1H NMR Study of the Enantioselective Binding of λ- and Δ-[Ru(bpy)2(m-bpy-GHK)]Cl2 to the Deoxynucleotide Duplex d(5'-C1G2C3G4A5A6T7T8C9G10C11G12-3')2

The interaction of the diastereomeric complexes Λ- and Δ - [Ru(bpy)2(m - GHK)]Cl2, (GHK = glycine-histidine-lysine) to the deoxynucleotide duplex d(5'-CGCGAATTCGCG-3')2 was studied by means of 1H NMR spectroscopy. The diastereomers interact with the oligonucleotide duplex differently. The Δ - [Ru(bpy)2 (m - GHK)]Cl2 is characterized by major groove binding close to the central part of the oligonucleotide, with both the peptide and the bipyridine ligand of the complex involved in the binding. The λ - [Ru(bpy)2 (m - bpy - GHK)]C2 binds loosely, approaching the helix from the minor groove. The NMR analysis shows that the peptide (GHK) binding has a determinative role in the interactions of both diastereomers with the oligonucleotide.


INTRODUCTION
Over the last decades, DNA-binding low molecular weight compounds have found considerable application as chemotherapeutic agents. On a molecular basis, their cytotoxic effect originates from their interaction with the DNA double helix, often in a non-covalent way. This type of.reversible interaction takes place in three primary ways/1/: (i) surface binding which is generally non-specific and primarily electrostatic in origin e.g. in the case of multiple charged simple cations such as magnesium and cations of simple organic amines/1/, (ii) groove binding interactions e.g. netropsin/2/, distamycin/3/, Hoechst 33258/4/and SN 6999 /5/, and (iii) intercalation of a planar or approximately planar aromatic ring system between base pairs as in the case of ethidium/6/, adriamycin /7/ and daunomycin/8/.
There is increasing interest in the chemical design of small molecular mimics of DNA-binding proteins playing an important regulatory role in controlling replication and transcription of genomic material. The sequence-specific binding of such molecules to DNA might affect replication, transcription or other physiological functions of the cell. The extremely high specificity upon recognition and binding to DNA target sites, of protein molecules such as restriction endonucleases, provides a very good basis for the design of sequence-specific DNA binders. The recognition process of the cognate DNA fragment by these proteins often involves base-specific interactions between the DNA bases and a recognition loop of amino acid residues, comprising mainly of hydrogen bonding schemes and Van der Waals interactions/9-11/.
DesPite their substantial contribution to sequence specificity, individual recognition peptide sequences lack the ability to bind tightly to DNA, a property sufficiently provided by the non-specific contacts made by the protein backbone/12-14/. On the other hand, positively-charged metal complexes can associate within the grooves of polyanionic DNA, with the binding being further stabilized by a variety of intermolecular forces such as Van der Waals, hydrophobic interactions and hydrogen bonding. Footprinting studies have shown that site-specific recognition by conjugation of small peptides to metal complexes can be successful/15-16/. Therefore, on the basis of the known high affinity of ruthenium polypyridine complexes towards DNA binding/17-19/, [Ru(bpy)3] 2+ was chosen for conjugation with the recognition peptide sequence.
As a starting peptide sequence, Gly-His-Lys (GHK), the growth modulating factor was chosen. Its water solubility as well as its capacity to facilitate transportation within the cell comprise essential characteristics for a DNA-targeted molecule. Moreover, its Cu(II) complex is known to adopt a specific orientation when interacting with the minor groove of DNA, thus introducing specificity in DNA binding/20/. The DNA binding propertie; of our designed complex were tested on the spectroscopically and crystallographically well-characterized Dickerson-Drew dodecamer, d(CGCGAATTCGCG)2 which forms a self-complementary duplex whose structure is understood in detail/21/.

EXPERIMENTAL
The measurements were made on a Varian Unity-500 MHz instrument. 1D-JHNMR spectra were recorded at 303 K into 4096 data points, with a 6024 Hz spectral width after 128 transients. 1H NOESY spectra were recorded in phase sensitive mode with a total 2048 X 256 points for mixing time 200 to 400 ms, while IH ROESY spectra at mixing times (rm) 60-120 ms. 1H DQF COSY spectra were collected using TPPI method, in a spectral width of 3125 Hz with total 2048 X 256 points and a relaxation delay of 1.5 s. The amounts of the oligonucleotide were estimated by weighing and the concentration of the sample was determined using its absorption at 260 nm. In all NMR experiments carried out, a 100 mM Na2HPOnfNaHPO4 (pH 7.00) buffer was used. The lyophilized samples were dissolved in DO (99.96) and lyophilized again to dryness.
The 1D H NMR spectra were recorded on sample concentration~100 OD260 units while the 2D NOE experiments in more concentrated samples (~300 OD260). JH NMR spectra of the labile oligonucleotide protons were recorded in 90% HzO 10% D20 (field-frequency lock). No internal chemical shift reference was added to the samples. A number of NOESY cross-peaks between protons of the metal complex and protons of the oligonucleotide were observed indicating interproton distances less than 5 A (Figures 3 and 4). In the case of A-[Ru(bpy)2(m-GHK)]C12, distances of less than 5/ were observed between the aromatic protons of m-bpy ligand and the H2" protons of the A5, A6 and C9 bases (in the complementary strand) all facing the major groove of the helix. The NH protons of the peptide backbone show cross-peaks with protons that are also accessible from the major groove, lntermolecular NOE's between the lysine aliphatic side chain and the oligonucleotide protons indicate that this part of the peptide is located close to the helix.

A [Ru(bpy)2(m-GHK)] 2+ and the
On the other hand, the intermolecular NOE contacts between the A-[Ru(bpy)2(m-GHK)]C12 and the oligonucleotide are significantly less compared to the Aenantiomer suggesting a looser binding of the former. A few of these take place between the ligand m-bpy protons H3', H5' and the A6HI 'and C9HI' and between the peptide backbone Gly-NH and the sugar proton HI' of T8, all located in the helix minor groove. In contrast to the Aisomer, no cross-peak was observed between the other two bpy ligand protons of the complex and the oligonucleotide indicating that only the m-bpy-GHK moiety of the complex binds to the d(5 '-CGCGAATTCGCG-3 ")2.
Major and minor grooves differ significantly in electrostatic potential, hydrogen bond characteristics, steric effects and hydration. Therefore, many proteins exhibit binding specificity primarily through major groove interactions while small groove binding molecules like netropsin and distamycin generally prefer the minor groove of DNA /1/. Unlike the other non-intercalative molecules, A-[Ru(bpy)z(m-GHK)] 2+ forms specific contacts with the walls of the major groove of DNA. This can be partially interpreted by the tendency of the bpy ligand to align itself close to the basepairs /2 8/, a fact already seen in the significant shifts of the aromatic bpy protons. This alignment is probably hindered in the case of the A-isomer where the bpy ligands do not participate in DNA binding, whereas the enantiomer binds from the minor groove.
In order to investigate the site-specificity of [Ru(bpy)/(m-GHK)] 2/ in DNA binding, its interaction with d(CGCGATCGCG)z, also a [3-type DNA, is under study. Preliminary results indicate weaker interaction between both isomers and the decanucleotide duplex compared to the dodecanucleotide. The binding of the A-isomer takes place from the major groove in the region of A5/T8, A6/T7 base extended until the ends of the dodecanucleotide. The A-enantiomer interacts very weakly with the double helix (shifts less than 0.05 ppm), probably through electrostatic interactions between the metal complex and the oligonucleotide backbone. Complementary molecular modeling studies are currently underway.

Conclusion
In conclusion, the NMR experiments presented show that the [Ru(bpy)2(m-GHK)] 2+ complex binds differently to oligonucleotides. Major groove binding to d(CGCGAATTCGCG)2 was observed for A-[Ru(bpy)z(m-GHK)]C12 with the histidyl-lysyl part of the peptide ligand recognising the adjacent C9G10Cll of the oligonucleotide sequence, thus placing the bpy ligands close to the central part. The A-isomer approaches the double helix from the minor groove, with the aromatic protons of ligand m-GHK interacting weakly ( Figure 5).

H NMR Study of the Enantioselective Binding
The site-specificity in DNA binding of the enantiomeric ruthenium complexes is indicated by preliminary results of their interactiofi with the nucleotide duplex d(CGCGATCGCG)2, where the change of the central part of the oligonucleotide sequence affects dramatically the ability of the complex to recognise and associate to its binding site.