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In order to explore the possibility and nature of the interactions of this new family of photoactivatable compounds with DNA, three complexes (1,4 and11) were selected for further studies with CT-DNA in cell-free media.

4.3.13.1 DNA Binding Kinetics

Reaction mixtures of DNA and complexes [(6-p-cym)Ru(bpm)(Py)]2+(1), [(6-p- cym)Ru(bpm)(4,4'-bpy)]2+ (4) and [(6-p-cym)Ru(phen)(Py)]2+(10) were prepared in three ways: (A) in the dark (henceforth referred to as non-irradiated), (B) following the addition to DNA of (beforehand) irradiated 1, 4 and 10 (pre- irradiated), or (C) by addition of 1,4and10 to DNA followed by photoirradiation of the resulting mixture (irradiated). The samples were photoirradiated with visible light (λirr = 400−660 nm) at selected photoirradiation times. The binding kinetics experiments with CT-DNA where then performed as described in Chapter 2. The results, summarised in Table 4.14, indicate that the non-irradiatedforms of

for1 and10 and ca. 20% of 4 after 48 h).Pre-irradiatedforms of complexes 1,4, and 10 reacted with DNA in ca. 40%, 54%, and 58%, respectively after 24 h; and irradiated forms of 1, 4 and 10 reacted in ca. 41%, 50%, and 54%, respectively after 24 h. It can be seen that pre-irradiated RuII arene complexes bind faster but to the same extent as theirradiatedDNA mixtures, Figure 4.27.

Table 4.14. Percentage of binding of non-irradiated, pre-irradiated, or irradiated forms of complexes 1, 4 and 10 to CT-DNA in 1.0 mM NaClO4 as determined by FAAS.

a_{Incubations with DNA were at 310 K. The concentration of DNA was 50g/mL. Data are the average} of two independent experiments.

b_{Photoirradiation was carried out in absence of DNA for24h, followed by a further incubation with} DNA in the dark.

c

Photoirradiation of was carried out in presence of DNA for24h. ND_{Not determined.}

Figure 4.27.Percentage of binding to CT-DNA bypre-irradiated(red) andirradiated (blue) forms of complexes [(6-p-cym)Ru(bpm)(Py)][PF6]2 (1), [(6-p- cym)Ru(bpm)(4,4'-bpy)][PF6]2 (4) and [(6-p-cym)Ru(phen)(Py)][PF6]2 (10) as a function of time.

Non-irradiateda Pre-irradiatedb Irradiatedc

Time (hours) 1 4 10 1 4 10 1 4 10

24 3.1 18.7 3.7 40.2 54.5 57.6 41.1 50.3 54.0

4.3.13.2 DNA Transcription by RNA Polymerase in Vitro

Cutting of pSP73KB DNA byNdeI andHpaI restriction endonucleases yielded a 212- bp fragment. This fragment contained the T7 RNA polymerase promotor. The RuII arene complexes were tested as their non-irradiated, pre-irradiated and irradiated forms. All samples were precipitated and redissolved after incubation with DNA (7.8×10–5 M, 0.5 μg/20 μL) to remove the unbound RuII arene complexes; rb of complexes 1,4and 10 was determined using EAS and FAAS. The autoradiogram of the inhibition of RNA synthesis by T7 RNA polymerase on pSP73KB DNA containing adducts of the RuII arene complexes or cisplatin is shown in Figure 4.28. The bands corresponding to the transcription of DNA modified by thenon-irradiated forms of complexes 1, 4, and 10 are rather faint in intensity indicating that RNA synthesis on the modified fragment do not stop the polymerase, except for complex [(6-p-cym)Ru(bpm)(4,4'-bpy)][PF6]2 (4), which was found to bind to DNA without photoirradiation (ca. 20%). The pre-irradiated, and irradiated forms of 1,4, and 10

yielded fragments of newly synthesized RNA of defined sizes, which indicates that RNA synthesis on these templates was prematurely terminated. The major stop sites produced occurred at similar positions in the gel and were exclusively at guanine residues, Figure 4.29 and are identical for the three RuII arene complexes. The total intensity of the bands on the autoradiogram corresponding to transcripts of single- ruthenated DNA fragments (modified to the same level (rb)) differed. The intensities of those corresponding to the transcription of DNA modified by the pre-irradiated forms of complexes 1, 4 and 10 are slightly stronger than those of those corresponding to the transcription of DNA modified by theirradiatedforms.

The profiles are similar to that obtained for DNA treated with the anticancer drug cisplatin and also to those reported previously for the RuII arene compounds, such as [(η6

-arene)Ru(en)Cl]+.17

Figure 4.28.Autoradiogram of 6% polyacrylamide/8 M urea sequencing gel showing inhibition of RNA synthesis by T7 RNA polymerase on pSP73KB DNA containing adducts of RuIIarene complexes or cisplatin. Lanes: chain terminated marker RNAs, cisplatin, at rb = 0.02; A, U, G and C, the template modified by RuII arene compounds.

Figure 4.29.Schematic diagram showing the portion of the sequence used to monitor inhibition of RNA synthesis by RuIIarene complexes. The arrows indicate the start of the T7 RNA polymerase, which used as template the upper strand of pSP73KB DNA, respectively. The numbers correspond to the nucleotide numbering in the sequence map of pSP73KB plasmid. (●) Indicates major stop sites for DNA modified by ruthenation.

4.3.13.3 Unwinding of Supercoiled pUC19 Plasmid DNA

The plasmid was incubated with the RuII arene complexes 1, 4 and 10 in 10 mM NaClO4, at pH ≈ 6 for 24 h at 310 K in their pre-irradiatedandirradiatedforms. All samples were precipitated and redissolved after incubation with DNA to remove free, unbound RuIIspecies. Therb(c) value was determined by EAS and FAAS. The native agarose gel resulting from DNA modified by the RuIIarene complexes1,4, and10in theirpre-irradiated forms are shown in Figure 4.30 and the correspondingirradiated forms is shown in Figure 4.31. The DNA unwinding angle produced by the adducts formed bypre-irradiated andirradiated forms of the RuIIarene complexes 1and4, and10was determined to be 6.6±1.7°, 5.2±2.2°, and 6.1±2.2°, respectively using this approach. The commigration point of the modified supercoiled and nicked DNA (rb(c)) was reached atrb= 0.16, 0.19 and 0.17 for complexes1,4, and10respectively, Table 4.15. From the autoradiogram it can also be noticed that an increasing amount of nicked (OC) form occurred during photoirradiation of the RuIIarene complexes in the presence of DNA (irradiated form), rb(c) for 1 and 10 is not changed whereas DNA is almost completely nicked with increasing ruthenation in the case of4.

Table 4.15.Unwinding of supercoiled pUC19 DNA by RuIIarene complexes1,4and

11.