Oxidation of Guanine at a Distance in Oligonucleotides Induced by Two-Photon Photoionization of 2-Aminopurine

Abstract

Photoionization of free 2-aminopurine (2AP) in aqueous solutions, or of 2AP residues in oligonucleotides, is observed upon excitation with 308 nm XeCl excimer laser pulses (fwhm = 12 ns, ≤100 mJ/cm2/pulse) and involves the consecutive absorption of two photons. The absorption of light by the normal DNA bases at this wavelength is negligible under the same conditions. The kinetics and transient absorption spectra of the hydrated electrons and of 2AP radicals resulting from the deprotonation of the 2AP radical cations formed initially, have been investigated using standard spectroscopic methods. The 2AP radicals in aqueous solutions reversibly oxidize 2‘-deoxyguanosine and guanosine 5‘-monophosphate but do not react with the other three DNA nucleosides or nucleotides. The efficiency of photoionization of 2AP decreases in the following order: free 2AP > 2AP incorporated into single-stranded oligonucleotide > 2AP in double-stranded oligonucleotides. Photoionization of single 2AP residues incorporated into 18-mer single- and double-stranded oligonucleotides results in the oxidation of guanine residues in GG doublets. Employing gel electrophoresis methods, strand cleavage at these GG sites and at the site of the 2AP residue is observed after treatment of the irradiated oligonucleotides with hot piperidine. Using nanosecond transient absorption techniques, it is shown that the unimolecular oxidation of guanine by 2AP radicals at a distance can also be monitored directly in single-stranded oligonucleotides containing GG-doublets. It is found that increasing the number of nucleic acid bases between the 2AP radicals and the GG-doublet from 0 to 5 results in a decrease in the rate constant of guanine radical formation by a factor greater than 103.

Oxidative DNA Damage Associated with Combination of Guanine and Superoxide Radicals and Repair Mechanisms via Radical Trapping

“…1). The laser flash photolysis experiments showed that the hydrated electrons are scavenged by molecular oxygen, and thus (i) the formation of superoxide radicals and (ii) the oxidation of guanine residues by 2AP radicals is complete within ϳ100 s, in agreement with previous observations (15)(16)(17). Therefore, the transient absorption spectra recorded on a millisecond time scale resemble the spectra of guanine neutral radicals (Fig.…”

“…Laser Flash Photolysis-The transient absorption spectra and kinetics of free radical reactions were monitored directly by using a fully computerized kinetic spectrometer system (ϳ7-ns response time) described elsewhere (15). Briefly, 308 nm nanosecond XeCl excimer laser pulses (ϳ12-ns pulse width, 1-Hz repetition rate, 60-mJ pulse Ϫ1 cm Ϫ2 ) were used to photolyse sample solutions (0.25 ml) of the oligonucleotides saturated with molecular oxygen under normal atmospheric conditions ([O 2 ] ϳ1.3 mM).…”

“…A laser flash photolysis method developed by our group (15)(16)(17) was employed to generate O 2 . and G(ϪH) ⅐ radicals in DNA.…”

“…Both the radical cation and the neutral radical forms are strong one-electron oxidants that are capable of oxidizing guanines even when the 2AP and guanine residues are separated by varying numbers of intervening bases (15)(16)(17). In the presence of molecular oxygen, the hydrated electrons derived from the photoionization of 2AP are rapidly and quantitatively scavenged by dissolved O 2 to generate superoxide radicals (10).…”

“…In this study, the oligonucleotide 5Ј-d(CC[2AP]TCGCTACC) was employed. The electron transfer step from G to the 2AP radicals occurs over a bridge of 2 bases and is rather efficient in oligonucleotides of analogous sequences (15,16). The formation of the G(ϪH) ⅐ radicals and their disappearance by reaction with O 2 .…”