Photocrosslinking in Ruthenium‐Labelled Duplex Oligonucleotides

Abstract

The formation of a photoadduct between a [Ru(1,4,5,8-tetraazaphenanthrene)(2)4,7-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.

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

“…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, photo-cross-linking has been proved to be resistant to the action of exonuclease III [62]. The demonstration of these inhibitions of DNA enzymes in in vitro systems represents a crucial milestone that highlights the interest in Ru-ODN conjugates as potential photoactivatable drugs in gene therapy.…”

“…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.…”

“…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).…”

“…The conjugation of ODNs to Ru-TAP complexes is obtained either by using a modified thymine or uracil residue bearing the complex [61,62] or by introducing into the Ru complex one ligand modified by an oxy-amine group. In this latter case, a highly specific oxime bond is then achieved in the presence of an ODN bearing an aldehyde function [63,64].…”

Highly Efficient Fluorescent Interstrand Photo‐crosslinking of DNA Duplexes Labeled with 5‐Fluoro‐4‐thio‐2′‐O‐methyluridine

“…Thus, ICL of DNA has attracted considerable interest in recent years, and much effort has been put into searching for highly efficient and selective crosslinking agents 3. ICL of DNA has been reported with various photochemically active chromophores, including an α‐chloroaldehyde derivative,4 a ruthenium complex,5 a p ‐carbamoylvinyl phenol nucleoside residue,6 a 3‐cyanovinyl‐carbazole nucleoside residue,7 5‐cyanovinyldeoxyuridine,8 a diazirine‐based nucleoside analogue,9 psoralens,10 and thionucleobases11 covalently attached to synthetic oligonucleotides.…”