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Nucleic Acids Research, 2003, Vol. 31, No. 15 4497-4502
© 2003 Oxford University Press

Oligoamine–acridine conjugates for promotion of gap-selective DNA hydrolysis by Ce(IV)/EDTA complex

Yoji Yamamoto, Wataru Tsuboi and Makoto Komiyama*

Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan

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

Received April 8, 2003; Revised and Accepted May 27, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Oligoamines (spermidine, dipropylenetriamine and propylenediamine) were covalently attached to acridine via a hexamethylene linker. These oligoamine–acridine conjugates were efficiently bound to gap sites in substrate DNA, and promoted the DNA hydrolysis by a homogeneous Ce(IV)/ethylenediamine-N,N,N',N'-tetraacetate (EDTA) complex at these sites. In contrast, the hydrolysis of the double-stranded portion in the DNA was little affected by these conjugates, although they were strongly bound thereto by the intercalation of their acridine moieties. As a result, the gap site was selectively and efficiently hydrolyzed by combining the Ce(IV)/EDTA complex with the oligoamine– acridine conjugate. Either the oligoamine or the acridine was only poorly active for the purpose, substantiating the essential role of cooperation between them. The promotion of gap-selective DNA hydrolysis by the conjugates has been ascribed to electrostatic stabilization of a negatively charged transition state by their positive charges.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
New tools for site-selective DNA scission are crucially important for further development of molecular biology and biotechnology (1–5). One of their promising applications is the manipulation of DNAs of higher animals and plants, which are too large to be selectively cut by naturally occurring restriction enzymes. Previously (6,7), artificial restriction enzymes were prepared by tethering metal complexes for DNA hydrolysis to the oligonucleotides which are complementary with substrate DNA near the target site. A similar strategy has been employed for the preparation of many ribozyme mimics for RNA hydrolysis (8–16). Recently, a new strategy involving non-covalent fixation of molecular scissors has been proposed (17–19). The target phosphodiester linkage in RNA is differentiated from the other linkages in terms of intrinsic reactivity and preferentially hydrolyzed, although the molecular scissors are freely moving around in the reaction mixtures. For example, the target linkage was activated by non-covalent interactions with acridine-bearing DNA, and was selectively hydrolyzed by various metal ions (19). How ever, non-covalent approaches to site-selective hydrolysis of DNA have been scarce.

A few years ago, we found that a homogeneous Ce(IV)/EDTA complex efficiently hydrolyzes DNA (20). This scission was further accelerated by spermine and other oligoamines (21). Interestingly, single-stranded DNA was hydrolyzed far more promptly than double-stranded DNA. By using this difference in the reactivity, gap sites in substrate DNA were selectively hydrolyzed by the Ce(IV)/EDTA complex (22). However, this gap-selective DNA hydrolysis is not sufficiently fast and some cocatalysts for its acceleration are desirable.

In order to promote the selective hydrolysis at the gap site, the cocatalysts must fulfill the following requirements: (i) they must be strongly bound to the gap site and (ii) the hydrolysis at the gap site must be accelerated more efficiently than the hydrolysis at the other portions. However, it is not easy to design these cocatalysts, since most of the DNA binders previously reported prefer double-stranded DNA to single-stranded DNA. Although oligoamines are bound to single-stranded DNA, they are also notably bound to double-stranded DNA since the binding is based on simple electrostatic interactions. In this paper, we show that covalent conjugates of oligoamine and acridine are strongly bound to the gap site in DNA substrates and selectively promote the hydrolysis of these sites by a Ce(IV)/EDTA complex. The hydrolysis at double-stranded portions is little affected. Either oligoamine or acridine is inefficient. The interactions of the conjugates with the gap site in DNA are quantitatively analyzed, and the mechanism for the acceleration of gap-selective DNA hydrolysis by the conjugates is proposed in terms of these results.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Materials
The conjugates 1–3 were synthesized according to the Supplementary Material (Scheme S1), and purified by recrystallization from ethanol/diethyl ether solution. Their structures and purities were confirmed by 1H-NMR. All the oligonucleotides were prepared on an automated synthesizer and purified by reversed-phase HPLC. Water was deionized by a Millipore Water Purification System and sterilized by an autoclave immediately before use. Commercially obtainable Ce(NH4)2(NO3)6 (Nacalai Tesque, Inc.) and EDTA·4Na (Tokyo Kasei Kogyo Co., Ltd) were used without further purification.

DNA cleavage experiments
The Ce(IV)/EDTA complex was prepared by mixing equal amounts of a 20 mM solution of Ce(NH4)2(NO3)6 in water and 20 mM EDTA·4Na in HEPES buffer. Then, the pH was adjusted to 7.0 with a small amount of NaOH. The substrate DNA (32P-labeled at the 5' end) and the DNA additives were incubated in NaCl solution at 95°C for 2 min and slowly (for 15 min) cooled to room temperature. Then, aqueous solutions of 1–3 were added. The cleavage reaction was started by adding an aqueous solution of the Ce(IV)/EDTA complex to the mixture. Typical reaction conditions were as follows: [substrate DNA]0 = 1 µM, [DNA additive] = 1.1 µM, [Ce(IV)/EDTA] = 500 µM, [oligoamine–acridine conjugate] = 30 µM, [NaCl] = 100 mM, [HEPES] = 5 mM, pH 7.0 and 50°C. After a predetermined time, the reaction was stopped by adding an excess amount of EDTA and inorganic phosphate, and the mixture was subjected to 20% polyacrylamide gel electrophoresis. The quantification of gel patterns was made using a Fuji Film FLA-3000G imaging analyzer.

Spectroscopy
Fluorescence, CD and UV/Vis absorption spectra were measured on a FP-750 spectrofluorometer, a J-725 spectropolarimeter and a V-530 UV/VIS spectrophotometer (from JASCO), respectively. Measurement conditions: [NaCl] = 100 mM, [HEPES] = 5 mM, pH 7.0 and 20°C. Scatchard analysis of the binding of 1 to DNAs, based on the neighbor exclusion model of McGhee and von Hippel, was achieved as described in the literature (23).


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
SUPPLEMENTARY MATERIAL
 REFERENCES
 
Gap-selective DNA hydrolysis by the combination of a Ce(IV)/EDTA complex and an oligoamine–acridine conjugate
In the oligoamine–acridine conjugates 1–3, spermidine, dipropylenetriamine and propylenediamine are bound to acridine via a hexamethylene linker (Fig. 1a). At pH 7.0, these conjugates have four, four and three positive charges, respectively. The ring-nitrogen atoms of the acridine are mostly protonated [the pKa of the ring nitrogen of 9-amino-6-chloro-2-methoxyacridine is 8.5 (24)] and all the aliphatic amino residues are also protonated. By using appropriate oligonucleotides (Fig. 1b), gap structures of the desired length were formed in the substrate 99mer DNA (32P-labeled at the 5' end). Figure 2a shows the typical gel electrophoresis patterns for the DNA hydrolysis at 50°C by the combination of the Ce(IV)/EDTA complex and the spermidine–acridine conjugate 1. The results of quantitative analysis of these lanes are presented in Figure 2b. Here, the reaction rate is expressed in terms of the conversion for each of the phosphodiester linkages in the gap, which is obtained by dividing the total conversion at the gap site by the number of linkages therein. In lanes 4 and 5, a 10-base gap ranging from T40 to C49 was formed by using DNA-L and DNA-R2. In the presence of 1, the gap site was preferentially hydrolyzed (lane 5). This gap-selective DNA scission assisted by 1 is far faster than that without it (lane 4) (22). Synergetic cooperation between the Ce(IV)/EDTA complex and 1 was also observed when a 5-base or 15-base gap was formed in the substrate DNA (lanes 2–3 and 6–7, respectively). The conjugate 1 itself is entirely inactive in the DNA hydrolysis, so that the cooperation of the Ce(IV)/EDTA complex and 1 is evident. The dipropylenetriamine–acridine conjugate 2 and the propylenediamine–acridine conjugate 3 also significantly enhanced the selective DNA scission at the gap site (lanes 4 and 5 in Fig. 3a). For all the conjugates, the scission was strictly restricted to the gap site (the standard markers for 39- and 49mers in lane M correspond to the site of the 10-base gap). The order in the efficiency of synergism with the Ce(IV)/EDTA complex (1, 2 >3) indicates that the number of positive charges in the conjugates is important for their cocatalysis (see the bar charts under the autoradiographs for quantitative data).



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  		Figure 1. (a) Structures of oligoamine–acridine conjugates 1–3 and quinacrine 4. (b) Sequences of the DNAs used for the site-selective scission. In order to form gap structures in the substrate DNA, the DNA oligomers in (b) were combined as follows: 5-base gap, DNA-L + DNA-R1; 10-base gap, DNA-L + DNA-R2; 15-base gap, DNA-L + DNA-R3; 20-base gap, DNA-L + DNA-R4; 30-base gap, DNA-L + DNA-R5.

 


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  		Figure 2. (a) Autoradiographs for the hydrolysis of gaps of different lengths by the combination of spermidine–acridine conjugate 1 and Ce(IV)/EDTA. Lane 1, control; lane 2, 5-base gap without 1; lane 3, 5-base gap with 1; lane 4, 10-base gap without 1; lane 5, 10-base gap with 1; lane 6, 15-base gap without 1; lane 7, 15-base gap with 1; lane 8, 20-base gap without 1; lane 9, 20-base gap with 1; lane 10, 30-base gap without 1; lane 11, 30-base gap with 1. (b) The conversions of scission at the gap sites with conjugate 1 (closed bars) or in its absence (open bars), evaluated by densitometry, are presented by the length of bar. These values were obtained by dividing the total conversion at the gap site by the number of linkages therein. Reaction conditions: [substrate DNA]0 = 1 µM, [DNA additive] = 1.1 µM, [Ce(IV)/EDTA] = 500 µM, [oligoamine–acridine conjugate] = [quinacrine] = [spermine] = [spermidine] = 30 µM, [NaCl] = 100 mM, [HEPES] = 5 mM, pH 7.0, 50°C and 16 h.

 


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  		Figure 3. (a) Autoradiographs for the hydrolysis of the 10-base gap by the combination of Ce(IV)/EDTA with oligoamine–acridine conjugates 1–3. Lane 1, control; lane 2, Ce(IV)/EDTA only; lane 3, Ce(IV)/EDTA + 1; lane 4, Ce(IV)/EDTA + 2; lane 5, Ce(IV)/EDTA + 3; M, authentic samples of 39-, 44-, 49mer DNA oligomers. (b) The results using the components of conjugates. Lane 1, control; lane 2, Ce(IV)/EDTA only; lane 3, Ce(IV)/EDTA + 1; lane 4, Ce(IV)/EDTA + quinacrine; lane 5, Ce(IV)/EDTA + spermine; lane 6, Ce(IV)/EDTA + spermidine. The bar charts under the lanes show the efficiency of scission at the gap site, which is expressed in terms of the conversion for each of the phosphodiester linkages (see text for details).

 
In contrast with the remarkable acceleration by the conjugates 1–3, quinacrine 4 (an acridine derivative having an alkylated exocyclic amino group; see Fig. 1a) showed no measurable effect on the hydrolysis at the gap site under the conditions employed (lane 4 in Fig. 3b). Spermidine (lane 6), dipropylenetriamine and propylenediamine without acridine were only poorly active. The efficient promotion of the Ce(IV)/EDTA complex-induced hydrolysis of the gap site absolutely requires intramolecular cooperation of oligoamine and acridine. It is noteworthy that spermine [H2N(CH2)3 NH(CH2)4NH(CH2)3NH2] was far less active than 1, although it has four positive charges at pH 7.0 as does 1 (compare lane 5 with lane 3 in Fig. 3b). Apparently, the acridine in 1 takes an essential role which cannot be executed by a simple positive charge.

The promotion of DNA hydrolysis by 1 was smaller for longer gaps (the 20-base gap system in lanes 8 and 9 in Fig. 2 and the 30-base gap system in lanes 10 and 11). The binding of 1 to these long gaps would be less favorable, since these sites are highly flexible and thus this binding should accompany a larger loss of entropy. Consistently, the hydrolysis of single-stranded 99mer DNA by the Ce(IV)/EDTA complex was hardly accelerated by 1 (data not shown).

Kinetic study on the gap-selective DNA hydrolysis by the combination of the Ce(IV)/EDTA complex and 1
By using both DNA-L and DNA-R1, a 10-base gap system was formed. As shown in Figure 4a (closed circles), the rate of hydrolysis at the gap site monotonously increased with increasing concentration of 1. The reaction rate is expressed in terms of the conversion for each of the phosphodiester linkages in the gap, and thus is directly comparable with the corresponding values (open circles) for the double-stranded region in which the number of linkages is different. When [1] = 50 µM and the reaction time was 16 h, the total conversion for the DNA hydrolysis at the gap site was ~15 mol%. Thus, the conversion for each of the linkages was 1.5 mol% (there exist 10 linkages in this gap site). In contrast with this significant promotion by 1 of the hydrolysis at the gap site, the hydrolysis of the double-stranded portion is little affected by 1 (open circles). Accordingly, the gap-selective scission becomes clearer with increasing concentration of 1.



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  		Figure 4. (a) Effect of the concentration of 1 on the DNA hydrolysis at the 10-base gap site (closed circles) or in the double-stranded portion (open circles). (b) Effect of the concentration of spermine on the DNA hydrolysis at the 10-base gap site (closed triangles) or in the double-stranded portion (open triangles). The efficiency of scission is expressed in terms of the conversion for each of the phosphodiester linkages, which is obtained by dividing the total conversion at the gap site by the number of linkages.

 
For the purpose of comparison, the results for the reactions using spermine (without acridine) are shown in Figure 4b. In the concentration range employed, the hydrolysis of either the gap site (closed triangles) or the double-stranded region (open triangles) is only marginally promoted by spermine. At the higher concentrations of spermine, the rate of hydrolysis at the gap site gradually increased with the increase in its concentration and attained a plateau when its concentration was ~800 µM. In order to obtain an acceleration of a similar magnitude, the concentration of spermine must be more than 10 times as large as that of 1. Strong binding of 1 to the gap site in substrate DNA is primarily responsible for its remarkable activity as a cocatalyst.

Spectroscopic analysis of the binding of 1 to DNA
In order to shed light on the role of the oligoamine–acridine conjugates for the present cooperative catalysis, the binding of 1 to the gap site was quantitatively analyzed. The DNA specimens in Figure 5 were used. DNA-1 has both a single-stranded portion (gap site) and two flanking double-stranded portions. Sufficiently long and flexible oligo(ethylene glycol) linkers are employed to stabilize these two double-stranded portions. DNA-2 and DNA-3 are exactly identical to the flanking double-stranded portions in DNA-1. DNA-4 is the non-gap version of DNA-1, and forms a stable duplex throughout the strand.



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  		Figure 5. Sequences of DNA oligomers used for the spectroscopic analysis of the binding of 1 to the gap site.

 

The binding of 1 to the double-stranded portion. First, the binding of 1 to the double-stranded DNA was investigated. Upon adding double-stranded DNA-4 to an aqueous solution of 1, the visible absorption spectrum of acridine residue in the 400–480 nm region showed a notable hypochromicity. A weak but significant negative sign circular dichroism (CD) was also induced in this region. Furthermore, the fluorescence from the acridine of 1 was strongly quenched by the DNA. All these results show that the acridine residue is intercalating to the DNA. By Scatchard-type analysis of the fluorescence change, the binding constant K and the exclusion number n for the intercalation were determined to be 4.1 x 106 M–1 and 2.1, respectively (the spectroscopic data and the Scatchard plots are provided in Supplementary Material). This binding constant is approximately 30 times as large as that for the binding of quinacrine to double-stranded DNA (1.3 x 105 M–1) (25). The binding of 1 to DNA is enhanced by the electrostatic interactions between the positive charges of 1 and the anionic charges of DNA.

The binding of 1 to the gap portion. When DNA-1 involving a 10-base gap was mixed with 1 at pH 7.0, both a hypochromicity in the 400–480 nm region and a weakly induced CD signal were observed (Fig. 6a and b, respectively). These changes are consistent with those in the DNA-4/1 system described above, and correspond to the intercalation of the acridine group of 1 to the double-stranded portions of DNA-1. However, the following analyses definitely substantiate that the conjugate 1 is efficiently bound to both the gap region and the double-stranded portions.



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  		Figure 6. (a) Absorption spectra of 1 with (dotted lines) or without (solid lines) DNA-1. (b) CD spectra of DNA-1 with (dotted lines) or without (solid lines) 1. The concentrations of DNA-1 and 1 are 5 and 50 µM, respectively.

 
The closed circles in Figure 7a show the plot of the change of absorbance at 421 nm as the function of the concentration of DNA. The open circles refer to the corresponding results for the 1:1 mixture of DNA-2 and DNA-3, which are identical to the double-stranded portions of DNA-1. If the binding of 1 to DNA-1 were taking place only at its double-stranded portion, these two plots should superimpose each other. However, the points for the DNA-1 (closed circles) enormously deviate in a positive direction from those for the 1:1 DNA-2/DNA-3 mixture (open circles). Similarly, the intensities of the induced-CD signal for DNA-1 are notably different from the corresponding values for the 1:1 DNA-2/DNA-3 mixture (Fig. 7b). Evidence for the binding of 1 to the gap site is conclusive. Its acridine group should show a stacking interaction with the DNA, whereas the positively charged oligoamine moiety is attracted by the negatively charged DNA. This binding is further promoted by the positive charge on the acridine. These arguments are supported by the fact that the plots for the 1:1 DNA-2/DNA-3 mixture, in both Figure 7a and b, fairly fit the theoretical plots (solid lines) obtained by using the binding constant K and the exclusion number n for the DNA-4/1 system (determined above by the Scatchard analysis).



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  		Figure 7. Changes of (a) the absorbance at 421 nm and (b) CD intensity at 455 nm on the addition of 1 to either DNA-1 (closed circles) or the 1:1 mixture of DNA-2 and DNA-3 (open circles). The solid lines are the theoretical lines calculated by using the binding constant (4.1 x 106 M–1) and exclusion number (n = 2.1) obtained by the Scatchard analysis on the DNA-4/1 system (see text for details). The concentration of 1 is 50 µM in (a) and the concentration of DNA oligomer is 5 µM in (b).

 
Proposed mechanism for the gap-selective DNA scission by the combination of the Ce(IV)/EDTA complex and oligoamine–acridine conjugates
The primary reason for the promotion of the scission at the gap site by these conjugates is their efficient binding to the gap site. The apolar interaction of the acridine cooperates with the electrostatic interactions of the oligoamine and the acridine. The negatively charged transition state for the DNA hydrolysis is stabilized by the electrostatic interactions with the positive charges of the conjugates (21). These positive charges are greatly accumulated at the gap site, and exhibit notable electrostatic catalysis. In DNA hydrolysis, the negative charges at the reaction center are increased in the transition state, although the net charges of the whole system are unchanged during the reactions. Thus, the positive charges near the reaction center promote the reaction, exactly as does the positive charge of Lys41 of ribonuclease A in the RNA hydrolysis (26). Furthermore, the formation of metal-bound hydroxide near the negatively charged DNA can be assisted by the positive charges. Both of these factors are favorable for DNA hydrolysis. Consistently, spermine (which is not attached to acridine) shows a significant acceleration only when its concentration is sufficiently high (e.g. >500 µM). Quinacrine 4 having two positive charges induces only a marginal effect.

In conclusion, the oligoamine–acridine conjugates are efficiently bound to the gap sites in DNA. Accordingly, the gap-selective DNA hydrolysis by the Ce(IV)/EDTA complex, reported previously (22), is accelerated due to the electrostatic catalysis by their positive charges. These findings should be useful for the preparation of still more effective catalysts for site-selective DNA scission and also for the design of new DNA-binding molecules.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at NAR Online.


   ACKNOWLEDGEMENTS
 
This work was partially supported by the Bio-oriented Technology Research Advancement Institution. The support by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, Culture and Technology, Japan, is also acknowledged.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

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