products useful for repair studies will include reference compounds and duplex DNA substrates containing identified lesions. Liquid Chromatography -Mass Spectrometry (LC-MS) utilizing an electrospray interface will be developed with this application especially in mind. PROPOSED COMMERCIAL APPLICATION: Reference materials, internal standards, and carriers for measuring oxidative DNA damage will be synthesized. These products will be offered commercially for facilitating DNA damage and repair by 32P-postlabeling or by LC-MS.

Thesaurus Terms:
DNA damage, DNA repair, bioassay, chemical synthesis, free radical oxygen, technology /technique development formamide, guanine, nuclease, stable isotope, thymine high performance liquid chromatography, liquid chromatography mass spectrometry, thin layer chromatography