Proximity Estimation and Quantification of Ionizing Radiation-induced DNA Lesions in Aqueous Media using Fluorescence Spectroscopy

Radiat Res. 2023 Dec 29. doi: 10.1667/RADE-23-00145.1. Online ahead of print.ABSTRACTClustered DNA damage (cluster) or a multiply damaged site, which is a region with two or more lesions within one or two helical turns, has a high mutagenic potential and causes cell death. We quantified fluorophore-labeled lesions and estimated their proximity through fluorescence anisotropy measurements depending on Förster resonance energy transfer (FRET) among the fluorophores close to each other. pUC19 plasmid DNA (2,686 base pairs) dissolved in water or 0.2 M Tris-HCl buffer at a concentration of 10 μg/μL was irradiated by several ionizing radiations with varying linear energy transfers (LET, 0.2-1890 keV/μm). Electrophilic carbonyls (aldehydes and ketones) at abasic sites (APs) produced in DNA were labeled with Alexa Fluor 488 fluorescent dyes with an O-amino functional group. Regardless of the presence or absence of the buffer, AP yields (the number of APs/base pair/Gy) tended to decrease with increasing LET, and the ratio of the AP yield (in 0.2 M Tris-HCl/in water) was less than 0.1 in the LET range of 0.2-200 keV/μm. However, in a higher LET range, the ratios were greater than 0.1. At a low dose, fluorescence anisotropy decreased with increasing LET in 0.2 M Tris-HCl, whereas, in water, this LET dependence was almost insignificant. These findings suggest that 1. the damage distribution on a DNA molecule formed by indirect effects (e.g., by hydroxyl radicals) does not depend on ...
Source: Radiation Research - Category: Physics Authors: Source Type: research