Olivier Lentzen

Photocrosslinking in ruthenium-labelled duplex oligonucleotides.

The formation of a photoadduct between a [Ru(1,4,5,8‐tetraazaphenanthrene)24,7‐diphenylphenanthroline]2+ complex chemically attached to a synthetic oligonucleotide, and a guanine moiety in a complementary targeted single‐stranded DNA molecule was studied for ten 17‐mer duplexes by denaturing gel electrophoresis. This photoadduct formation leads to photocrosslinking of the two strands. The percentage quenching of luminescence of the complex by electron transfer was compared to the resulting yield of photocrosslinked product. This yield does not only depend on the ionisation potential of the guanine bases, which are electron donors, but also on other factors, such as the position of the guanine bases as compared to the site of attachment of the complex. The photocrosslinking yield is higher when the guanine moieties are towards the 3′ end on the complementary strand as compared to the tethering site. Computer modelling results are in agreement with this preference for the 3′ side for the photoreaction. Interestingly, the photocrosslink is not alkali labile. Moreover, a type III exonuclease enzyme is blocked at the position of photocrosslinking.

Determination of DNA guanine sites forming photo-adducts with Ru(II)-labeled oligonucleotides; DNA polymerase inhibition by the resulting photo-crosslinking.

The influence of the distance between the anchoring site of the tethered [Ru(TAP)2dip]2+ complex (TAP=1,4,5,8-tetraazaphenanthrene; dip=4,7-diphenyl-1,10-phenanthroline) on a probe sequence and the guanines of the complementary target strand was studied by (1) the luminescence quenching of the complex (by electron transfer) and (2) the oligodeoxyribonucleotide adduct (ODN adduct) formation which results in photo-crosslinking of the two strands. Moving the guanine moieties away from the complex induces an important decrease of the efficiency of both processes, but clearly affects the ODN adduct formation more specifically than the quenching process. From these results, we determined the positions of the guanine bases in the duplex ODN that are able to form a photo-adduct with the tethered complex. We also examined the possible competition between a long-range hole migration in the duplex ODN and the formation of a photo-adduct by using a sequence labeled with the complex at the 5′-phosphate end. Such a hole migration appears to be inefficient as compared to the ODN adduct formation. Finally, we studied the influence of the photo-crosslinking on the function of two different DNA polymerases. A 17-mer Ru(II)-labeled ODN was hybridized to its complementary sequence located on the 5′-side of a 40-mer matrix. After illumination, the elongation of a 13-mer DNA primer hybridized to the 3′-extremity of the same matrix was stopped at a position corresponding to the formation of the ODN adduct.

Photoadduct Leading to Crosslinking in RuII-Derivatized Oligonucleotides

The use of modified oligonucleotides that may recognize messenger RNA and react with the target RNA sequence is a promising strategy in the development of new drugs that control or block gene expression. [1] In this prospect we have exploited the photochemical properties of Ru(II) complexes able to form adducts with DNA. Under illumination some complexes are able to abstract an electron from the guanine, the most reductive base of DNA. This process, evidenced by luminescence quenching of the metallic species, gives rise to the formation of radicals that may recombine to form a covalent bond between a guanine and one ligand of the complex. In order to cumulate this photoreactivity with a sequence specificity, we have prepared different 17-mer duplex oligonucleotides derivatized by the [Ru(TAP)2dip] 2þ complex. Visible illumination of these duplexes induces an electron transfer with