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Lanthanide-mediated DNA hydrolysis Sonya J Franklin Lanthanide ions are remarkably effective catalysts for the hydrolytic cleavage of phosphate ester bonds, including the robust bonds of DNA. This makes Ln(III) and Ce(IV) ions attractive candidates for developing selective and efficient artificial nucleases, which could have many biochemical and clinical applications. Both small-molecule-based and biopolymer-based lanthanide complexes are being pursued. Addresses Department of Chemistry, University of Iowa, Iowa City, Iowa 52242, USA; e-mail: [email protected] Current Opinion in Chemical Biology 2001, 5:201–208 1367-5931/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations Bis–Tris 2,2-bis(hydroxymethyl)-2,2′,2′-nitrilotriethanol BNPP bis(p-nitrophenyl)phosphate BTP (bis-tris propane) 1,3-bis[tris(hydroxymethyl)methylamino]propane DOTA 1,4,7,10-tetraazacyclododecane-N,N,N′,N′-tetraacetic acid HTH helix-turn-helix MRI magnetic resonance imaging

Introduction With the recent burgeoning growth in gene-sequencing and genomics, new opportunities to explore and exploit this information arise which require the facile regulation and understanding of transcription. There has recently been much interest in the development of efficient and catalytic hydrolases to mimic the enzymatic function of endonucleases [1,2,3••,4–6]. The ability to manipulate genes and cleave DNA efficiently and selectively by synthetic systems is fundamentally of great interest as a mechanism by which the interaction of natural hydrolases with DNA can be studied. Also, synthetic hydrolases would have utility as conformational probes and customized restriction enzymes for molecular biology applications. These functions would be particularly useful for sequencing and other applications if the designed system recognized longer sequences than physiological enzymes. Clinically, these agents could be designed to block gene-specific transcription for antibiotic and chemotherapy applications, potentially even allowing customized targeting of oncogenic mutations. The hydrolytic cleavage of DNA is particularly challenging because of the stability of its phosphate ester bond. Unlike RNA, whose 2′-hydroxyl group serves as an internal nucleophile to promote backbone strand scission, DNA is extremely resistant to hydrolysis under physiological conditions. The inertness of the DNA backbone toward decomposition is paramount for its effectiveness as the storage mechanism for the genetic code, but poses problems for living systems, which must manipulate these

stable bonds on physiologically relevant timescales. Cells must be able to cut and repair DNA lesions, control strain and overwinding during DNA production, and destroy foreign DNA to prevent its incorporation into cellular processes. Biological systems have developed nuclease and topoisomerase enzymes to catalyze DNA strand cleavage and repair. Many of these hydrolytic enzymes are metalloenzymes, utilizing hard Lewis acids such as Ca(II), Mg(II), and Zn(II) to activate both the P–O bond for cleavage and the water nucleophile. From this perspective, lanthanide ions, as hard Lewis acids with high coordination numbers, fast ligand exchange rates, and no accessible redox chemistry are ideally suited to mimic the reactivity of these biological metal cofactors. For nearly half a century, Ln(III) have been known to promote Lewis acid cleavage of phosphate esters. Only in the past decade, however, has this reactivity been demonstrated for the phosphate backbone of DNA [7]. In this relatively short time there has been much work on encapsulating Ln(III) with polyaminocarboxylate, Schiff base, and glycol ligands, packaging the metal for biochemical applications, while retaining the catalytic efficiency of the free ions. These small-molecule complexes have been shown to enhance the rate of DNA-phosphate hydrolysis by seven orders of magnitude [8,9,10]. Further, lanthanide metals provide excellent NMR, fluorescent, and luminescent spectral markers for studying structure and molecular interactions of biomolecules. Just as with natural nucleases, a hydrolytic rather than oxidative cleavage mechanism is desirable for several reasons. Oxidative cleavage of DNA and RNA produces diffusible free radicals. For molecular biology applications, radical abstraction results in strand ends that cannot be enzymatically religated. For clinical applications, oxidative cleavage can cause indiscriminant peripheral damage to the cell, and radical diffusion may significantly hinder the specificity of cleavage that can be achieved. There has been a great deal of interest and success in designing transition-metal-based nucleases to promote DNA hydrolysis, though these systems must always be tuned to promote hydrolytic over oxidative mechanisms [5]. For these reasons, there is an open field for the development of lanthanide-based nucleases. This review highlights progress toward the goal of non-enzymatic DNA cleavage by Ln(III) complexes, and reasons for the catalytic prowess of these ions. Both small-molecule lanthanide complexes and biopolymer-based antisense and protein–DNA recognition mechanisms are discussed.

Hydrolytic activity of lanthanide ions The ability of lanthanide ions to readily catalyze the hydrolysis of DNA is notable, particularly in comparison to

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biologically relevant transition metal or alkaline earth Lewis acids such as Zn(II), Ca(II), or Mg(II). This efficiency results from the conjunction of higher oxidation state and charge density, coordination number, and rapid ligand exchange rates. These characteristics make the Ln(III) ions well-suited to be catalytic centers in the development of artificial enzymes. A recent discussion of the metal ions that naturally occur in hydrolases points out a truism of bioinorganic chemistry, namely that some metals are ideally suited, and thus commonly used, for given functions [5]. There is a limited subset of physiologically accessible metals that is consistently used in different contexts to promote hydrolysis. Hydrolysis requires a metal cofactor capable of binding hard oxygen atoms, polarizing bonds (Lewis acidity), and rapid ligand exchange for catalytic turnover. Lewis acidity correlates with metal ionization potential, which serves to lower the pKa of a metal-bound water nucleophile. Ligand substitution rates are dependent on ligand field stabilization energy, which provides an activation barrier to ligand exchange. Based on the criteria of substitutional lability and Lewis acidity, Cu(II), Zn(II), Ca(II), and Ln(III) ions would be the expected catalytic centers of natural hydrolases. The facile redox behavior of Cu(II) precludes its function in native hydrolases, but has made the ion an intriguing target for artificial nuclease design, along with the environmentally scarce Ln(III) ions. Had the lanthanides been more abundant as early hydrolases were evolving, we might now require our daily Eu(III) supplements to maintain our health. The hydrolysis of deoxynucleotide phosphates proceeds by the nucleophilic attack at phosphorus by a water oxygen, to give a five-coordinate phosphate intermediate.[1,5] Subsequent cleavage of either the P-O3′ or P-O5′ (dependent on the catalytic system) causes a strand break yielding R–OH and R–O–PO3H2 termini. For DNA, the breakdown of the intermediate is rate-limiting. The hydrolysis is assisted by metal ions in predominantly two ways. The Lewis acid can serve to activate a water nucleophile by lowering the pKa of water to near neutrality, thus generating a reactive metal-bound hydroxyl group. In addition, the metal ion can activate the electrophile or stabilize leaving groups, by polarizing a P–O bond and withdrawing electron density from the phosphorus. Further, bimetallic complexes can act cooperatively to accelerate both these mechanistic steps, and therefore are often more reactive than their mononuclear counterparts.[4,11] Thus, Ln(III) or Ln(III)2 complexes that have open coordination sites and can lower the barrier to both formation and collapse of the five-coordinate intermediate are effective hydrolytic catalysts.[1,12••] The relative catalytic efficiency of the Ln(III) ions can be determined by comparing the rates of bis-(p-nitrophenyl)phosphate (BNPP) cleavage by the aqueous ions. A commonly used model system for nucleases, BNPP is an

activated diphosphate ester, which is hydrolyzed to yield two equivalents of a yellow nitrophenylate product (λmax = 400 nm). The uncatalyzed BNPP hydrolysis rate is too slow to directly measure at room temperature, but at 80°C, the uncatalyzed rate was found to be kobs = 1.0 × 10–8 s–1 [13]. From this and thermodynamic parameters determined from measurements at a range of higher temperatures, the rate of BNPP hydrolysis at 37°C and pH = 7.0 is estimated to occur at kobs = 10–11 s–1. In the presence of Ln(III) ions (5 mM), BNPP is cleaved approximately seven orders of magnitude faster [10], by a dioxygen-independent mechanism. It should be noted that although the monophosphate NPP cleavage is not rate-limiting, it may be slow enough to perturb simple first-order dependence on BNPP for some systems [10,14]. Schneider and co-workers [10] found the aqueous Ln(III) ions exhibited saturation behavior in the cleavage of BNPP, which they fitted to the Michaelis–Menten equation. They found little difference in KM across the series (1600–3300 M–1). However, the catalytic rate constants (La kcat = 1.3 × 10–5 s–1; Er kcat = 8.6 × 10–4 s–1) increase systematically across the series as a function of decreasing ionic radius, except for an unexpected lower efficiency of Yb(III) and Lu(III) attributed to aggregation. The activation of both the water and phosphoryl group is dependent upon higher charge density of the cations, which apparently causes the later lanthanides (up to Er(III)) to be more effective catalysts. Interestingly, the opposite effect was observed for nicking supercoiled DNA. For that substrate, the aqueous ions exhibited little difference in kcat values, but KM decreased from La(III) to Dy(III). This may suggest that the kinetic parameters are reflecting different mechanistic steps for DNA and BNPP, as the rates of substrate capture and product release need not necessarily be the same. Increased ionic strength was also found to have an impact on catalysis rates for both BNPP and DNA substrates. Hydrolysis rates of the aqueous Eu(III) ions decreased with added Na+ or Mg2+, suggesting an ion-pair competition between these ions and Eu(III) for the phosphate substrate. This may have implications for ligand design, as positively charged amine ligands also slow rates. The nuclease behavior of lanthanides in complexes, however, is not necessarily the same as that of the free hydrated ions. When complexed by BTP ([bis-tris propane] 1,3-bis[tris(hydroxymethyl)methylamino]-propane; Figure 1a), the early Ln(III) ions were more reactive than the late Ln(III) toward BNPP cleavage [14]. This activity seemed to correlate with increasing pKa (and thus increasing nucleophilicity of the bound OH) across the series, and rates were in fact faster at higher pH as would be expected. Additionally, this system had a very weak dependence on ionic strength, and no Michaelis–Menten type satur0ation effects. Thus, unlike the aqueous ions, the dominant catalytic barrier for this system is apparently water activation, rather than polarizing the electrophile. Conversely, bimetallic

Lanthanide-mediated DNA hydrolysis Franklin

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Figure 1 Small-molecule lanthanide nucleases. Representative ligands for lanthanidemediated oligonucleotide hydrolysis. (a) BTP. (b) Bis–Tris. (c) Macrocyclic Schiff base Ln(III)-complexing ligand of Morrow and co-workers [19]. (d) Larger Schiff-base macrocycle of Zhu et al. [20], with two metalbinding sites and hydroxyl groups to provide general base functionality. (e) Azacrown compound of Martell and co-workers [22]. (f) Azacrown scaffold of Janda and coworkers [25]. (g) DOTA–amide ligand of Akkaya and co-workers [29]. (h) Polyaminocarboxylate ligand of Branum and Que [27•]. (i) Hydroxamic acid group appended to an intercalator ligand [32].

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systems are particularly effective in enhancing the stability of the intermediate and leaving groups. Tuning these two effects is the next challenge in rational nuclease design. Because of the lability of the Ln(III) ions toward ligand exchange, designing complexes with high kinetic and thermodynamic stability, while retaining the necessary open coordination sites for solvent and substrate, can be demanding. However, years of work and lessons learned in the development of magnetic resonance imaging (MRI) contrast agents and lanthanide shift reagents have led to quite stable and inert complexes [15]. The trivalent lanthanide ions have very high charge density, similar to Mg(II), but are comparable in size to Ca(II) (size decreases across the series; La(III)→Lu(III) = 1.17 Å→1.00 Å, whereas Ca(II) = 1.06 Å) [16]. As such, they prefer hard oxygen ligands over nitrogen donors, and coordination numbers of eight and nine. Because their valence 4f orbitals are both diffuse and of an inner shell, the metals experience very little ligand field splitting effect. Thus the coordination geometry and number is primarily dependent on ligand–ligand repulsion and steric factors. Cerium is unique among the lanthanides in its ability to access a tetravalent oxidation state under aqueous conditions. The Ce(IV) ion has been found to be particularly effective in promoting nuclease activity, enhancing BNPP hydrolysis by a factor of >1010 and DNA hydrolysis by >1011 [8,12••,17]. These rates are 20–1000 times faster than in the presence of the trivalent Ln(III) ions. This striking activity has been ascribed to the ability of Ce(IV) to make covalent bonds with a phosphate substrate and promote formation of the pentacoordinate intermediate.

An EXAFS (extended X-ray absorption fine structure) and XANES (X-ray absorption near-edge structure) study of the Ce(IV)–BNPP complex showed that the metal 4f orbitals were involved in a weak covalent bond to the phosphate oxygen [18]. This bond covalency, coupled with greater charge density and a tendency to form bimetallic hydroxo-clusters in solution, causes Ce(IV) to be significantly better in binding and activating nucleotide substrates than other Ln(III) ions, which only withdraw electron density electrostatically. A significant D2O solvent isotope effect (kH2O/kD2O = 2.2–2.4) indicates that the cleavage proceeds via a rate-limiting proton transfer, which is ascribed to the protonation of the P-O5′ (or P-O3′) leaving group during dissociation by a metalbound water. Despite the fact that product release remains rate-limiting, as it is for Ln(III) ions, the overall Ce(IV) hydrolysis rates are quite rapid. The activity of Ce(IV) offers hope that enzyme-like activities could soon be achieved.

Small-molecule nucleases Although hydrated Ln(III) ions have been shown to be effective DNA-cleavage agents, the free ions have a tendency to precipitate from solution as the hydroxides around pH = 9, and are toxic to biological systems because of their similarity to Ca(II). This necessitates the encapsulation of these ions to tune and target their reactivity. In the past few years, great progress has been achieved in developing lanthanide–smallmolecule complexes that retain the hydrolytic capacity of the free metal towards phosphate esters, RNA, and even the more robust bonds of DNA. The most successful compounds include Schiff base macrocycles, polyhydroxyl ligands, crown ethers and azacrowns, polyaminocarboxylate derivatives,

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DOTA (1,4,7,10-tetraazacyclododecane-N,N,N′,N′-tetraacetic acid) derivatives, hydroxamic acids, polymeric sugars, micelles, and various mixed Ln–M systems (where M = transition metals or other Ln(III)). Schiff base and azacrown macrocycles

Schiff-base-containing macrocycles were among the first examples of Ln(III) complexes developed as nucleases. Because of the preference of Ln(III) for harder oxygen donor atoms, complexes involving N-ligation must utilize the stabilization and kinetic inertness afforded by the chelate effect. This results in neutral or positively charged complexes with open coordination sites, which are thus well-suited to promote oligonucleotide hydrolysis. Morrow et al. [19] first showed that Schiff base Ln(III) complexes could hydrolyze RNA bonds catalytically (Figure 1c). Recently, Zhu and co-workers [20,21] have built on the Schiff base design by synthesizing larger macrocycles that can accommodate two metal ions, improving the reactivity toward phosphates. Additionally, the incorporation of hydroxyl groups into the ring provides a base functionality to assist in proton transfer steps. The Ho2 and Er2 complexes of this ligand (Figure 1d) were found to catalyze double-stranded cleavage of supercoiled plasmid DNA. This was the first example of such linear products by a Ln(III) complex. Crown ether and azacrown lanthanide complexes have also been shown to catalyze phosphate hydrolysis. These macrocyclic complexes are similar to those of the Schiff base ligands in that they typically have open coordination sites and a tendency to aggregate as dimers or higher-order species in solution. Martell and co-workers [22] reported mono- and di-nuclear Ln(III) complexes (Figure 1e) that have an unusual third order dependence of the BNPP hydrolysis rates on dinuclear complex concentration. Intriguingly, the catalysis rates as a function of pH track remarkably well with the formation of an azacrown-Ln2(OH)2 species (pKa2 = 10.03). It is suggested that the adjacent metal ions promote the deprotonation of the second bridging water, which serves as an active and proximal nucleophile. Schneider and co-workers [23,24] have described phosphate and DNA hydrolysis by mono- and binuclear Ln(III) azacrown complexes (despite very weak formation constants), and have shown that the cleavage is hydrolytic by enzymatically religating the product ends. Janda and coworkers [25•] have also utilized azacrown scaffolds (Figure 1f) to develop a library of Ln(III) nucleases by combinatorial means. They have varied the appended ether sidechains and type of Ln(III) ion to identify several highly active DNA nucleases.

affinities. Clinically approved MRI contrast agents have Gd(III) affinities in the range of log Kf = 16.9–25.3 [15]. However, they have been typically less effective as nucleases because of their overall negative charge or few open coordination sites. In fact, [Eu–EDTA]–1 is not appreciably hydrolytically active toward BNPP or DNA, though the more reactive Ce(IV) complex does promote cutting of oligonucleotides [26]. Recently, these versatile scaffolds have provided a framework with high Ln(III) affinity to bring two metal ions into proximity for concerted double-strand DNA hydrolysis. Around the time Zhu and co-workers reported linearized products of supercoiled DNA, Branum and Que [27•] independently reported a bimetallic polyaminocarboxylate complex that was capable of generating double-strand cuts of both supercoiled and oligonucleotide DNA (Figure 1h). This was significant, as linearized oligonucleotides are not activated by internal strain as is supercoiled DNA, and are thus more difficult to hydrolyze. It is becoming apparent in the design of MRI contrast agents that functionalizing carboxylates as amides offers a way to design neutral or positive complexes without significant loss of metal affinity. In addition, the amide linkages can be used to append biomolecules for antisense applications, such as described for RNA nuclease applications. As such, it is likely that this type of ligand system will remain important for the design of nucleases as well as MRI contrast agents. Another polyaminocarboxylate ligand scaffold used effectively for MRI applications, and now in nucleases, is the DOTA backbone. This macrocyclic ligand is quite attractive as it has very high Ln(III) affinity, but is also remarkably kinetically inert to metal exchange. Morrow and co-workers [28] have demonstrated that Eu(III) is not lost to exchange from their DOTA derivatives over three days at 37°C, using Cu(II)-trapping experiments. Like the linear polyaminocarboxylate ligands, the Ln–DOTA species are more effective nucleases as neutral or positive complexes. Several groups [28–30] have therefore explored substituting amide or alcohol groups for the carboxylate arms (for example, Figure 1g). These carboxylate derviatives also provide a point of covalent attachment to append oligonucleotides or other recognition elements, and DOTA–DNA conjugates have been shown to cleave RNA with high selectivity [31]. A series of ligand-modification studies have illustrated that ribonuclease or phosphate hydrolase activity requires two open coordination sites for the most effective cleavage [30]. To date, however, this scaffold has been applied only to Ln-mediated hydrolysis of RNA and RNA models. Heterogeneous systems

Polyaminocarboxylate ligands

Lanthanide complexes of polyaminocarboxylate derivatives (such as EDTA [ethylenediaminetetraacetic acid] or DTPA [diethylenetriaminepentaacetic acid]) have enjoyed great success as MRI contrast agents, because of their high Ln(III)

Along with the well-characterized small-molecule nucleases being developed, there has been interest in promoting Ln–oligonucleotide interactions of a less defined and perhaps heterogeneous nature. These systems are quite simple and often very effective at catalyzing phosphate

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Figure 2 Mechanisms of cleavage by biopolymer-based nucleases. (a) Schematic representation of antisense recognition and hydrolysis of singlestrand DNA by a Ln–DNA conjugate. (b) Schematic representation of proteinduplex DNA recognition and cleavage by a Ln-chimera.

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and DNA hydrolysis. It has been found that oligonucleotides can be readily cleaved by a wide variety of structures and systems, provided the Ln(III) ion can approach the DNA substrate, and does not precipitate from solution at neutral or slightly basic pH. This led Hashimoto and Nakamura [32] to consider appending a hydroxamic acid group to an intercalator ligand (Figure 1i), to deliver a Ln(III) to the DNA. Although the affinity of a single hydroxamic group for Ln(III) is low, the metal presumably binds to both this chelate and the DNA phosphates simultaneously, creating a ‘pocket’ binding site along the DNA backbone. Cleavage efficiency was found to be dependent on the linker, with 5-methylenes between intercalator and hydroxamic group being the correct length to orient the Ln(III) ion within close proximity of an oligonucleotide phosphate. The intercalator did not impart any specificity, but the Ce(IV) complex was found to be quite active as a DNA nuclease [33]. Among the simplest solutions to the high pH solubility problem is the use of polyalcohols to keep the active Ln(III) ions in solution. Even the common chelating buffer Bis–Tris (2,2-bis(hydroxymethyl)-2,2′,2′-nitrilotriethanol; Figure 1b) has been shown to significantly enhance Ln(III) activity toward BNPP cleavage at high metal concentrations and pH [34]. As with BTP (Figure 1a), this effect is apparently a result of improved Ln(III) ion solubility, and perhaps a cooperative function of ligand hydroxyl groups as general bases. The importance of including proximal basic functional groups to assist in metal-bound water deprotonation was systematically studied by Schneider and co-workers [8,9]. They tested the hydrolytic activity of a series of polyalcohol ligands toward BNPP and supercoiled DNA, finding that glycerol and gluconate (though only weakly coordinated) were most effective as catalysts. Schneider also found the late lanthanides to be the most reactive catalysts (Yb(III) > Eu(III) > La(III)). Polymeric sugars (dextrans) also solubilize and activate Ln(III) for hydrolysis [35,36]. Intriguingly, Ce(IV)–Ln(III)–dextran ternary systems showed that cooperativity of the very active

Ce(IV) species with another Lewis acid (various Ln(III)) generated a more effective nuclease than either Ce(IV) or Pr(III) dextrans alone. The dextran sugars served to bring the two Lewis acids and the substrate into proximity for phosphate and hydroxyl activation. Micellular or bilayer surfaces have also been shown to promote hydrolytic DNA cleavage in the presence of Ca(II), Mg(II), Cu(II) and Ln(III) ions [37,38]. The phosphate surface provides a two-dimensional matrix on which the metal ions can bind and cooperatively interact with the oligonucleotide phosphate backbone. No evidence for hydrolysis of the surfactants’ phosphate head-groups was found.

Biopolymer-based nucleases Lanthanide conjugates to biomacromolecules have myriad uses, including MRI contrast agents, fluorescent probes, and ribonucleases. A great deal of progress has been made on the latter front, utilizing an antisense approach. This methodology involves the recognition of a single-strand RNA or DNA target sequence by a complementary oligonucleotide sequence (usually DNA) to which a nuclease or metal ion has been appended (Figure 2a). The antisense approach is extremely selective in recognition and cleavage of a target sequence. Recently, protein and peptide-based Ln(III) nucleases, which could utilize protein–DNA recognition, have begun to be explored as well (Figure 2b). Oligonucleotide-based nucleases

The antisense strategy has been primarily employed to target and cleave RNA, utilizing the more stable DNA backbone as the delivery scaffold [31,39]. Because cleavage of the RNA strand of the resultant DNA–RNA hybrid is much more facile, the catalyst is not significantly degraded in the reaction. However, the high affinity of the complementary strands means that product release can be very slow, hindering catalytic turnover for these systems. Strategies to append the metal site such that smaller fragments are produced (for example, in the center of a recognition sequence rather than the end) are being explored for ribonucleases, and could be utilized for DNA nuclease in the future.

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Figure 3

A docked overlay of the crystal structures of engrailed homeodomain, cocrystallized with DNA (Protein Data Bank code 2HDD), and one EFhand of calmodulin (residues 52–79; Protein Data Bank code 1OSA). The model shows the similarity in the two protein domains, which was utilized to design hydrolytically active Ln-binding chimeric peptides [45,48•].

In the mid-1990s, Komiyama [40] demonstrated that the lanthanide-mediated antisense strategy could be employed to hydrolyze DNA site-specifically as well. An iminodiacetate ligand was appended to the 5′-terminus of a 19-mer singlestrand DNA oligonucleotide. The Ce(IV)–DNA complex promoted highly selective cleavage two bases to the 3′-side of the complementary strand-binding site, as expected from the molecular design. The cleaved 3′-fragment had a 5′-OH terminus, with no dependence on molecular oxygen, confirming the hydrolytic mechanism of the Ce(IV) cleavage. Despite this early success, RNA cleavage has garnered more attention in the past few years, and thus the antisense approach for DNA nucleases remains relatively unexplored. Protein- and peptide-based nucleases

An alternative approach to the antisense strategy, which involves specific interactions with single-stranded RNA or DNA, is to design an agent to specifically recognize doublestranded DNA in the manner of transcription factors and

restriction enzymes. This strategy employs the same flexible, yet sequence-selective, protein–DNA recognition that cells themselves use to regulate their growth. By incorporating both a metal site and a protein-based DNA-binding element into a nuclease design, the artificial enzyme utilizes the protein–DNA interactions to deliver a hydrolytic metal to the backbone of DNA for cleavage. This approach is only now being explored as a means to exploit the extremely effective hydrolytic cleavage potential of the Ln(III) and Ce(IV) ions, already demonstrated with small-molecule complexes. Variations on the duplex-DNA-recognition strategy that have been employed for oxidative DNA-cleavage include appending a Cu(II)- or Ni(II)-binding tripeptide (His–Gly–Gly) onto the amino terminus of a DNA-binding domain [41] and incorporating an Fe(III) ion into the Zn(II)-binding site of the estrogen receptor element, a DNA-binding transcription factor [42]. This ‘iron-finger’, in the presence of peroxide, causes oxidative damage to DNA in the vicinity of the binding site. Hydrolytic cleavage and recognition of duplex DNA have also been accomplished by systems containing physiological dications. A fusion protein generated by splicing a known endonuclease domain (Fok I, a Mg(II)-dependent type II restriction endonuclease) onto DNA-binding zinc-finger domains cleaves double-strand DNA with high specificity [43]. Another strategy involved appending a Zn(II)-binding peptide onto an ancillary ligand of a rhodium metallointercalator [44]. The Rh(III) complex provides the DNA affinity, delivering the Zn(II)–peptide to the major groove of DNA for hydrolytic cleavage. The hydrolytic mechanism was confirmed by enzymatic religation of the products. Although this system incorporates a potentially redox-active heavy metal complex, it shows that effective hydrolytic cleavage can be achieved at low concentrations and under mild conditions. Metallopeptides and metalloproteins are now being developed to utilize native-like protein–DNA interactions to promote lanthanide-mediated DNA hydrolysis as well. In our laboratory, we have developed peptide nucleases that incorporate a Ca(II)-binding site into a helix-turn-helix (HTH) DNA-binding domain [45]. These peptide designs are based on the remarkable similarity of two unrelated physiological folds. Surprisingly, the calcium-binding EF-hand and the HTH motifs are virtually superimposable, and thus allow us to incorporate both a Ln(III)-binding pocket and DNA-binding helices into a chimeric system. A docked model of engrailed (cocrystallized with DNA) and calmodulin crystal structures (Protein Data Bank files: 2HDD [46] and 1OSA [47]) shows the similarity of the two turns and the potential proximity of the metal ion to the DNA backbone (Figure 3). However, the structure of the chimera as it interacts with DNA has not been established. We have designed several 33- and 34-residue peptides, which comprise helix 2 and 3 of engrailed homeodomain (the HTH region), and the 12-residue calcium-binding loop of an EFhand from calmodulin. These peptides bind Ln(III) ions, folding as a function of added metal. The chimeric peptides

Lanthanide-mediated DNA hydrolysis Franklin

have Ln(III) affinities similar to native EF-hand peptides (log KLnL = 5–6), and bind strongly to DNA. The Eu(III)–peptide complexes are hydrolytically active toward supercoiled plasmid DNA, causing single-strand breaks at lower concentrations than free Ln(III) [48•]. The cleavage of the model BNPP phosphate was also followed spectroscopically. The observed initial pseudo-first order rate constants as a function of calculated Eu–peptide concentration could be fitted to a second order rate constant of 0.1–0.3 M–1s–1 at low catalyst concentrations (≤ 25 µM [Eu–peptide]calc). Importantly, the Ln(III) affinities allow us to calculate the amount of free metal ion and metal–peptide complex present in solution, and show that the observed hydrolysis rates are not attributable simply to aqueous Ln(III). At higher concentrations (≥ 50 µM [Eu-peptide]calc), one metallopeptide (EuP3) shows a tendency to dimerize, as do peptides with native EF-hand sequences. The hydrolytic activity of this metallopeptide toward both BNPP and DNA is drastically reduced at these concentrations, suggesting that the native-like dimerization buries the active metal site and prevents hydrolysis. We have used the DNA affinity of a transcription protein to deliver a Ln(III) for phosphate hydrolysis. This exciting result shows that structural protein loops can be hydrolytically active with Ln(III) ions, provided that accessible open coordination sites and adjacent base ligands are incorporated, as was found with small-molecule systems. This design opens a new and potentially selective mechanism to deliver a hydrolytic Ln(III) ion to duplex DNA.

Conclusions and future challenges The past few years have demonstrated that there are several emerging themes in the design of effective lanthanide–DNA nucleases, and several systems are showing great versatility and promise for biomolecular applications. Binuclear systems are particularly effective in promoting DNA hydrolysis, as the metals can function in tandem to both activate the P–O bond and the water nucleophile. Even with single-metal systems, adjacent hydroxyl ligands that can serve as general bases greatly enhance the efficiency of phosphate ester cleavage. Finally, it is becoming increasingly clear that Ce(IV) is among the most effective hydrolytic metal ions available for DNA hydrolysis, though its structural and complexation behavior is similar to that of other Ln(III) ions and Ca(II). The exquisite sequence specificity of native restriction enzymes has yet to be realized in vivo (or even ex vivo), nor has their catalytic efficiency been approached. With the lessons learned, however, the future is promising for the development of small-molecule and biopolymer-based Ln(III) complexes to target DNA sequences at will, and to offer insights into the mechanism of native nucleases.

Acknowledgements Our work is supported by the University of Iowa Carver Research Foundation and a National Science Foundation CAREER Award. The author thanks Kinesha Harris and Joel Welch for assistance in preparing the manuscript.

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