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

Abstract

The influence of the distance between the anchoring site of the tethered [Ru(TAP)(2)dip](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.

Highly DNA‐Photoreactive Ruthenium 1,4,5,8‐Tetraazaphenanthrene Complex Conjugated to the TAT Peptide: Efficient Vectorization inside HeLa Cells without Phototoxicity – The Importance of Cellular Distribution

“…On the basis of previous results, this DNA photodamaging action should result in cell death [30] through a mechanism quite different from the classical oxygen photosensitization used in photodynamic therapy (PDT). Indeed, as explained above, the Ru-TAP compounds are able to photoinduce irreversible covalent binding of the TAP ligand to the G bases of DNA under visible-light irradiation (Figure 1, a).…”

Ru–TAP complexes and DNA: from photo-induced electron transfer to gene photo-silencing in living cells

“…The distance between the Ru complex anchored on the probe sequence and the guanine in the targeted ODN is also of critical importance for both the PET and the photocross-linking. The maximum distance reachable by the complex depends on its linker and on the direction followed by the tethered complex (3 or 5 direction) in the duplex configuration [65]. For example, with a previously examined complex and linker [62], a photo-adduct could be formed with a G up to the fourth base towards the 3 side and the fifth base towards the 5 side.…”

“…In vitro experiments within this framework have demonstrated that this photo-process with a Ru-ODN probe leads to complete inhibition of the enzyme DNA polymerase [65]. Indeed, although this enzyme in the absence of or even in the presence of the complementary Ru-ODN probe but in the dark is able to produce DNA from the DNA template, after illumination, the elongation of the primer is completely blocked at the site of the photo-cross-linking ( figure 15).…”

“…Moreover, the ASO sequence had to respond to the criteria elaborated from the earlier-described results, i.e. two C bases in positions 3 and 4 of the attachment site of the Ru-ASO complex [62,65], so that the two Gs of the complementary target gene (sequence (b) of figure 19) would be close to the photoreactive Ru complex, and one G would be more or less in the middle of the ASO sequence so that the seppuku process would be possible. Another Ru-ASO has also been tested and corresponds to a non-complementary sequence (Ru-nc) (sequence (c) of figure 19).…”

[Ru(TAP)3]2+‐Photosensitized DNA Cleavage Studied by Atomic Force Microscopy and Gel Electrophoresis: A Comparative Study

“…[3] These metallic complexes have also been examined as potential phototherapeutic drugs, [4] probes for nucleic acids, or sequence-specific photoreagents. [5] The possibility of using atomic force microscopy (AFM) to observe directly conformational transitions of plasmid DNA due to, for example, a single-strand cleavage, is very attractive because the topology of plasmid DNA plays an important role in genetic processes and in the interaction of DNA with enzymes. However, the understanding of conformational transitions of plasmid DNA as a result of a photocleavage is still rather limited.…”