DNA and RNA Cleavage Mediated by Phenanthroline ... - Springer Link

DNA and RNA Cleavage Mediated by PhenanthrolineCuprous Oligonucleotides: From Properties to Applications J.-C. FRANCOISl, M. FARIA2, D. PERRIN 3, and C. GIOVANNANGELI l

1 Introduction Several metallic ions when bound to a ligand bringing them in close proximity to nucleic acids are able to cleave phospho diester bonds, in the presence of oxygen. Redox-active iron and copper ions induced DNA cleavage in the presence of bleomycin, a well-known antitumoral antibiotic interacting with DNA double helix. Small molecules such as ethylene-diamine tetraacetic acid (EDTA), porphyrin, and 1,10-phenanthroline (OP) chelate redox-active divalent metal cations to form EDTA-Fe(II), porphyrin-Fe (I!), and OP-Cu(I) complexes, and also induce DNA cleavage with production of hydroxyl radicals or metal-oxo derivatives leading to hydroxylation via oxidative insertion of deoxyribose or ribose moieties that in turn leads to cleavage of phosphodiester bonds via a variety of elimination reaction schemes. Since their discoveries, the cleavage properties of these metal complexes have been used in footprinting experiments to analyze the structure of protein-nucleic acid complexes. These cleavage reagents have also been tethered to larger molecules such as oligonucleotides, proteins, and intercalating agents, which confer additional specificity. This review will focus on the 1,1O-phenanthroline-copper complex and its cleavage properties when conjugated to macromolecules, especially in the case of oligonucleotides. For other use of OP-Cu complexes, one can refer to previous reviews (Sigman et al. 1993b; Chen et al. 2001; Hermann and Heumann 2000; Milne et al. 2001; Muth and Hill200l). We lLaboratoire de Biophysique, Museum National d'Histoire Naturelle, INSERM U.565, CNRS UMR8646, 43 rue Cuvier, 75231 Paris Cedex OS, France 2Department of Biochemistry, Instituto de Quimica, Universidade de Sao Paulo, caixa postal 26077, Sao Paulo-SP, Brasil 3Chemistry department, Room A337,Chem/Phys Building, 2036 Main Mall, UBC Vancouver, Canada Nucleic Acids and Molecular Biology, Vol. 13 Marina A. Zenkova (Ed.) Artificial Nucleases © Springer-Verlag Berlin Heidelberg 2004

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present here a few examples of the utilization of these cleaving 1,10-phenanthroline-copper conjugates in biochemical studies as structural probes, as artificial ribo- and deoxyribonucleases and as modulators of gene expression.

2 The 1,10-Phenanthroline-Cuprous Complex 2.1 Background The nucleolytic activity of 1,1O-phenanthroline (OP) in the presence of cuprous ions was discovered in 1979 by D. Sigman and coworkers. Initially, they thought that the inhibitory effect of 1,10-phenanthroline, observed on DNA polymerase activity of the E.coli DNA polymerase I was simply due to the chelation effects of phenanthroline as Zn2+, was supposed to be a metal ion essential for polymerase catalytic activity. Past studies had employed phenanthroline as a specific chelator for Zn2+ and to a lesser extent for Mg2+, Mn 2+ and Co2+ to probe the specific roles of various active site metals in metalloendoprotease activities. In the case of DNA Pol I, Sigman eventually demonstrated that 1,1O-phenanthroline inhibited polymerase activity not by chelating Zn2+ metal ions, but by degrading DNA template in the presence of contaminating amounts of copper ions that had engaged in redox-cycling in the presence of DTT giving rise to short DNA fragments presenting 3'-phosphate extremities (Sigman et al. 1979) that were nonextendable termini for DNA polymerase. This insight not only explained the observed inhibition of polymerase activity but opened a new field based on its use as artificial nucleases. In the following report, we have summarized several aspects concerning cleaving properties of phenanthroline-copper complexes and their applications in biological studies. The mechanism of nucleic acid cleavage by the phenanthroline-cuprous chelate is quite well characterized. Nucleic acid scission by 1,10-phenanthroline requires the activation of copper ions by reducing agents and oxygen, in order to drive the Cu(I)Cu(II) redox cycle, generating activated cupryloxo species. Cleavage could be carried out by adding various reducing agents such as ascorbate and thiols (mercaptopropionic acid, dithiothreitol). Kinetics rates of cleavage may depend on the reducing conditions (Veal et al.1991). Hydrogen peroxide, which is an essential co-reactant for the chemical nuclease activity, was generated in situ by the redox cycle but can also be added exogeneously along with thiols enhance DNA scission (Reich et al. 1981). Among the nucleic acid conformations, B-DNA is the most efficiently cleaved by phenanthroline cuprous complexes. The first step in the nucleolytic reaction of phenanthroline with DNA involves the reversible binding of the active complex formed by two phenanthrolines and one cuprous ion to the double helix (Fig.I). This (OP)2CU+ chelate, which exhibits tetrahedrical structure, could partially intercalate into DNA, first suggested by spectroscopy and

DNA and RNA Cleavage Mediated by Phenanthroline-Cuprous Oligonucleotides

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Fig. 1. Phenanthroline derivatives. (1) Tetrahedral 2:1 1,1O-phenanthroline-cuprous complex, (2) 2,9 dimethyl-phenanthroline known as neocuproine, (3) AoP and (4) MoP. For (1), arrows designate the positions used for conjugation (see Fig. 2). Compounds (3) and (4) are mainly used as footprinting reagent on rRNA targets

viscometry studies (Veal et al.1991; Veal and RillI99I). By contrast, a derivative of (OP)2CU+' the 2,9 dimethyl-phenanthroline-copper chelate associated with double helix by binding at the surface of DNA in a non-intercalating mode (for detailed discussion about structures of the reactive intermediate see Sigman et al.1993b; Fig. I). The second step is the oxidation of the DNA-bound cuprous ion by hydrogen peroxide resulting in non-diffusible copper-oxo species that attacks the deoxyribose predominantly atthe CI' -position via hydroxide insertion. A series of reactions occur resulting in strand scission and in production of cleavage fragments bearing 5'- and 3'-phosphomonoester termini, bases and 5-methylene furanone (5-MF) (Goyne and Sigman 1987; Sigman et al. 1993b). Several reaction pathways for cleaving DNA with copper-oxo species were proposed involving either oxidative attack of Cl'and/or C4'-position of deoxyribose. Recently, mass spectrometry studies of DNA scission by 1,10phenanthroline copper complex have demonstrated that carbonyl oxygen of one cleavage product, 5-MF, resulting from Cl' attack, was derived from water and not from hydrogen peroxide (Meijler et al.1997). The authors proposed a chemical mechanism where the first appearing cleavage product bearing 3'phosphate resulted from strand scission and oxidative attacks of copper-oxo species on CI'-carbon of deoxyribose. The second product which exhibited a

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5' -phosphorylated terminus was obtained after water attack that incorporate oxygen into the Cl'-position of desoxyribose further leading 5-methyl furanone (Meijler et al. 1997). Copper complex of phenanthroline and its derivative could also produce direct DNA strand scission via C5'-oxidation (Oyoshi and Sugiyama 2002; Pitie et al. 2000a). Nevertheless, it was recently confirmed by kinetic studies that the major pathway for DNA cleavage induced by phenanthroline-copper complex involves C'l-oxidation as previously proposed by D. Sigman and co-workers (Bales et al. 2002). In addition to B-DNA as the preferred substrate for (OP)2CU+' Z-DNA and single-stranded DNA are also cleaved but much less extensively (Sigman et al. 1993b). Because the C1' hydrogen, the preferred cleavage site, extends into the DNA minor groove, the chemical mechanism of the cleavage reaction requires that (OP)2CU+ complex attacks DNA from a binding site within this DNA domain. Double-stranded RNA is not labile to scission by (OP)2CU+ since a much shallower minor groove is present on A form compared to B form (Muth and Hill 2001). Nevertheless, this metal complex has been used for cleavage of RNA molecules as mentioned in the following.

2.2 Structural Probes for Nucleic Acid-Containing Complexes Ligand binding to nucleic acids as well as structures of nucleic acids themselves may locally influence the cleavage activity of nucleases. Various techniques have been used to study binding sites of proteins or drugs as well as to monitor - at single nucleotide resolution - induced structural changes in nucleic acids. These studies are based on the use of either natural endonucleases, such as DNAse I, or chemical nucleases such as EDTA-Fe and phenanthroline-Cu. Indeed, the phenanthroline-copper chelate was extensively used as a cleavage reagent for DNA since it was shown that DNA cleavage efficiency is sequence- and structure-dependent. DNA lesions such as bulged bases within hairpin structures are recognized and preferentially cleaved by (OP)2CU+ (Williams et al.1988). Despite the fact that copper chelate does not exhibit any clear nucleotide preference, the sequence most sensitive to scission appears to be the trimer TAT predominantly cleaved at the central adenosine (Sigman et al. 1993b; Veal and Rill 1989). Consequently, (OP)2CU+ complex was used as footprinting reagent to study chromatin or DNA structural dis torsions induced by protein or oligonucleotide binding (Basak et al. 2001; Chen et al. 2001). Several years ago, we demonstrated that phenanthroline-copper complex could be used to monitor binding of an oligopyrimidine third strand to a double helix target via triplex formation (Francois et al. 1988). The triplex-forming oligonucleotide recognizes DNA in the major groove of an oligopyrimidine-oligopurine sequence by forming base triplets, T.AxT and C.GxC+. DNA cleavage occurred only on double-stranded sequence and at the junction


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between the double and triple helices leading to a triplex footprint. Moreover, we observed that the cleavage pattern on opposite strands of the duplex target obtained with the (OP)2CU+ footprinting reagent were asymmetric. The cleavage sites were staggered to the 3' -side, indicating that the metal chelate was bound in the minor groove of DNA but was sensitive to subtle conformational changes within the minor groove induced by third strand binding within the major groove (Francois et al. 1988). More recently, a highly specific triplex ligand (a benzoquinoquinoxaline derivative), previously reported to be a strong triplex stabilizer, has been linked either to an EDTA molecule (Zain et al. 1999) or to an OP moiety (Zain, pers. comm.). In the presence of Fe(II) or Cu(I), respectively, these conjugates were able to specifically cleave DNA at a triplex site. This kind of conjugate may prove useful as a specific probe of triple helical structures in DNA. Phenanthroline-copper can be used to probe structural nuances or accessibility of single-stranded regions of DNA interacting with protein machineries (Sigman et al. 1993b). For example, it was shown that the chemical nuclease activity of (OP)2CU+ cleaves single-stranded or strained DNA at transcription start sites of active genes. When transcription initiation occurs, the so-called open complex formed within the RNA polymerase bound to DNA was recognized and cleaved by phenanthroline-copper chelate (Mazumder et al. 1993). No scission of the non-template strand is observed. The redox inactive 2:12,9dimethyl-I, 1O-phenanthroline copper(I) complex protected the transcription start site from scission by competing with the binding site of (OP)2CU+ on the promoter sequence (Gallagher et al. 1996b). Interestingly, it was recently demonstrated in prokaryotic cells that (OP)2CU+ was a versatile probe to assess the in vivo interaction of proteins with DNA (Basak and Nagaraja 2001). This metal complex in presence of exogenous thiols was successfully used to study structural differences in DNA, interaction of RNA polymerase with promoter, interaction of specific DNA-binding proteins with their cognate sites and recruitment of transcription machinery. Chelates of OP-Cu and EDTA-Fe have been also extensively used to study protein-RNA interactions and to probe structures of biologically important RNAs at single nucleotide resolution. In addition, the folding kinetics of higher-order RNA tertiary structures could also be monitored using timeresolved (OH·) footprinting methods as demonstrated with EDTA-Fe complexes (Hampel and Burke 2001). Phenanthroline-copper was also a suitable probe to determine nucleotide distance in RNA due to the fact that diffusion of copper-oxo species will only permit the cleavage of very neighboring nucleotides (Hermann and Heumann 1995). Phenanthroline and its derivatives have been adapted for use in RNA structural studies, especially the structures of ribosomal RNA in situ (Muth and Hill 2001, Fig. 3). It was shown that phenanthroline-copper complexes cleaved phospho diester bonds in rRNA in specific regions of a constrained nature.

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Specific cleavage using two untethered phenanthroline-Cu(II) derivatives, 5acylamide-I,IO-phenanthroline (AoP) and N-acetyl-2-aminomethyl-I,10orthophenanthroline (MoP) were observed in selected regions in E. coli 23S rRNA (containing nucleotides 60-66 and 87-100), suggesting that these regions were involved in dynamic structures allowing phenanthroline intercalation in the intact ribosome (Muth et al.1999).AoP which is a better cleaving agent than MoP, exhibited increased discrimination among structures compared to MoP (Fig. 1). Secondary structures of the iron responsive element (IRE) located in the 5' untranslated region of ferritin mRNA were analyzed in vitro using both ribonucleases and chemical nucleases such as phenanthroline-copper (Wang et al. 1991, Fig. 3). The advantages of (OP)2CU+ include accessibility of singlestranded regions too small for bulky enzymes and the ability to cleave nucleic acids under a wider range of experimental conditions than ribonucleases. Indeed, this chemical nuclease was able to detect looplbulge structures in IRE of ferritin mRNA in cells, demonstrating that secondary structures detected previously in vitro did also form in vivo (Ke and Theil 2002). Importantly, Cuphenanthroline has been successfully used in cellular footprinting studies as it has the advantage of being relatively hydrophobic and then should cross easily cell membranes. In addition, an IRE-binding protein expressed from a cotransfected plasmid protected the hairpin structure from (OP)2CU+ cleavage in vivo, demonstrating its binding to this secondary structure insides cells (Ke and Theil 2002).

3 Phenanthroline Conjugates 3.1 Conjugation of Biomolecules to Phenanthroline The chemical nuclease activity of 1,10-phenanthroline-copper could be directed to various targets, such as DNA and RNA and even proteins. Indeed, the cleavage activity of phenanthroline-copper complex, which had been discovered as nucleolytic activity, could be re-directed towards protein in order to obtain proteolytic activity. The first example of this was observed when 1,10-phenanthroline was linked to a single cysteine of E. coli lactose permease, producing protein cleavage in the presence of reductant, 02 and Cu(II). Up to 30 % of scission of the protein backbone was observed (Wu et al. 1995). More recently, phenanthroline conjugated to a sulfonamide inhibitor of carbonic anhydrase has been used to cleave specifically within the active site cavity of the enzyme (Gallagher et al. 1998) (compound 5, Fig. 2). With regard to nuclease or ribonuclease activities, in order to achieve specific recognition of nucleic acid targets, different conjugates between phenanthroline or its derivatives were tethered to various ligands that recognize DNA or RNA. For example, 1,10-phenanthroline has been conjugated to oligonu-


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Fig. 2. Phenanthroline conjugates. 1,10 phenanthroline was linked to 4-carboxybenzene-l-sulfonamide, an inhibitor of carbonic anhydrase (5) and to an A. T specific minor groove ligand, the Hoechst dye 33258 (6). Clip-Phen, built by conjugation of two phenanthrolines in order to favor a 2:1 OP:Cu ratio, was tethered to a distamycin derivative (7). RNA could be derivatized internally using 5-allylamine-UTP and 5-iodoacetamido-l, 10phenanthroline (8) and could induce cleavage in R-Ioop structures. Neocuproine internally linked to oligonucleotide could act as a ribozyme mimic (9)

cleotides, minor groove binders such as Roescht -33258 (Chen et al. 1993b) or DNA binding proteins such as E. coli Trp repressor (Chen et al. 1993b; Sutton et al. 1993), Fis (factor for inversion stimulation) (Gallagher et al. 1996a; Pan et al.I994), and M13 gene V protein (Chen et al.I998).All these chimeric compounds, which are targeted to RNA or DNA, lead to indirect oxidative cleavage of the phospho diester bonds of the nucleic acid. Various OP derivatives have been used for conjugation. A new ligand with two phenanthrolines linked via their C2-carbon by a short flexible arm, named Clip-Phen was synthesized and linked to spermine (Pitie and Meunier 1998; see Clip-Phen part in compound 7, Fig. 2). The arm of Clip-Phen was adapted for having readily both phenanthrolines on the same copper ion, in order to achieve a Phen/Cu ratio of 2:1, which was supposed to be the cleaving active metal complex. Clip-Phen exhibited more efficient DNA cleavage activity than a single 1,10-phenanthroline moiety. We have also previously shown that an octathymidylate covalently linked via its 3' -end to two phenanthrolines through their C5-carbon has been able to cleave a single-stranded DNA target containing a d(A)8 sequence at specific sites more efficiently than an octathymidylate linked to only one phenanthroline (Francois et al.1989b).

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The linking of DNA binders to phenanthroline derivatives confers additional and often highly specific recognition properties compared to the otherwise non-specific activity of the phenanthroline nucleus. For example, untethered phenanthroline which has apparently no strict sequence specificity within B-DNA could recognize A.T rich regions in this B-DNA when it was covalently linked to a minor groove binder, Hoechst dye 33258 (Chen et al. 1993b) (compound 6, Fig. 2). Clip-Phen was also tethered to distamycin, a tri-N-methylpyrrole derivative which tightly binds to the minor groove of double stranded DNA, via various length of linker arm and was targeted to a DNA restriction fragment (Pitie et al. 2000b) (compound 7, Fig. 2). It was recently shown that coupling of Clip-Phen to the intercalators acridine and 6chloro-2-methoxyacridine increased DNA cleavage efficiency of the copper complexes (Boldron et al. 2002). In the following, we will focus on the phenanthroline chimeric compounds derived from oligonucleotides which permit targeting to RNA and DNA molecules.

3.2 Neighborhood Sensors Conjugation of oligonucleotides to chemically active phenanthroline provides opportunities to combine the recognition properties of oligonucleotides with the metal chelating and redox-cleavage properties of phenanthroline. The conjugate consists of three important parts : the oligonucleotide moiety which provides sequence-specific recognition, the linker and the specific agent which is 1,10-phenanthroline (Arimondo et al. 2002). The arm length and the position of linkage on the phenantholine rings played important roles in the efficiency and accuracy of target cleavage (Francois et al. 1989a; Gallagher et al. 1996a; Sigman et al.1996). In general, 5' -substituted phenanthroline gave better cleaving reagent than 2' -substituted phenanthroline. As (OP)2CU+ was successfully used to probe ribosomal RNA structure in situ, Hill and coworkers targeted the activity of various phenanthroline bioconjugates to elucidate structures involved in ribosomal complexes (Muth and Hill 2001; Fig. 3). Firstly, tRNA-Phe was linked to phenanthroline via the introduction of 4-thiouridine in the tRNA sequence. This tRNA-Phe-OP caused cleavage mainly in the 23S rRNA when it was bound to precise sites in the ribosomal complex (namely, to P and E sites of 70S ribosome; Hill et al. 1995). This phenanthroline-conjugate allowed an analysis of the neighborhood of the rRNA surrounding the tRNA binding site (Bullard et al. 1995, 1998). Secondly, one could modify mRNA with phenanthroline in such a way that cleavage would occur at the closest nucleotides in rRNA. In the 16S rRNA, specific nucleotides (528-532, 1053-1055, 1196, and 1396-1397) were cleaved by phenanthroline and therefore appeared very close to mRNA position +5 where OP was linked within ribosomes (Bucklin et al. 1997). Thirdly, using DNA oligonucleotide tethered via their 5' -end to phenanthroline and comple-

DNA and RNA Cleavage Mediated by Phenanthroline-Cuprous Oligonucleotides Fig. 3. Untethered phenanthroline derivatives, as AoP, were used as structural probes for ribosome (a) or for mRNA (e). OP incorporations at random sites into tRNA (b), or at a specific site into the mRNA (d) gave also structural information about rRNAs in the ribosome machinery.Oligonucleotidephenanthroline conjugates were targeted to decoding region of rRNAs (c) or to hairpincontaining nucleic acids (f,g)

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mentary to several nucleotides in the decoding regions of rRNA, it was demonstrated that ribosome conformation was altered by the binding of tRNA activating the ribosome (Muth et al. 2000). Dynamic movements as well as regions in close proximity in the 30S ribosomal subunit could thus be studied by the targeted chemical nuclease. Similar experiments were conducted with EDTA-Fe(II)-derivatized oligonucleotides that gave more delocalized cleavage than OP-oligomer (Bowen et al. 2001). Oligonucleotides tethered to phenanthroline also have been used in order to identify nucleic acid structures including determination of proximities. They have been targeted to single-stranded DNA containing hairpin structures (Francois et al. 1994). Depending on the choice of the target site, the oligonucleotide could be designed to bind two single-stranded regions. Consequently, specific DNA cleavage took place, on the complementary single-stranded sequence demonstrating that sequence flanks of the hairpin could be accommodate to bind an oligonucleotide forming a three-way junction as shown in Fig. 3. We also used the cleavage properties of OP-oligomers to demonstrate that oligonuclotides could recognize a hairpin structure and form contiguous double- and triple-stranded regions by simultaneously binding to single- and double-strand regions (Francois and Helene 1995; Fig. 3). The phenanthrolineoligonucleotide which was obtained by the addition of the 5(w-bromohexanamido)-I,lO-phenanthroline to a 5'-thiophosphate lead to efficient cleavage on both strands of the hairpin duplex. Moreover, the cleavage sites were shifted toward 3' -end of the target, suggesting that cleavage took place in the minor groove. In this case, the oligonucleotide was bound to hairpin major groove, and the phenanthroline ring was intercalated within the stem structure as described for a triple helical complex (see below).

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3.3 Oligonucleotide-Phenanthroline as Site-Specific Ribonuclease A great variety of artificial chemical ribonucleases has been described to produce specific cleavage of RNA and could therefore be useful in the antisense field (Haner and Hall 1997). Specific inhibition of gene expression by antisense oligonucleotides targeted to complementary RNA sequences has been demonstrated in a variety of in vitro models. One of the most efficient mechanisms of action of these antisense oligomers requires cellular recruitment of RNase H, a specific ribonuclease. This enzyme recognized the hybrid formed by antisense oligodeoxynucleotides bound to mRNA and cleaved the RNA moiety (for review see Myers and Dean 2000). Since the objective of antisense strategy is to inhibit RNA translation in cells, antisense oligomers should be chemically modified in order to be resistant to cellular nucleases. Chemists have designed many intracellularly stable oligonucleotides useful in cell applications, such as phosphorothioates, methylphosphonates, phosphoramidates. Most modified antisense oligonucleotides, except the phosphorothioate ones, have lost their ability to induce ribonuclease H, despite the fact that they still recognized their complementary sequences on mRNA, but could not induce specific cleavage. Some of these modified oligonucleotides were effective as inhibitors of protein synthesis acting as translation physical blockers, mainly when they were targeted to non-coding region (5'UTR, 3'UTR, and splicing sites), but were totally ineffective when targeted to coding regions. In order to overcome this problem, the cleaving reagent, 1,10-phenanthroline, could be covalently linked to antisense oligonucleotides. Activation of these conjugates should irreversibly cleave the mRNA target and inhibit its translation. In other words, one would like to replace ribonuclease H activity by using the chemical nucleolytic properties of phenanthroline. From pioneering work in Sigman's group, it appears that phenanthroline tethered to 21-nucleotide-long oligonucleotides cleaved both RNA and DNA with similar kinetics targets (Chen and Sigman 1988). Nevertheless, the efficiency of these OP-oligonucleotide was quite low: 20 % of the mRNA is specifically cleaved after incubation for 2 h at 37°C following heat denaturation for increase of hybridization. We have previously shown that octathymidylates tethered to phenanthroline which were able to cleave r(A)10 target in a specific manner, allowed us to determine relative orientation of the binding of IX oligomers on RNA targets (Sun et al. 1988). In a more recent study, we have tried to cleave mRNA of Harvey ras oncogene using modified oligonucleotides covalently linked at their extremities to 1,10 phenanthroline (Godard et al. 1994). Dodecadeoxyribonucleotides built with por IX anomers and targeted to the codon 12 region present in human Ha-ras mRNA were unable to cleave RNA targets containing their complementary sequence due to their stable secondary structure. Indeed, these phenanthroline-oligomer conjugates induced efficient cleavage of the corresponding DNA targets. We have succeeded in specifically degrading Ha-ras mRNA by addition of a 20-


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nucleotide-long "helper" oligonucleotide targeted to immediately neighboring sequences (Godard et al. 1994). It is likely that cooperative binding of the helper oligonucleotide increases the binding affinity of the phenanthrolinedodecamers towards RNA target, allowing the cleavage reaction to take place. A 17-nucleotide-Iong oligonucleotide tethered to phenanthroline was also targeted toward ~-globin messenger RNA, and found to induce efficient specific cleavage in the absence of any helper oligonucleotide, probably reflecting a sufficient accessibility of the targeted RNA sequence. More recently, it has been shown that the replacement of 1,1 O-phenantholine by the ligand 2,9-dimethyl-phenanthroline (neocuproine) into a 17nucleotide-long oligomer exhibits at least 30-fold improvements in cleavage efficiency of RNA targets (Putnam et al. 2001). Around 80 % of specific cleavage of a large RNA target was achieved after 10 h at 37°C. In these conjugates, phenanthroline derivatives were internally incorporated in the oligonucleotides using a serinol-neocuproine building block (Fig. 2). These new kinds of efficient ribozyme mimics appear very promising for use on ribonucleic acids. It remains to be determined if OP-oligonucleotide-induced RNA cleavage could occur in cells and how efficient it could be compared to RNAse Hinduced RNA degradation.

3.4 Oligonucleotide-Phenanthroline Targeted to Double-Stranded DNA

3.4.1 Artificial Endonucleases for in Vitro Applications Site-specific nucleases that cleave double-stranded DNA at any selected sequence would be of interest for analysis of chromosome and gene structures. Different ways to achieve specific cleavage at any DNA sequence are based on the use of oligonucleotides tethered to 1,10-phenanthroline targeted to DNA via either R-Ioop or triplex formation (Chen et al. 1993a; Francois et al. 1989a; Fig. 4). In order to obtain sequence-specific scission of DNA, Sigman's group has proposed to induce the formation of partially denatured DNA by treating DNA with 70 % formamide in the presence of a 50-nucleotide-Iong RNA linked to phenanthroline-copper complex to form the so-called R-Ioop structure (Chen et al. 1993a). The cleaving reagent was introduced internally into in vitro transcribed RNA using of 5-(3-aminoallyl)-UTP, which was thereafter acylated with mercaptoproprionic acid and then subsequently alkylated with 5-iodoacetamido-l,1O-phenanthroline (Fig. 2). Despite moderate yields of double stranded cleavage, this R loop method has been applied to map physical distance between two marker genes with the use of two 1,10-phenanthroline-modified RNAs targeted to distinct sequences on genomic DNA (Sigman et al. 1993a; Fig.4). In order to increase clevage efficiency, a slightly different method always based on R-Ioop formation and on use of OP conjugates was proposed. An R-


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Fig. 4. Targeting double-stranded DNA using phenanthroline-conjugates. The utilization of R-Ioops formed between DNA and RNA internally modified by phenanthroline (thick bar) was proposed for cleaving and mapping large DNA fragments (a). Tethering 1,lO-phenanthroline to a single-strand-specific DNA binding protein targeted to these R-Ioops allowed efficient DNA strand cleavage (b). On the other hand, phenanthrolineoligonucleotide could be used as artificial endonucleases or transcription inhibitors thanks to triplex formation on native DNA duplexes. An oligopyrimidine linked to phenanthroline could be targeted via triplex formation to a double-stranded DNA containing an oligopyrimidine-oligopurine sequence and induced specific cleavage on both strands (c). Cleavage reaction took place in minor groove as cleavage sites were shifted toward 3'-end of the target (d). The formation of the "open complex" after the RNA polymerase binding could be monitored by cleavage induced by untethered phenanthroline (e) or by using phenanthroline-oligomers (j) that recognized the locally single-strand region

loop structure was formed with an unmodified RNA oligomer following by a glyoxal treatment (Landgraf et al. 1995). The RNA was then removed from Rloop by RNase treatment. Originally, micrococcal nuclease, a single-strand specific nuclease, was added to cleave single-stranded region in the modified R-loop structure. With this method, single strand scission yield approached 70 %, but double stranded cutting did not exceed 10 % (Landgraf et al. 1995). The micrococcal nuclease could be replaced by a single-strand-specific DNA binding protein of M13, the gene V protein, tethered to 1,10-phenantholine (GVP-OP) via cysteine residues (Chen et al.1998; Fig. 4). High yield of double strand DNA cleavage (95 %) is obtained with this approach, likely due to the cooperative binding of GVP-OP to unwound DNA. It should be noted that all fragments produced by this GVP-OP mediated specific cleavage are easily clonable after alkaline phosphatase treatment to remove phosphate group from 3' -ends followed by fill in and 3' adenylation using Taq polymerase to generate 3'overhang products (Chen et al. 1998).


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Selective recognition of sequences in double helical DNA could be achieved

via triple helix formation. Oligonucleotides can bind to oligopyrimidineoligopurine sequences in duplex DNA involving hydrogen bond formation with the purine strand of the duplex target (Praseuth et al. 1999). Information about triple helices and their applications in molecular biology has been reviewed elsewhere (Faria and Giovannangeli 2001; Giovannangeli and Helene 2000; Praseuth et al. 1999). Here we will describe their use to position ubiquitous molecules such as OP at selected genomic sites. In order to obtain sequence-specific endonucleases, we have linked the 1,10-phenanthroline molecule at the 5' -end of oligopyrimidines using various length of linkers (Francois et al. 1989a). Specific and efficient DNA doublestrand cleavage (>70 %) was achieved using these phenanthroline-conjugates after activation by reducing agents and copper ions. We have found that cleavage efficiency was not equivalent on both strands (Fig. 4). The analysis of the cleavage sites on both strands of the duplex target revealed that phenanthroline-mediated reactions took place in the minor groove of duplex DNA, suggesting that phenanthroline intercalated at the junction between double and triple helices (Francois et al. 1989a; Shimizu et al. 1996). When a short linker was used to tether phenanthroline, no cleavage was observed, demonstrating that copper chelate resting in the major groove of DNA - where oligopyrimidine is bound - was unable to cleave deoxyribose. The efficiency of doublestranded DNA cleavage obtained with these TFO-OP conjugates is high enough to suggest biological applications in different fields such as sitedirected mutagenesis or artificial control of gene expression at the DNA level (see below). Their applications for gene mapping oflong DNA fragments were also proposed as oligopyrimidine-oligopurine target sequences were found over-represented in genomes. It should be noted that no DNA melting was required to induce the triple-stranded structures formed by TFO-OP bound to DNA, contrary to R-Ioop formation mentioned above. We have succeeded in cleaving large genomes using triplex-forming oligomers conjugates to phenanthroline (unpubl. results). This kind of phenanthroline-oligonucleotide conjugate also represented a good alternative to rare-cutting restriction enzymes, useful in molecular biology.

3.4.2 Biological Activities of Oligonucleotide-Phenanthroline We and others have demonstrated that phenanthroline-copper chelates tethered to various ligands induced in vitro DNA double-strand cleavage in presence of reducing agents as thiols and Cu{II) ions. Could these copper-complexes cleave DNA intracellularly and interfere with DNA metabolism?

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3.4.2.1 Phenanthroline Cleavage Activity Inside Cells A few years back, it was shown that 1,10-phenanthroline exhibited antibacterial, antifungal, antiviral and antineoplastic activities, likely by acting as a metal chelator. Indeed, the addition of divalent metal ions could reverse these effects. Nevertheless, the copper-phenanthroline chelates have been reported to be cytotoxic likely by inducing DNA cleavage intracellularly. Using the Ames mutagenesis test, 1,10-phenanthroline was found to be mutagenic in bacterial cells when mercaptopropionic acid (MPA) and Cu(II) were added exogenously, suggesting that cleavage reaction could at least take place in a cellular environment (Feig et al. 1988). When performing RNA footprinting studies within ferritin mRNA in Hela cells, it was found that cuprous-phenanthroline induced specific cleavage of an intracellular mRNA target with the same efficiency in the absence or in the presence of exogenous MPA, suggesting that cleavage redox chemistry of (OP)2CU+ could occur using endogenous reductants (Ke and Theil 2002). Nevertheless, it was also found that this copper-chelate dimished cell survival (60% viability) likely because of cutting intracellular tRNAs. In isolated ratliver nuclei, it has been demonstrated that 1,10-phenanthroline induced internucleosomal DNA fragmentation, a hallmark of apoptosis (Burkitt et al. 1996). The addition of ascorbate and hydrogen peroxide potentiated the level of DNA cleavage by phenanthroline, suggesting a scission mechanism related to the nucleolytic properties of cuprous-phenanthroline. Copper ions, which existed naturally in chromosomes, were chelated by 1,10-phenanthroline that then promoted hydroxyl-radical-dependant DNA fragmentation. An internucleosomal DNA fragmentation was also observed when a human hepatocarcinoma cell line, HepG2, was treated with copper(II) complex of 1,10-phenanthroline, suggesting that (OP)2CU+ penetrated and induced apoptosis in cells (Tsang et al. 1996). On another hand, it was shown that phenanthroline inhibited the apoptosis of rat thymocytes induced by dexamethasone or etoposide treament, likely because of its metal chelating properties (Wolfe et al. 1994). All these results concerning in vivo footprinting and induction of cell apoptosis by untethered phenanthroline complexed to copper ions strongly suggest that nuclease activities of (OP)2CU+ may occur inside cells. The activation of phenanthroline was achieved using endogenous reducing agent, and binding of endogenous or exogenously added copper ions to phenanthroline. 3.4.2.2 Modulation of DNA Metabolism Induced by OligonucleotidePhenanthroline Synthetic oligonucleotides could be used as specific inhibitors of gene expression, in the so-called antisense or antigene strategies. In the antisense approach, oligonucleotides have been targeted to mRNA in order to specifically block RNA translation into protein by ribonuclease H recruitment or by physical blockade of ribosome machinery (see above). Another approach


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consisted in specific inhibition of gene transcription by interfering with RNA polymerase via binding of oligonucleotides to DNA. Double stranded DNA could be recognized by oligonucleotides via either triple helix formation or locally R-loop formation. The initiation of transcription by RNA polymerase (RNAP) required the formation of an open complex which is a transient structure characterized by a single-stranded DNA of about 12 nucleotides, close to the transcription start site. It was shown in vitro that short oligoribonucleotides (ORNs) with non extendable 3'-termini targeted to this region were able to inhibit transcription starting from two well known promoters, E. coli lac UV5 and trp EDCBA (Milne et al. 2000; Fig. 4). Among the various chemically modified derivatives designed, the most efficient to inhibit transcription contained phospho rothioate linkages (Milne et al. 2001). The chemical nuclease 1,10-phenanthroline was linked to these oligoribonucleotides in order to demonstrate an antiparallel orientation for ORN binding to the template strand within the promoter "open region". These ORNs were gene specific and bound to promoters only in the presence of RNA polymerase that locally melt the double helix. The linking of phenanthroline to previously described ORNs and therefore cleavage of template strand were not necessary to specifically inhibit in vitro transcription. In the previously mentioned studies, these phenanthroline oligomers were used only as tool for orientation studies. As triplex-forming oligonucleotides covalently linked to phenanthroline induced specific cleavage of their DNA target, we wonder whether these conjugates could specifically inhibit gene transcription inside cells. The target sequence of these TFO-OP could be located within transcribed or regulatory regions. Double-stranded DNA scission induced in a selected gene will inhibit gene transcription either by cleaving the chromosome, or by inducing localized mutation or deletion around the cleavage site consequent to break repair. In fact, it was shown that double-strand breaks provided by non targeted DNA cleavage induced by either restriction endonucleases or bleomycin, were highly mutagenic and led to translocation, inversion, deletion or insertion of larger chromosomal fragments (Bennett et al. 1993; Phillips and Morgan 1994). Experiments are in progress in order to demonstrate that phenanthroline-oligonucleotide conjugates could interfere with transcription elongation inside transfected cells, inducing specific cleavage of DNA target due to the activation of phenanthroline-copper complex tethered to the oligonucleotide.

4 Conclusion The 1,10-phenanthroline-cuprous complex cleaves nucleic acids in a reaction that requires oxygen and reducing conditions. Covalent linkage of 1,10phenanthroline to an oligonucleotide or to a DNA-binding protein brings this cleaving reagent in close proximity to the complementary target sequence

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and therefore confers a targeted nucleolytic activity. Phenanthroline-oligonucleotides induce efficient in vitro cleavage of phospho diester backbone of either single-stranded or double-stranded nucleic acids containing their target sequences. These oligomer conjugates are now extensively used in probing structures of nucleoproteic complexes. For induction of RNA cleavage, the use of 2,9-dimethyl-phenanthroline compound instead of 1,10-phenanthroline should be promising for further applications as it was demonstrated that neocuproine-oligonucleotide cleaved RNA targets very efficiently. The 1,10phenanthroline-oligonucleotides achieved very efficient cleavage of doublestranded DNA when bound to oligopyrimidine-oligopurine sequences via triplex formation. These triplex-forming oligonucleotides linked to 1,10phenanthroline could be used as sequence-specific artificial endonucleases in molecular biology to cleave large genomes as well as in the antigene strategy to cleave DNA templates, providing that copper-phenanthroline cleavage chemistry occurs inside cells. The possibility to induce targeted doublestrand cleavage opens perspectives for the characterization of associated cellular processing and for the design of novel anticancer drugs. Acknowledgements. We would like to honor the memory of Professor David Sigman (1939-2001), who accomplished extensive pioneering work on the nucleolytic properties of 1,10-phenanthroline-copper complexes. We also wish to thank Ulysse Asseline, Alexander Boutorine, Claude Helene, and Tula Saison-Behmoaras for helpful discussions concerning our work with phenanthroline-oligonucleotides.

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