Two novel “release-on-demand” fluorescent biosensors for probing UV-induced DNA damage induced in single stranded and double stranded DNA: Comparative study

Abstract

Light in the UVC spectral region damages both single-strand (ssDNA) and double-strand DNA (dsDNA), and contributes to the formation of mutagenic photoproducts. In-vivo studies show greater damage for ssDNA compared to dsDNA. However, excited-state spectroscopy shows that dsDNA has longer excited-state lifetime than ssDNA, which increases the probability of damage for dsDNA. However, lack of a direct comparison of in-vitro ssDNA and dsDNA damage rates precludes the development of a model that elucidates the molecular factors responsible for damage. In this work, two novel sensitive “release-on-demand” biosensors are developed for the selective probing of DNA-damage and comparing the rate of DNA damage in ssDNA and dsDNA. The two biosensors involve the use of EvaGreen and Hoechst dyes for the sensitive probing of DNA-damage. The results show that ssDNA is damaged at a faster rate than dsDNA in the presence of UVC light (200–295 nm). Furthermore, we examined the effect of G/C composition on the damage rate for mostly A/T ssDNA and dsDNA oligonucleotides. Our results show that DNA damage rates are highly dependent on the fraction of guanines in the sequence, but that in-vitro dsDNA always exhibits an overall slower rate of damage compared to ssDNA, essentially independent of sequence.