title: Chemically-enzymatic synthesis of photosensitive DNA.
abstract: The sensitizing propensity of radio-/photosensitizing nucleoside depends on DNA sequence surrounding a sensitizer. Therefore, in order to compare sensitizers with regard to their ability to induce a DNA damage one has to study the sequence dependence of damage yield. However, chemical synthesis of oligonucleotides labeled with sensitizing nucleosides is hindered due to the fact that a limited number of such nucleoside phosphoramidites are accessible. Here, we report on a chemically-enzymatic method, employing a DNA polymerase and ligase, that enables a modified nucleoside, in the form of its 5'-triphosphate, to be incorporated into DNA fragment in a pre-determined site. Using such a protocol two double-stranded DNA fragments - a long one, 75 base pairs (bp), and a short one, 30bp in length - were pin-point labeled with 5-bromodeoxyuridine. Four DNA polymerases together with DHPLC for the inspection of reaction progress were used to optimize the process under consideration. As an ultimate test showing that the product possessing an assumed nucleotide sequence was actually obtained, we irradiated the synthesized oligonucleotide with UVB photons and analyzed its photoreactivity with the LC-MS method. Our results prove that a general approach enabling precise labeling of DNA with any nucleoside modification processed by DNA polymerase and ligase has been worked out.
authors.affiliation: Faculty of Chemistry, University of Gdańsk, Wita Stwosza 63, 80-308 Gdańsk, Poland.
authors.forename: Kinga
authors.initials: K
authors.lastname: Westphal
authors.affiliation: Faculty of Chemistry, University of Gdańsk, Wita Stwosza 63, 80-308 Gdańsk, Poland.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Zdrowowicz
authors.affiliation: Faculty of Chemistry, University of Gdańsk, Wita Stwosza 63, 80-308 Gdańsk, Poland.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Zylicz-Stachula
authors.affiliation: Faculty of Chemistry, University of Gdańsk, Wita Stwosza 63, 80-308 Gdańsk, Poland. Electronic address: janusz.rak@ug.edu.pl.
authors.forename: Janusz
authors.initials: J
authors.lastname: Rak
mesh_terms: D001973:Bromodeoxyuridine; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000138:chemical synthesis* / Q000528:radiation effects; D004249:DNA Damage; D004798:Enzymes / Q000737:chemistry*; D008027:Light; D013058:Mass Spectrometry; D058105:Polymerization
chemical_list: D004798:Enzymes; D004247:DNA; D001973:Bromodeoxyuridine
keywords: DNA strand breaks; Modified nucleosides; Sensitizers; UV irradiation