.I. Photochem. Photobiol. B: Biol., 23 (1994) 69-78

Photoaddition of ruthenium(II)-tris-1,4,5&tetraazaphenanthrene to DNA and mononucleotides

Martin M. Feeney, John M. Kelly+ and Alessandro B. Tossi Chemishy Department, Trinity College, University of Dublin, Dublin 2 (Ireland)

AndrCe Kirsch-de Mesmaeker and Jean-Paul Lecomte Universitk Libre de Bwelles, Chimie Organique Physique, CP 16OfO8, B-1050, Brussels (Belgium)

(Received October 15, 1993; accepted December 14, 1993)

Abstract

Formation of adducts between Ru(TAP), ‘+ (TAP = 1,4,5,8_tetraazaphenanthrene) and DNA has been monitored by, gel electrophoresis, UV-vis spectroscopy and dialysis methods. Adduct formation is found for both single- and double-stranded nucleic acids. The reaction with double-stranded DNA is found to be insensitive to solution pH or aeration. Spectroscopic changes similar to those for DNA are found with GMP in oxygen-free pH 5 solution. However, different reactions occur with GMP at higher pH or when the solution contains oxygen. Comparative experiments with double-stranded poly[d(G-C)] or poly[d(A-T)] indicate that the adduct with DNA involves binding to the guanosine moiety. It is proposed that the product is formed by the reaction of the reduced ruthenium complex and oxidised guanine species produced by photo-induced electron transfer.

Key words: Photo-adduct; Ruthenium; DNA; Guanine oxidation

69

1. Introduction

It has been widely demonstrated that the photophysical properties of ruthenium polypyridyl complexes are sensitive to binding to DNA [14] and that these metal complexes can be used as conformational probes for nucleic acids. However, much less is known about the photochemical re- actions of these complexes with nucleic acids and especially about how the reaction depends on the type of ligand bound to the ruthenium. Early studies indicated that photoexcitation .of either Ru(phen),2+ or Ru(bpy),2+ could cause strand breaks in plasmid DNA [S, 61, although subsequent work [7] revealed that the quantum yield for this process is low and affected by the presence of oxygen. (For example, for Ru(bpy)32+ at a nu- cleotide/metal complex molar ratio (P/D) of 18 the quantum yields are 6.6 x 10d6 in air-saturated solution and 1.2 x 10m6 in argon-saturated solution [7].) Studies with 32P-labelled oligonucleotides or restriction fragments have shown that a much more efficient, oxygen-dependent reaction causes dam-

‘Author to whom correspondence should be addressed.

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age to the guanine bases and that subsequent alkali treatment leads to cleavage of the ,DNA strand at these sites [8].

Ru(TAP)3*+

Our initial studies with Ru(TAP),~+ showed that it induced direct strand-breaks in DNA with a substantially higher efficiency than either Ru(phen)32+ or Ru(bpy)32+ [9]. Ru(TAP),~+ dif- fers from these complexes as its excited state emission is strongly quenched by guanine-con- taining DNA and it was proposed that the DNA strand cleavage was induced by the reaction of the guanine radical cation formed by photo-induced electron transfer

70 M.M. Feeney et al. / Photoaddition of RutTAP):+ to DNA and mononucleotides

Ru(TAP)~*+* + G j

[Ru(TAP),(TAP--)] + + G’+ (1)

Direct evidence for electron transfer has since been obtained by flash photolysis studies [lo]. Electrophoretic experiments with samples of Ru(TAP)~*+ photolysed in the presence of single- stranded radio-labelled 27-mer oligonucleotide have shown that under these conditions the most important reaction is the formation of a photo- adduct between the metal complex and the DNA [ll]. Adduct formation between metal complexes and DNA is, of course, well known for labile platinum group metal compounds (such as the important anti-tumour drug Pt(NH3)2C12), but there are few reports for kinetically stable metal complexes. Thus, photo-induced binding of the sensitiser to the oligonucleotides is not found with Ru(phen),*+ or most other ruthenium polypyridyl complexes, although recently Morrison et al. have reported the photoaddition of Rh(phen)*Cl*+ to DNA [12]. Covalent binding of photosensitisers to DNA is well known for the [2+2] cycloadducts of psoralens and related compounds and is held to be the mode of action of these compounds in controlling psoriasis [13]. Photo-adducts have been reported for a few other cases, including chlor- promazine [14] and porphyrins [15], although in these cases the precise mechanism is uncertain.

In the present publication we present spectro- scopic, dialysis and electrophoretic data which show that visible light induces addition of Ru(TAP),*+ to double-stranded as well as to single-stranded DNA. The effects of solution pH and aeration on this photoreaction are reported, and the role of base type is investigated by em- ploying synthetic polynucleotides and nucleotides. The spectroscopic data strongly suggest that the adduct formed from Ru(TAP)~*+ is of a type quite unlike those produced from cis-platin or Rh(phen),Cl,+.

2. Experimental details

The synthesis and the purification of the [Ru(TAP)$12 (c(H,O) = 1.7x 104 M-l cm-’ at 407 nm) were as described previously [16]. Deoxy- guanosine-5’-monophosphate (Sigma), guanosine- 5’-monophosphate (Sigma) as the sodium salt or free acid, poly[d(A-T)] (mol. wt. =9 X 105) and poly[d(G-C)] (mol. wt. = 5 X 105) (Pharmacia-BL Biochemicals) were used without further purifi- cation. Buffer solutions were prepared with

K21-IP04 and KH,PO,. Water was purified with a Milli-Q (Millipore) system.

High molecular weight calf thymus sodium salt (CT-DNA) (Sigma) solutions were purified by standard procedures [17] to remove histone pro- teins (by phenol extraction) and small nucleic acids (by dialysis) as previously reported [5]. Single- stranded DNA was prepared by heating double- stranded DNA for 10 min at 90 “C, rapidly cooling and then using immediately. The 27-mer oligon- ucleotides (a gift from Prof. C. Helene and Dr. T. LeDoan) were 5’-end-labelled with Y-~*P ATP by the standard methods described in ref. 15. Photolysis of 10 ~1 samples of these 27-mer samples was carried out in Eppendorf tubes using 436 nm light (isolated from a PTI 200 W Hg-Xe lamp source by NaN02 and Cu(NH3)42+ filters [18]). Vertical 20% polyacrylamide denaturing gel elec- trophoresis was used to separate oligonucleotide fragments after photolysis and the radioactive oli- gonucleotide fragments were detected by auto- radiography at -20 “C [17].

Steady state photolysis with CT-DNA, poly[d(A- T)], poly[d(G-C)], dGMP and GMP were carried out in a standard 1 cm* quartz cuvette using visible light from a 250 W Hg lamp (MEDmom EMI) filtered with a NaNO, solution to remove the UV part of the lamp emission. Where required, so- lutions were deoxygenated by bubbling argon for at least 20 min before the measurements. Ab- sorption spectra were obtained on Pye-Unicam SP8200 or 8800 W-vis spectrophotometers.

A typical dialysis experiment was carried out as follows. The solution (typically 3 ml) was trans- ferred to the dialysis tubing (Spectra/par No. 132678, molecular weight cutoff 12000, 14000) and dialysed with gentle stirring against 100 ml of buffer in the dark, with three changes of buffer over a 24 h period.

3. Results

3.1. Gel electrophoresis experiments with 27-mer oligonucleotide

In this series of experiments, a 32P-5’-end- labelled, single-stranded deoxyoligonucleotide 5’- *TGAGTGAGT AAAAAAAATGAGTGCCAA- 3’ (I) was irradiated with 436 nm light in the presence of Ru(TAP)~*+. Figure 1 (lane 3) shows the result of a typical electrophoresis experiment carried out with a sample photolysed in an aerated 10 mM phosphate pH 6.9 buffered solution con- taining 27-mer I (5.4~ lO-‘j M nucleotide) and Ru(TAP)~*+ (2.7~ lo-’ M). The photolysed sam-

M.M. Feeney et al. / Photoaddition of Ru(TAP),~+ to DNA and mononucleotides 71

123 4

Fig. 1. Photoadduct formation of Ru(TAP),‘+ and nP-labelled 27-mer I in the presence and absence of complementary oli- gonucleotide II monitored by gel electrophoresis. Lane 1, for double-stranded 27-mer I and II; lane 2, for 27-mer I and II in no salt buffer; in both cases [I] and [II] = 2.7 x 10m6 M, lane 3, single stranded I, [I] =5.4x 10m6 M nucleotide. All samples contained Ru(TAP)s’+ (2.7 X 10e5 M) in 10 mM phosphate buffer pH 6.9 and were irradiated for 5 min at 436 nm. Samples in lanes 1 and 3 contain additionally 100 mM NaCl. Lane 4 is the product of a standard Maxam-Gilbert G +A reaction [17].

ple shows two predominant bands, one due to the single-stranded 27-mer and the other to a less mobile species. Samples which had not been ir- radiated show only a single band with mobility identical to that of the single-stranded 27-mer (data not shown), indicating that the effect is due neither to a dark reaction nor to non-covalent binding of the metal complex to the oligonucleotide. The amount of the less mobile band increases with increasing period of illumination (Fig. 2(a)). More extended illumination leads to the formation of further lower mobility bands (Fig. 3 of ref. 11). The absence of bands of greater mobility than the parent oligonucleotide indicates that the produc- tion of frank breaks is inefficient. Treatment of Ru(TAP),‘+ -photosensitised samples with piper- idine reduced the amount of adduct and yielded weak cleavage bands at each G-residue (data not shown). This behaviour may be contrasted with that found for Ru(phen),” where no low mobility bands are observed and where piperidine treatment induces efficient cleavage at guanine residues [8].

Cl 12 345

J

Fig. 2. The effect of (a) irradiation time, (b) buffer concentration and (c) type of buffer on the formation of photoadduct from Ru(TAP),‘+ and “P-1abelled 27-mer oligonucleotide I followed by acrylamide gel electrophoresis. (a) lane 1, 0.5 mm, lane 2, 1 min; lane 3, 5 min; lane 4, 10 min; 10 mM phosphate pH 6.9 buffer; (b) lane 1,20 mM, lane 2,50 mM; lane 3,100 mM, lane 4, 500 mM; phosphate butfer pH 6.9; samples irradiated for 5 min. (c) lane 1, no irradiation; lane 2, 10 mM phosphate; lane 3, 10 mM tris; lane 4, 10 mM EDTA, lane 5, 10 mM tris/l mM EDTA. In all samples [Ru(TAP)~“] =2.7X lo-’ M, [oligonu- cleotide 11=2.7x 10m6 M nucleotide irradiated for 5 min with 436 nm light. The figure shows only that part of the autoradiogram of the gel containing the 27-mer and lower mobility species.

The proportion of single-stranded oligonucleo- tide I converted to the adduct is dramatically reduced when the phosphate buffer concentration is increased from 20 mM to 500 mM (Fig. 2(b)). This is consistent with the need for the excited Ru(TAP),~ + to be associated with the oligonu- cleotide if the adduct is to be efficiently formed. When a 10 mM tris/l mM EDTA buffer was used, the adduct yield decreased substantially. With 10 mM EDTA, no adduct was observed (Fig. 2(c)). EDTA will efficiently quench the excited state of

72 M.M. Feeney et al. J Photoaddition of Ru(TAP)z+ to DNA and mononucleotides

Ru(TAP)~*+ [19], and it is probable that this is the cause of the reduced adduct yield.

To test whether adduct formation is also found with double-stranded oligonucleotides, experi- ments were carried out with the 27-mer I in the presence of an equal concentration of its complementary strand 5’-TTGGCACT- CA-ACTCAC3’ (II) under con- ditions of 5 “C and 10 mM phosphate buffer/O.1 M NaCl, where a double-stranded 27-mer should form. The Ru(TAP)~*+ is expected to bind re- versibly and non-covalently to double-stranded 27- mer [3]. As can be seen in Fig. 1 (lane 1) irradiation with 436 nm light and subsequent electrophoresis on the denaturing polyacrylamide gel shows clear evidence for the formation of the low mobility bands, which are associated with the photo-adduct covalently bound to the 32P-labelled 27-mer. Figure 1 (lane 2) confirms that adduct formation also occurs under conditions of a 10 mM phosphate buffer (no NaCl), where double-strand formation is not favoured. These results strongly suggest, therefore, that adduct formation may occur with both single- and double-stranded DNA. To test this, spectroscopic and dialysis experiments have been carried out with high molecular weight DNA.

3.2. Spectroscopic monitoring of the photoadduct with DNA, synthetic polynucleotides, and (deoxylguanosine monophosphate

In order to determine whether formation of the adduct can be monitored by its W-vis absorption, steady state illumination was carried out under various conditions with the complex both alone and in the presence of CT-DNA, the synthetic double-stranded polynucleotides poly[d(G-C)] and poly[d(A-T)], and mononucleotides dGMP and GMP.

3.2.1. Ru(TAP),‘+ in the absence of DNA It is known that Ru(TAP),*+ can undergo photo-

substitution and photoanation reactions in aqueous [20] and organic solvents [21] resulting in loss of one of the chelating ligands. To study the effect of pH on this reaction, samples of Ru(TAP),*+ (2.5~10-~ M) in 5 mM pH 5, pH 7 and pH 9 phosphate buffers were irradiated with visible Hg- lamp radiation. Before photolysis the visible ab- sorption spectrum of Ru(TAP),*+ is identical in each of the buffers used. In all three cases, the 400 nm absorption band is bleached on irradiation and is replaced by a new band at about 500 run (Fig. ,3(a)). Absorption in this wavelength region is expected for [Ru(II)(TAP),XY]“+ complexes (X and Y are H,O or possibly Cl- or phosphate).

360

(a)

560 Wavelength (nm)

e t ~_ __~-H /

5 0.0 2 ,,,,, ,,,, ,,,, ,,,, ,,,, ,,,,

d 5 6 7 8 9 10 1

(b) PH

Fig. 3. (a) Changes in the absorption spectra of Ru(TAP)~‘+

(2.5 X lo-’ M) following visible light irradiation at pH 5, 7 and 9 (5 mM phosphate buffer). (b) Plot of the rate of dechelation of Ru(TAP)~‘+ following 10 min irradiation at various pHs relative to that at pH 5. Absorbance monitored at 550 nm.

The rate of dechelation increases markedly with pH. Figure 3(b) shows the solution absorption at 550 nm after 10 min of illumination in the pH range 5-10. Oxygen was found to make little difference to the dechelation process (experiments were performed in the absence of buffer).

3.2.2. Ru(TAP)~~+ and calf thymus DNA Changes in the UV-vis spectra following visible

light (A>400 nm) irradiation of Ru(TAP),*+ (2x low5 M) in the presence of calf thymus DNA were monitored in 5 mM phosphate buffers at pH 5, pH 7 and pH 9 with a DNA (nucleotide) concentration of 2 mM (P/D = 100). (The high P/ D ratio was used in order to minimise the amount of unbound Ru(TAP),‘+.) Figure 4 shows the changes in spectra for an argon-deoxygenated so- lution at pH 5. Closely similar results are observed at pH 7 and pH 9, indicating that the course of reaction is not affected by pH. It may be observed that the photo-reaction of the DNA-bound Ru(TAP),*+ is quite different from that of the complex alone (Fig. 3(a)). A clear hyperchromic effect is observed (the final absorption is 40%

M.M. Feeney et al. / Photoaddition of Ru(TAP)~” to DNA and mononucleotides 73

120 mins irradiation

0.5 -

6 P

% 0.4 :

$ ;: 0.3 -

a!

0.2 1

0.1 -

0.0

300 350 400 450 500 550 600 650 700

Wavelength (nm)

Fig. 4. Changes in the absorption spectra of Ru(TAP)~*+ in the presence of calf thymus DNA following visible light irradiation in 5 mM phosphate buffer, pH 5, deoxygenated solution for 0, 10, 30, 60, 90 and 120 min. [Ru(TAP),*+] =2x lo-’ M, [DNA] =2x 10m3 M nucleotide.

greater after 90 min illumination) and the maximum shifts to slightly shorter wavelengths. This ab- sorption band (centred at 392 nm) maintains its shape even after an illumination time as long as 2 h. The lack of a distinct absorption band at 500 nm indicates that the dechelation process is sup- pressed in the presence of DNA; this effect is particularly evident at pH 9 as dechelation occurs with a greater efficiency at this pH. The absorption spectra were compared for both argon- and oxygen- purged solutions (data not shown). No difference was found for the final spectra nor for the evolution of those spectra. This behaviour was found at both pH 5 and pH 9. Thus it appears that the presence or absence of oxygen has no effect on the product formation.

In order to determine if the product having an absorption maximum at about 400 nm is bound to the DNA, dialysis experiments were performed on photolysed samples. Figure 5(a) compares the spectra of samples either illuminated for 120 min or kept in the dark, both before and after dialysis. In the non-illuminated case, the absorption de- creased markedly, owing to the expulsion of the complex out of the bag. With the illuminated sample, the absorption decreased by only lO-15%, showing that most of the product is hindered from diffusing through the dialysis membrane as would be expected if it is chemically bound to the polynucleotide. Similar results are obtained when the irradiation is carried out in either aer- ated or deaerated solution.

In order to compare these results with those obtained with the single-stranded 27-mer oligo- nucleotide, photolysis experiments were carried out in the presence of denatured calf thymus DNA, where a substantial portion will be single-stranded.

0.6

300 350 400 450 500 550 600 650 700

(a 1 Wavelength (nm)

0.6

0.5

i L 0.4

x

k 0.3

z 0.2

0.1

0.0

300 350 400 450 so0 550 600 650

(b) WZ+VelC”~th (nm)

0.7

0.6

0.5

t 5 0.4

e f 0.3 4

0.2

0.1

0.0

700

300 350 400 450 500 550 600 650 700 (c) Wavelength (nm).

Fig. 5. Changes in the absorption spectra of Ru(TAP),*+ in the presence of double stranded (a) calf thymus DNA, (b) poly[d(G- C)], (c) poly[d(A-T)] following visible light irradiation (0) and subsequent dialysis (I) compared with unirradiated samples dialysed (0) and not dialysed (0). [Ru(TAP)~*‘] =2.5 x lo-’ M, [DNA] =5 x lo-“ M nucleotide in 10 mM phosphate buffer pH 7 solution. Sample (a) was argon-degassed, samples (b) and (c) were aerated.

The same kind of hyperchromic effect is observed as with ds-DNA but with a more important am- plitude, the absorption at the maximum increasing by 70% in argon-flushed solution. In the presence of oxygen, the general behaviour is qualitatively the same as in deaerated solution (hyperchromic and small hypsochromic effect). The exact con- centration of ss-DNA in the denatured ds-DNA samples is difficult to control during the experiment, so that precise quantitative comparisons are dif-

74 MM. Feeney et al. / Photoaddition of Ru(TAP)t’ to DNA and mononuckotides

ficult to make. However, within experimental error it is apparent that there is little oxygen effect.

3.2.3. Ru(TM),~+ and synthetic poiynucleotides Double-stranded poly[d(G-C)] and poly[d(A-T)]

were used to determine how photoadduct for- mation depends on the base type in DNA. In both cases the polynucleotide concentration was 5 X 10T4 M nucleotide, Ru(TAP)~~+ = 2.5 X 10m5 M (P/D = 20), phosphate buffer = 10 n&I, pH 7.

The behaviour with poly[d(G-C)] is quite similar to that found with CT-DNA, in that increased absorption is found around 400 nm (maximum at 388 mu). Dialysis indicates that most of the visible- absorbing compound cannot diffuse through the membrane (Fig. S(b)). Compared with the be- haviour with CT-DNA there is extra absorption (although not a resolved band) around 500 nm. It is particularly significant that the species ab- sorbing near 500 nm is retained upon dialysis. A probable explanation is that more than one type of adduct is formed with this polynucleotide. As discussed above, absorption in the 500 nm region would be expected for a product of dechelation [19] and the dialysis results indicate that, if this is the case, the Ru(TAP)22+-fragment is irreversibly bound to the polynucleotide.

In the presence of poly[d(A-T)], the absorption evolution is quite different from that observed in the presence of CT-DNA or poly[d(G-C)]. Instead of hyperchromism there was bleaching of the band near 400 run and the appearance of a new band centred on 486 nm (Fig. 5(c)). These spectral changes are similar to those observed with free Ru(TAP),~ + in solution, although there is some- what more absorption at about 400 nm. Dialysis of the irradiated sample leads to a substantial decrease in the absorption of the solution but not as much as that of the non-irradiated sample. The spectrum still retains a strong absorption at about 500 mu. This would suggest that an adduct has formed in which a Ru(TAP)22+-fragment has be- come covalently coordinated to the polynucleotide. It is also interesting to note that upon irradiation the absorption at 500 nm increases more slowly in the presence of poly[d(A-T)] than for free Ru(TAP),’ + , indicating that the dechelation pro- cess is partially quenched by the polynucleotide.

3.2.4. Ru(TM)),‘+ and deoxyguanosine-5’- monophosphate The above studies suggest that the main adduct

formed with DNA involves the metal complex and guanine sites. To test this further, experiments have been carried out with dGMP or GMP

(1 X 10m2 M) in 50 mM phosphate buffer. (These conditions were used to parallel those employed earlier for emission quenching studies [lo].) No significant differences were found between GMP and its deoxy analogue. The course of reaction was monitored at different pHs and in argon- and oxygen-flushed solutions. Unlike that observed for DNA it was found that variation of these conditions has a significant effect on the reaction.

At pH 5 in argon-deaerated solution, photolysis (436 mu) induces a hyperchromic effect (with an increase of = 90%), and a small blue shift of the maximum (to 398 nm) (Fig. 6(a)). The resulting spectrum is quite similar to that observed in the presence of DNA. At pH 9, by contrast, the spectrum evolution in deaerated solution is qual- itatively the same as in the absence of dGMP. Thus the 400 mu band is bleached, and a new band appears at 500 mn (Fig. 6(c)). The rate of transformation, however, is not as rapid. This is consistent with the quenching of the excited state by dGMP [lo] reducing the yield of dechelated product.

The spectroscopic changes are also very different if the reaction is carried out in oxygenated solutions. Thus at pH 9 the band at 500 nm (expected for the dechelated complex) is accompanied by extra absorption in the 300-450 nm range (Fig. 6(d)). At pH 5 the absorption evolution in the 45Ck500 nm is not significantly dependent on the presence of oxygen, but a large increase of the absorption in the 350-450 nm region is recorded (Fig. 6(b)). The nature of the species responsible for this near- W absorption is not known at present.

4. Discussion

The lowest emissive state of Ru(TAPX’+ is of MLCT-type and has a lifetime of 220 ns in aqueous solution. Photodecomposition of the complex, how- ever, appears to proceed from a metal-centred (MC) excited state and leads to the loss of one of the TAP ligands [21]. Ru(TAP)~~+ is one of the complexes with the highest quantum yield of formation of the 3MC state and therefore deche- lation is relatively efficient [22]. As with other polypyridyl complexes, the Ru(II)(TAP),XY”+ complexes, where X and Y are water or anionic ligands, absorb at much longer wavelengths than Ru(TAP)~~+. The strong enhancement of this photo-dechelation reaction at pH > 7, is presum- ably a consequence of a ligand substitution by hydroxide ions either in the 3MC-excited state or

M.M. Feeney et al. I Photoaddition of Ru(TAP)~” to DNA and mononucleotides

300 350 400 450 500 550 600 Wavelength (nm)

C pH 9, Argon ~

300 350 400 6110

B

PH 5, O2

1

37.5 450 525 600 Wavelength (nm)

400 500 Wavelength (nm)

600

Fig. 6. Effects of pH and solution oxygenation on the absorption spectra following visible light irradiation of Ru(TAP):+ in the presence of GMP or dGMP(1 X lo-’ M). [Ru(TAP)r”] =5X 10m5 M. (a) GMP in deoxygenated 50 mM phosphate buffer pH 5 solution. Irradiation times 0, 10, 30, 60 and 90 min. (b) GMP in oxygenated 50 mM phosphate buffer pH 5 solution. Irradiation times 0, 10, 20 and 30 min. (c) dGMP in deoxygenated 50 mM phosphate buffer pH 9 solution. Irradiation times 0, 10, 30 and 60 min. (d) GMP in oxygenated 50 mM phosphate buffer pH 9 solution. Irradiation times 0, 5, 10, 20, 30, 40 and 60 min.

in a transient monodentate intermediate formed from this state.

The non-covalent binding of ruthenium poly- pyridyl complexes to DNA has been a matter of considerable attention [l, 3, 231. The prototypal Ru(phen)32+ complex unwinds the double helix when it binds to double-stranded DNA and this has been interpreted as indicating that the phen- anthroline is inserted between the base pairs of the nucleic acid (partial intercalation) [l, 5, 24, 251. However, more extensive recent studies [23] have suggested a revision of this model to one where both enantiomers of the metal complex bind to DNA by an entropically driven process and the A enantiomer induces kinking of the polynucleotide chain [23]. As a result of the binding of Ru(phen),*+ to the polynucleotide the complex excited state is protected both from molecular oxygen and from water. This leads to a substantial lengthening of the lifetime of the excited state [3, 261. The structural similarities of Ru(TAP),*+ and Ru(phen),*+ might imply that they would interact

with DNA in a similar fashion. While the bio- physical studies carried out with Ru(TAP),*+ are not nearly as extensive as those with Ru(phen),*+, it would appear (especially from thermal dena- turation studies [3]), that there is less interaction with the base pairs in double-stranded DNA and that Ru(TAP)~*+ does not show the partial pref- erence for binding to poly[d(A-T)] compared with

poMW-C)l exhibited by Ru(phen)S2+. Ru(phen),*+ and Ru(bpy),*+ have been shown to bind to single-stranded nucleic acids by a process which is substantially electrostatic [4], and the same mechanism may be presumed for Ru(TAP)~*+ . Increasing the ionic strength of the medium removes the ruthenium complex from the polynucleotide chain. Higher ionic strength is re- quired to displace Ru(phen),*+ from double- stranded DNA, because of the stronger hydro- phobic interactions discussed above, but the binding is still substantially more ionic-strength sensitive than classic intercalators.

76 MM Feeney et al. I Photoaddition of Ru(TAP)~~’ to DNA and mononucleotides

From the combined data of the electrophoretic and spectroscopic experiments presented in Section 3, it is apparent that visible light can cause irreversible photoaddition of Ru(TAP),‘+ to oli- gonucleotides, polynucleotides, or DNA. Forma- tion of adducts is found for both single- and double- stranded nucleic acids. The fact that the yield of photo-adducts is reduced by increasing the ionic strength of the medium suggests that an efficient reaction requires prior non-covalent binding of the metal complex to the nucleic acid. The relative broadness of the electrophoresis bands could imply that adducts are being formed at more than one site on the 27-mer-oligonucleotide chain. Such species should have slightly differing mobility and a population of such mono-adducts would be ex- pected to produce the observed broadening of the electrophoresis trace. The presence of multiple low-mobility bands upon prolonged irradiation is consistent with the formation of adducts at several sites on the polynucleotide chain, probably at different guanine bases.

The spectroscopic changes upon photolysis of Ru(TAP),‘+ in the presence of DNA are quite different from those in its absence. The product absorption band at about 400 nm (rather than at about 500 nm) is inconsistent with the formation of a dechelated product and more in accord with the formation of a substituted Ru(TAP)~*+ com- plex. A similar absorption band dominates the spectrum of the reaction product with poly[d(G- C)], but not that with poly[d(A-T)]. This strongly suggests that the DNA-product responsible for the 400 nm absorption band is formed by reaction with guanine. It is known that photo-excited Ru(TAP),‘+ is efficiently reduced by guanine but not by adenine or pyrimidines [lo], and it is probable that the DNA-product is formed by re- action of the reduced metal complex and an ox- idised guanine species (see below). The absence of similar products from the photochemical re- action of Ru(phen),*+ and DNA (where no elec- tron transfer takes place) is consistent with this. We have previously reported that Ru(TAP),*+ photosensitises strand breaks in closed-circular plasmid DNA with a higher efficiency than does Ru(phen),2+, and we attributed this to cleavages induced by the guanine radical cation. The absence of cleavage bands in the 32P-labelled 27-mer sam- ples shows that the quantum yield for adduct formation is much higher than that of strand cleavage.

The rate of formation of the 500 nm absorbing species is greatly reduced when Ru(TAP),*+ is photolysed in the presence of poly[d(A-T)]. This

feature may be attributed to reduced access of H20 and anions to the metal complex, when it is bound to the polynucleotide, and consequent suppression of dechelation. Alternatively it may be a consequence of a decreased quantum yield for formation of the 3MC state due to the rigidity imposed on the complex ion by the polynucleotide. Such an effect has been observed for Ru(TAP)~*+ in other media [27] and more recently in poly[d(A- T)] [28]. It may be noted, however, that the dialysis experiment suggests that some of the Ru(TAP),*+- fragment formed is firmly anchored to the nucleic acid. Thus it is probable that in this case photolysis has caused one TAP ligand to be substituted by an adenine. Coordination complexes with adenine ligands (especially via the Nl position) have been reported in the literature [29].

The UV-vis spectrum of the reaction product with calf thymus DNA is not affected by whether the reaction is carried out at pH 5 or pH 9 or by the presence of oxygen in the solution. This spectrum is very similar to that obtained upon reaction of Ru(TAP),~+ with dGMP in deaerated solution at pH 5. Under these conditions the Ru(TAP),* + is known to undergo dynamic electron transfer quenching. The [Ru(TAP),(TAP’-)]+ formed subsequently protonates (pK, = 7.6) [lo] and the guanine radical cation deprotonates (pK, = 3.9) [30].

[Ru(TAP),(TAP’-)]+ +H+ -

[Ru(TAP),(TAPH’)12+ (2)

G’+ - G(-H)‘+H+ (3)

Coupling of these two radical species may be responsible for the observed photoadduct. This could occur either by reaction of the isolated species

[Ru(TAP),(TAPH’)12+ + G( -H)’ -

Photoadduct (4)

or directly within the ion pair formed after initial photo-electron transfer

{[Ru(TAP),(TAP--)] +, G’+} -

{[Ru(TAP),(TAPH-)I’+, G( -H)‘} (5)

{[Ru(TAP),(TAPH’)]~ +, G( - H)‘} -

Photoadduct (6)

The proton transfer (reaction (5)) will be in com- petition with back electron transfer.

M.M. Feeney et al. / Photoaddition of Ru(TAP)t’ to DNA and mononucleotides 77

{[Ru(TAP),(TAP’-)] +, G+} j

{[RW’AW2+, Gl (7)

On DNA the initially-formed reduced ruthenium complex and guanine radical cation would be expected to remain close together for a longer period than in the ion pair formed from Ru(TAP),‘+ and dGMP. This would allow the back electron transfer (eqn. (7)) to be more efficient (as has been verified by the lower yields of transient photoproducts found by laser flash photolysis) [lo]. The observed independence of the nature of the photo-products with DNA on pH and solution oxygenation can be explained by the protection of the reduced ruthenium complex and oxidised guanine species from reactions with oxygen or buffer ions. In contrast, the photoproducts from dGMP, when they have escaped from the ion pair, may react with these reagents.

Ru(TAP)~‘+ is known to photosensitise strand breaks in plasmid DNA with a significantly higher efficiency than Ru(phen),‘+ [3, 91 and this higher yield has been attributed to reaction of the guanine radical cation. The absence of direct cleavage bands in the gels of the 27-mer samples indicates that adduct formation proceeds with a much higher quantum yield.

The precise molecular structure of the photo- adduct is unknown at present and will require the isolation and characterisation of the product by NMR, fast atom bombardment (FAR) mass spec- trometry etc. Such studies are planned along with experiments to determine the effect of adduct formation on the molecular biological function of DNA.

Acknowledgments

The authors thank Prof. C. Helene and Dr. T. LeDoan for the gift of oligonucleotides. The Trinity College group are grateful to the EC Science programme (Project SC1 009) for financial support. J.-P.L. thanks the FNRS (Fonds National pour la Recherche Scientifique) for a Fellowship.

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