Photochemical formation of a fluorescent thymidine-pterin adduct in DNA

SANDRA ESTÉBANEZ; MIGUEL A. MIRANDA; MARISA MARÍN; ANDRÉS H. THOMAS; CAROLINA LORENTE; VIRGINIE LHIAUBET

Villa Carlos Paz , Córdoba, Argentina.

Congreso; XIII Encuentro Latinoamericano de Fotoquímica y Fotobiología (XIII ELAFOT); 2017

ELAFOT

Most solar radiation incident on the surface of Earth inthe UV-range corresponds to wavelenghts between 320 - 400 nm (UV-A). Thisfraction of radiation is poorly absorbed by the DNA biomacromolecule, andtherefore it is responsible for only low amounts of directly formedphotolesions. Nevertheless, UV-A radiation acts indirectly by photosensitizedreactions and is recognized as a class I carcinogen [1]. A photosensitizedreaction is a photochemical modification occurring in a molecular entity as aresult of the initial absorption of radiation by a photosensitizer [2]. Pterinsbelong to a family of heterocyclic compounds present in a wide range of livingsystems and participate in relevant biological functions. Under UV-A excitation,pterins can fluoresce, undergo photooxidation and generate reactive oxygenspecies (ROS) [3]. In the presence of oxygen, pterin (Ptr) act asphotosensitizer through type I (electron abstraction) and/or type II (1O2-mediatedoxidation) mechanisms [4-5].Thephotosensitized degradation of the pyrimidine nucleotide thymidine5?-monophosphate (dTMP) by Ptr under anaerobic conditions generates an adduct, wherethe pterinic moiety is attached to the nucleobase. The spectroscopic propertiesof the adduct are similar to those of Ptr itself [6].The main objective of this work was to attach aphotosensitizer to an oligonucleotide chain. Single stranded oligonucleotide ofdTMP with the sequence 5´-d(TTTTT)-3´ (dT5) was exposed to UV-Aradiation in the presence of Ptr in neutral aqueous solutions at roomtemperature, under different experimental conditions. The samples were analyzedby UV-Vis spectrophotometry, HPLC coupled to a photodiode array andfluorescence detector and UPLC coupled to a mass spectrometry system.Chromatographic peaks corresponding to both dT5and Ptr decreased with irradiation time. Several products presented absorbancein the UV-A region, and their absorption spectra showed a band centeredapproximately at 340 nm, which is similar to the typical low-energy band ofPtr. Moreover, some of these products were fluorescent and emitted at 450 nmwhen excited at 350 nm, which is compatible with the emission properties of Ptr.Products were analyzed by mass spectrometry, and the mass spectra showed agroup of signals with m/z » 808.65 Da, which correspond to a di-charged ion of a compound bearingboth of the photosensitizer and the oligonucleotide moieties