Bioquímica e Bioquímica Analítica

Abstrato

Double-Stranded DNA Damage Assessed with Raman Spectroscopy

Auner AW and Thomas JC

Double stranded DNA breaks [DSBs] and subsequent repair can correct DNA damage or may mistakenly cause mutations leading to cell damage and disease. DSBs can be by measured with Raman spectroscopy, using inelastic scattered light resulting from distinct molecular vibrations from purified DNA samples. With an exposure time of 20 s and 2 accumulations, Raman analysis found circular pBS KS+ plasmid DNA vibrations were similar to the water blank control. Restriction of the pBS KS+ single EcoR1 site created linear DNA and significant increases in Raman peaks at 880, 1044, 1084, and 1458 cm^-1. To further explore Raman detection of DNA damage, human Jurkat lymphocytes were grown in +/- 16 μg/ml of Bleocin™. DNA from the Bleocin^™ treated cells demonstrated enhanced Raman absorption at 880, 1044, 1084, and 1458 cm^-1 versus untreated cells. Jurkat cells are deficient in the ability to express pro-apoptotic Bax protein and p53, yet Bleocin^™ exposure increased TAp73 levels, and subsequently cell death. Due to low interference in biological materials and high sensitivity, Raman spectroscopy is a rapid and simple method to comparatively estimate the extent of relative DSBs. Unlike comet assays, which require analysis of living and dying cells, isolated DNA can be easily recovered from virtually any cell, stored, and DSB analysis done later.