2002 Oxford University Press

Nucleic Acids Research, 2002, Vol. 30 No. 19 e102

Preferential hydrolysis of gap and bulge sites in DNA by Ce(IV)/EDTA complex

Yoshihito Kitamura and Makoto Komiyama*
Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan
Received June 18, 2002; Revised and Accepted July 19, 2002

ABSTRACT A new strategy for site-selective DNA hydrolysis, which takes advantage of the difference in reactivity between the phosphodiester linkages at the target site and the others, is presented. As the molecular scissors, homogeneous Ce(IV)/ethylenediamineN,N,N ,N -tetraacetate (EDTA) complex is used without being bound to any sequence-recognizing moiety. When a gap structure is formed at the target site by using two short oligonucleotides and the composite is treated with the Ce(IV)/EDTA complex at pH 7.0 and 37C, the gap site in the substrate DNA is preferentially hydrolyzed over the doublestranded portion of the DNA. Site-selective DNA scission is also achieved by forming a bulge structure at the target site with the use of the appropriate oligonucleotide. These site-selective scissions are based on the following two factors: (i) the phosphodiester linkages in a single-stranded DNA are far more susceptible to the hydrolysis by the Ce(IV) complex than are the linkages in double-stranded DNA, and (ii) the phosphodiester linkages in the bulge sites are still more reactive than those in single-stranded DNA. In both cases, the addition of spermine signicantly accelerates the scission. INTRODUCTION Site-selective hydrolysis of DNA has been one of the most attractive and challenging themes for chemists and biochemists (15). Versatile applications to biotechnology and molecular biology have been proposed. For example, manmade restriction enzymes can show far higher sequence specicity than naturally occurring ones, and pave the way to the manipulation of huge DNA of higher animals and higher plants. Articial enzymes for selective cleavage of the ribose at the target site were successfully prepared by attaching oxidation catalysts to oligonucleotides or their equivalents (6). Later, site-selective DNA hydrolysis was achieved by conjugating Ce(IV) ion [a catalyst for DNA hydrolysis (710)] with oligonucleotides (11,12). Use of a Fe(III) complex as molecular scissors was also reported (13). These articial enzymes selectively hydrolyze the target phosphodiester

linkage, since the molecular scissors are xed near this linkage and thus the scission is promoted by a favorable activation-entropy term. Most ribozyme mimics reported to date for site-selective RNA scission also employ this strategy (1422). However, in these covalent strategies, the intrinsic activities of molecular scissors are often diminished upon their covalent xation to oligonucleotides. Furthermore, the synthesis of conjugates is in some cases troublesome and time consuming. Recently, a new strategy for site-selective RNA scission, which requires no covalent xation of molecular scissors, has been proposed (2325). There the target phosphodiester linkages are discriminated from the others in terms of intrinsic reactivity. For example, bulge sites in RNA, formed by using appropriate oligonucleotides, are preferentially hydrolyzed by unbound lanthanide complexes, since the RNA in the bulges takes a favorable conformation for the transesterication by the 2-OH group (23). Furthermore, highly efcient siteselective RNA scission was accomplished by using acridinetethered oligonucleotides, which interact with the RNA and activate the target phosphodiester linkages (24,25). However, there has been no report on the application of non-covalent strategy to DNA hydrolysis, which employs an entirely different mechanism from RNA hydrolysis. Here we present the rst success of this approach, which takes advantage of the remarkable substrate specicity of the Ce(IV)/EDTA complex. As reported previously (26), this complex is effective for the hydrolysis of polynucleotides and oligonucleotides that are longer than tetranucleotides. However, dinucleotides and trinucleotides are never hydrolyzed. Accordingly, gap structures and bulge structures are formed at the target sites in substrate DNA to differentiate between these sites and the others. Exactly as designed, the gap sites and bulge sites are preferentially hydrolyzed by the Ce(IV)/EDTA complex, even when this complex is never bound anywhere covalently. MATERIALS AND METHODS The sequences of substrate DNAs and oligonucleotide additives are presented in Figure 1. All of them were prepared on an automated synthesizer and puried by the usual method. Homogeneous Ce(IV)/EDTA complex was prepared immediately before use by mixing equimolar amounts of Ce(NH4)2(NO3)6 and EDTA (4Na salt) in HEPES buffer (26). The substrate DNA (32P-labeled at the 5-end) was treated with this complex at pH 7.0 and 37C, where

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*To whom correspondence should be addressed. Tel: +81 3 5452 5200; Fax: +81 3 5452 5209; Email: komiyama@mkomi.rcast.u-tokyo.ac.jp

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Figure 1. Sequences of substrate DNAs and oligonucleotide additives.

[substrate DNA]0 = 1.0 mM, [each of oligonucleotide additives]0 = 1.1 mM and [NaCl]0 = 100 mM in 2.5 mM HEPES buffer. Unless otherwise noted, 100 mM spermine was added to the reaction mixtures in order to accelerate the reactions as described below. The typical specimen preparation was as follows. First, the substrate DNA and oligonucleotide additives were dissolved in 8 ml of aqueous NaCl solution. The mixture was heated to 90C for 1 min and slowly cooled to room temperature. To this solution was added pH 7.0 HEPES buffer solutions of Ce(IV)/EDTA complex (1 ml) and of spermine (1 ml). The hydrolytic character of DNA scission by the Ce(IV)/EDTA complex, as well as by the relevant Ce(IV) complexes, was already substantiated (2628). After a predetermined time, the DNA scission was stopped by adding the water containing 10 mM EDTA and 70 mM inorganic phosphate (in 1/2 volume of the reaction mixture). The reaction mixtures were then subjected to denaturing 20% polyacrylamide gel electrophoresis. Authentic oligonucleotides having the same sequences as the corresponding fragments (prepared by the synthesizer) were used as the markers. The scission fragments were quantied with a Fuji Film FLA-3000G imaging analyzer. RESULTS AND DISCUSSION Gap structures for site-selective DNA scission Figure 2 shows the gel electrophoresis patterns for the DNA hydrolysis by Ce(IV)/EDTA complex at pH 7.0 and 37C. In the absence of oligonucleotide additive, the substrate DNA1(S) (50mer) is cut almost randomly without any specic base preference (lane 2). Hydrolytic character of the scission has been denitely conrmed by the previous HPLC analysis (26). However, when gap structures are formed in this DNA by using two oligonucleotide additives, the scission is strictly restricted to the corresponding gap site. In lane 6, the nucleotides C28, A29 and C30 in DNA1(S) are unpaired, and the others are forming WatsonCrick base pairs (the gap site is shown by the bar at the right-hand side of the gel). The scission by the Ce(IV) complex selectively occurs at these

three unpaired nucleotides, although the scission efciency is rather low. The scission at A29 is the most efcient. With the 5-base gap from T21 to G25 (lane 7), the scissions at T22 and A23 are dominant, and minor bands are at T21, T24 and G25. For the 10-base gap, the scission also takes place within the gap (lane 8). It is noteworthy that the scission site moves concurrently with the shift of the gap site (compare the bands in lanes 68 with the bar for the corresponding lane). Apparently, the site of selective scission by the Ce(IV) complex, which is not covalently bound anywhere, can be modulated by using an appropriate combination of oligonucleotides. The fragments of these scissions show the same mobilities as authentic samples obtained by a synthesizer (this conclusion has been conrmed by using longer gels). It is indicated that the phosphates at their 3-termini are removed by the phosphomonoesterase activity of the Ce(IV)/EDTA complex (29). The weak bonds between the strong ones are probably associated with the fragments bearing the terminal phosphate. Quantitative analysis by densitometry on these electrophoresis patterns has further conrmed the overwhelmingly preferential scission of the gap site over the doublestranded portion. The typical result (on lane 8) is presented in Figure 3A. Almost all the scissions occur within this 10-base gap, and the total conversion of scission in this region is 12% under the conditions employed. The double-stranded region is not hydrolyzed to a measurable extent, except for the minor scission near the gap edges. Site-selective scission by using gap strategy was also successful for all other DNA substrates investigated, and both the site selectivity and the scission efciency are not much dependent on the substrate sequence. However, the site selectivity is slightly lower when too many A-T pairs exist near the edges of the gap. For example, a 10-base gap is prepared in DNA2(S) by combining DNA2(G)-L and DNA2(G)-R2. Here, three consecutive A-T pairs are formed at one edge of the gap, and six consecutive A-T pairs are at the other edge. As depicted in Figure 3B, even non-gap region, especially in the 3-side of the gap, is notably hydrolyzed [the gel electrophoresis pattern is presented in the Supplementary Material (Fig. S1), together with those for

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Figure 3. (A) Relative intensities of the scission of DNA1(S) by the Ce(IV)/ EDTA complex with the DNA1(G)-L1/DNA1(G)-R1 combination (10-base gap): the quantitative analysis of lane 8 in Figure 2. (B) The pattern is for the scission of DNA2(S) by the Ce(IV)/EDTA complex in the presence of the combination of DNA2(G)-L and DNA2(G)-R2, where consecutive A-T pairs exist near the site of the 10-base gap.

Figure 2. Gap strategy for site-selective hydrolysis of DNA1(S) by the Ce(IV)/EDTA complex. Lane 1, control; lane 2, DNA1(S) alone; lane 3, with DNA1(F); lane 4, with DNA1(G)-L1; lane 5, with DNA1(G)-R1; lane 6, 3-base gap (DNA1(G)-L2 + DNA1(G)-R1); lane 7, 5-base gap (DNA1(G)-L1 + DNA1(G)-R2); lane 8, 10-base gap (DNA1(G)-L1 + DNA1(G)-R1). The bars in the right-hand side designate the sites of gaps. Reaction conditions: [DNA1(S)]0 = 1.0 mM; [each of oligonucleotide additives]0 = 1.1 mM; [NaCl]0 = 100 mM; [Ce(IV)/EDTA complex] = 500 mM; and [spermine] = 100 mM at pH 7.0 (2.5 mM HEPES buffer) and 37C for 4 days.

10-base bulge in lanes 4, 5, 6 and 7, respectively), the scission efciency monotonously increases but the site distribution becomes wider. However, the scission is sufciently restricted to the bulge site even with large bulges in lanes 6 and 7. The addition of a 3-base gap at the joint of a bulge causes no signicant effect (Fig. 4, lane 8). However, when the bulge is smaller than 4 bases, almost no DNA scission occurs (data not presented). The scission bands in the middle of bulges in lanes 6 and 7 are ~2-fold more intense than the corresponding bands for the hydrolysis of single-stranded DNA in lane 2. These phosphodiester linkages are activated for the hydrolysis, in comparison with those in single-stranded DNA. Some of the strain energy at the bulge site could serve to lower the activation energy for the hydrolytic scission, and/or the conformation of DNA backbone there is suitable for the catalysis by the Ce(IV) complex, as was previously observed in metal-mediated RNA hydrolysis (23). Origin of the preferential scission of gap and bulge sites In the presence of DNA1(F), which is complementary with the whole part of DNA1(S), the DNA scission is virtually nil (compare lane 3 with lane 2 in Fig. 2). Similarly, DNA2(F) almost completely inhibits the hydrolysis of DNA2(S) (see lane 3 in Fig. 4). The phosphodiester linkages at unpaired nucleotides are far more reactive for the hydrolysis by the Ce(IV)/EDTA complex than are those at paired nucleotides. This is the primary factor for the present site-selective scission at the gap and bulge structures. These arguments are further conrmed by the experiments in lanes 4 and 5 in Figure 2, where either DNA1(G)-L1 or DNA1(G)-R1 is added so that a predetermined portion of the substrate DNA forms the duplex. Consistently, the single-stranded portion is efciently hydrolyzed by the Ce(IV)/EDTA complex, whereas the double-stranded portion remains intact.

the scissions involving the gaps of various lengths in DNA2(S)]. Since A-T pairs are intrinsically less stable than G-C pairs, the corresponding terminal parts of the two oligonucleotide additives are more weakly bound to the substrate DNA. Thus the selective scission is made less clear cut. Bulge structures for site-selective DNA scission Site-selective DNA scission by the Ce(IV)/EDTA complex is also successful when bulge structures are formed at the target site by using appropriate oligonucleotide additives (Fig. 4). As depicted in lanes 47, the scission selectively occurs at the bulge site. The scission efciency is the largest at around the center of bulge. With increasing bulge length (5-, 6-, 8- and

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Figure 4. Bulge strategy for the site-selective hydrolysis of DNA2(S) by the Ce(IV)/EDTA complex. Lane 1, control; lane 2, DNA2(S) alone; lane 3, with DNA2(F); lane 4, 5-base bulge [with DNA2(B)-1]; lane 5, 6-base bulge [DNA2(B)-2]; lane 6, 8-base bulge [DNA2(B)-3]; lane 7, 10-base bulge [DNA2(B)-4]; lane 8, 10-base bulge + 3-base gap [with DNA2(B)-5]. The reaction conditions are presented in Figure 2.

As shown previously (26), the catalysis by the Ce(IV)/ EDTA complex is specic to polynucleotides which are longer than tetranucleotides. Trinucleotides are not hydrolyzed as they are, but are subject to the catalysis when a phosphate is attached to the terminus. Apparently, the catalysis of this complex requires three or more phosphoesters in the substrate. On the basis of this remarkable substrate specicity, it was proposed that two (or more) phosphodiester linkages of DNA substrate are simultaneously coordinated to the Ce(IV) ion. As a result, these linkages are activated and hydrolyzed. These catalytic processes, which are accompanied by notable conformational change of DNA backbone, are responsible for the structurereactivity relationship in the present gap and bulge induced site-selective DNA scission. The gap strategy is successful only when the number of unpaired nucleotides is greater than 3 (see Fig. 2), since otherwise the conformational change induces unacceptably large strain. The doublestranded portion is too rigid for this conformational change

and is hardly hydrolyzed. In the bulge strategy, the bulge length must be 5 bases or longer to provide the required molecular exibility (Fig. 4). These arguments are in fair agreement with the 1H-NMR analysis on the bulge composed of ve adenine residues (30). It was shown that the bulge loop is localized at intrahelical positions within the double helical stem and the unpaired adenine bases efciently stack on the double strand stem. Similar arguments should hold in the bulges involving both purines and pyrimidines, although the stacking interactions should be weaker than that in the homopurine bulge. For catalysis by the Ce(IV) complex, these stacking structures must be (at least partially) destroyed, and this is energetically unfavorable. Interestingly, heterogeneous gels of Ce(IV) hydroxide, obtained by the addition of Ce(NH4)2(NO3)6 to buffer solutions in the absence of EDTA, hardly distinguish between single-stranded and double-stranded DNA, and hydrolyze both of them at almost the same rates (12). Even when gap or

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Nucleic Acids Research, 2002, Vol. 30 No. 19 e102 hydrolyzed by the Ce(IV)/EDTA complex even in the presence of spermine. Accordingly, all the experiments in Figures 24 were carried out in the presence of 100 mM spermine. In the absence of the Ce(IV) complex, spermine is completely inactive for DNA hydrolysis. Similar synergism of Ce(IV)/EDTA complex with ethylenediamine, diethylenetriamine and other oligoamines was previously reported for the hydrolysis of DNA which involved neither gap nor bulge structures (27). However, spermine is advantageous over these oligoamines in that it is effective at much smaller concentrations. The concentration of ethylenediamine, for example, must be b10 mM in order to accelerate the reaction sufciently. In a proposed mechanism, the positive charges of oligoamines stabilize the negatively charged transition state of DNA hydrolysis through Coulomb interactions. In conclusion, either gap structures or bulge structures in DNA are preferentially hydrolyzed by homogeneous Ce(IV)/ EDTA complex under physiological conditions. Notable site specicity in DNA hydrolysis is for the rst time substantiated by non-covalent strategy which involves no xation of molecular scissors to sequence-recognizing moieties. The sites of selective scission can be chosen rather freely. These ndings should be useful for the design of second-generation articial restriction enzymes, although the present scission is rather slow. Improvements of site selectivity and scission efciency by appropriate chemical modication of additive oligonucleotides are currently underway in our laboratory. SUPPLEMENTARY MATERIAL Supplementary Material is available at NAR Online. ACKNOWLEDGEMENTS This work was supported by the Bio-oriented Technology Research Advancement Institution. Financial support from a Grant-in-Aid for Scientic Research from the Ministry of Education, Science, Sport, Culture, and Technology, Japan, is also acknowledged. REFERENCES
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Figure 5. Effect of spermine on the hydrolysis of DNA2(S) by the Ce(IV)/ EDTA complex at pH 7.0 and 37C. Lane 1, control; lane 2, no additive; lane 3, with DNA2(F) (without spermine); lane 4, 5-base gap without spermine; lane 5, 10-base gap without spermine; lane 6, 5-base gap with 100 mM spermine; lane 7, 10-base gap with 100 mM spermine. The 5-base gap is formed by using DNA2(G)-L and DNA2(G)-R1, whereas the 10-base gap is formed with DNA2(G)-L and DNA2(G)-R2.

bulge structures are formed, no site-selective scission takes place. Homogeneous Ce(IV) solutions are absolutely necessary for the present site-selective reactions. Spermine for the acceleration of the site-selective DNA hydrolysis The gap-induced site-selective DNA hydrolysis by the Ce(IV)/ EDTA complex is notably accelerated by spermine (Fig. 5, compare lane 4 with lane 6 for the 5-base gap, and lane 5 with lane 7 for the 10-base gap). The acceleration by spermine on the bulge-induced site-selective DNA hydrolysis is also evident. In both cases, the site selectivity of scission remains unchanged. The hydrolysis of single-stranded DNA (in the absence of any additive oligonucleotide) is also accelerated by spermine. In contrast, double-stranded DNA is hardly

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