Site-selective and hydrolytic two-strand scission of double-stranded D
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《核酸研究医学期刊》
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@mkomi.rcast.u-tokyo.ac.jp
ABSTRACT
By combining Ce(IV)/EDTA with two pseudo-complementary peptide nucleic acids (pcPNAs), both strands in double-stranded DNA were site-selectively hydrolyzed at the target site. Either plasmid DNA (4361 bp) or its linearized form was used as the substrate. When two pcPNAs invaded into the double-stranded DNA, only the designated portion in each of the two strands was free from Watson–Crick base pairing with the counterpart DNA or the pcPNA. Upon the treatment of this invasion complex with Ce(IV)/EDTA at 37°C and pH 7.0, both of these single-stranded portions were selectively hydrolyzed at the designated site, resulting in the site-selective two-strand scission of the double-stranded DNA. Furthermore, the hydrolytic scission products were successfully connected with foreign double-stranded DNA by using ligase. The potential of these artificial systems for manipulation of huge DNA has been indicated.
INTRODUCTION
Preparation of artificial restriction enzymes has been attracting significant interests of chemists and biochemists, mainly because they are necessary to manipulate huge DNAs of animals and plants (1–7). Site-specificity of naturally occurring restriction enzymes is too low for this manipulation. Thus, artificial enzymes, which have much higher site-specificity and can selectively hydrolyze huge DNA at predetermined position, are crucially important for further developments. To date, a number of challenging attempts for the development of artificial restriction enzymes have been already made. Single-stranded DNA was selectively cleaved at the target site by conjugating catalysts for DNA hydrolysis with sequence-recognizing oligonucleotides or their equivalent (8–10). However, only little has been known on site-selective scission of double-stranded DNA that is more widely spread in nature. Conjugation of metal complexes with DNA-binding protein has been proposed as one of the solutions (11). Alternatively, the target site in double-stranded DNA (homopurine–homopyrimidine sequences) was made single-stranded through triple-helix formation by peptide nucleic acid, and selectively digested by single-strand specific nuclease S1 (12).
Recently, it was found that Ce(IV)/EDTA complex preferentially hydrolyzes the phosphodiester linkages in gap-sites in single-stranded DNA substrates (13–15). This scission is selective because gap-sites are more susceptible to the catalysis by the complex. Thus, no covalent fixation of the complex near the target site is necessary. These results have indicated that site-selective two-strand scission of double-stranded DNA should also be possible if one can form gap-like structure in both strands of double-stranded DNA. In this paper, gap-like structures are formed at the designated sites in double-stranded DNA substrate by using invasion of two pseudo-complementary peptide nucleic acids (pcPNAs) additives (16). Upon treatment of these systems with Ce(IV)/EDTA, each of the strands in the substrate DNA is selectively hydrolyzed at the target site, and thus the substrate is divided into desired two fragments. This site-selective two-strand scission is also applicable to supercoiled plasmid DNA. Furthermore, the double-stranded fragments obtained by the scission are connected with foreign double-stranded DNA by using DNA ligase to provide recombinant DNA. A promising applicability of the present DNA scission to molecular biology is indicated.
MATERIALS AND METHODS
Materials
PBR322 plasmid DNA was linearized by EcoRI (Takara). The 408mer double-stranded DNA (T1651-T2058 in the linearized DNA) was prepared by PCR using the following two primers; 5'-TGCACCATTATGTTCCGGATCTG-3' and 5'-AAGCTCATCAGCGTGGTCGTG-3', and purified by QIAquick PCR Purification Kit (Qiagen). Fluorescein-labeled 408mer double-stranded DNA of the same sequence was also prepared by PCR using the corresponding fluorescein-labeled primers. For the preparation of pcPNAs, Boc-protected 2-thiouracil and 2,6-diaminopurine were used together with commercially available Boc-protected monomers and (4-methylbenzhydryl)amine resin (both from ABI) (18). The DNA oligomers (primers and foreign DNAs) were prepared on an ABI 394 DNA/RNA synthesizer using the phosphoroamidite monomers (Glen Research). All these PNAs and DNAs were purified by reversed-phase high-performance liquid chromatography and characterized by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) (Shimadzu, KOMPACT MALDI II). The Ce(IV)/EDTA complex was prepared by mixing 20 mM solution of Ce(NH4)2(NO3)6 (Nacalai Tesque) in water and 20 mM EDTA·4Na (Tokyo Kasei Kogyo) in HEPES buffer, and then the pH was adjusted to 7.0 with small amount of NaOH.
Site-selective scission of double-stranded DNA by Ce(IV)/EDTA
At pH 7.0 (5 mM HEPES buffer containing no NaCl), the linearized PBR322 was incubated with 1:1 mixture of pcPNA(1) and pcPNA(2) (100 nM each) or pcPNA(3)/pcPNA(4) mixture at 37°C for 3 h. Then, NaCl was added to a final concentration of 100 mM. The DNA hydrolysis was started by adding aqueous solution of Ce(IV)/EDTA. Typical cleavage conditions: = 4 nM, = 100 nM, = 20 μM, = 100 mM, = 5 mM and pH 7.0. After a predetermined time, loading buffer (adjusted to pH 7.0) containing bromophenol blue (0.05%), glycerol (30%) and ethylenediaminetetramethylenephosphonic acid (5 mM) in 0.5x TBE buffer was added. The mixture was further incubated for 2 h at 37°C, and then subjected to 0.8% agarose gel electrophoresis. The bands were detected by staining with GelStar (Cambrex). The direct site-selective hydrolysis of supercoiled plasmid DNA was accomplished under exactly the same conditions.
Gel-shift assay for the formation of invasion complex
Invasion complex composed of the 408mer double-stranded DNA (T1651-T2058 in the linearized DNA), pcPNA(1) and pcPNA(2) was prepared as described in the above section. Invasion conditions: = 20 nM, = 200 nM, and = 5 mM at 37°C and pH 7.0 for 3 h. Loading buffer containing bromophenol blue (0.05%) and glycerol (30%) in 0.5x TBE buffer was added to the mixture and then subjected to 5% non-denaturing PAGE. In order to investigate the stability of the invasion complex under the conditions for site-selective DNA scission, NaCl was added to the invasion mixture to a final concentration of 100 mM, incubated at 37°C for 66 h and then subjected to PAGE. The bands were detected by staining with GelStar.
Detailed analysis of the scission sites
Either strand of the 408mer double-stranded DNA (T1651-T2058) was labeled with fluorescein at the 5'-end, and treated with Ce(IV)/EDTA in the presence of 1:1 mixture of pcPNA(1) and pcPNA(2). The reaction conditions were the same as described above. After the reaction, ethylenediaminetetramethylenephosphonic acid was added to a final concentration of 500 μM and the mixture was purified by ethanol precipitation. The resultant pellet was dissolved in loading buffer (Amersham), heated for 10 min at 95°C, cooled on ice and then subjected to 6% denaturing PAGE at 50°C. The Sanger standard lanes were prepared by Thermo Sequenase Primer Cycle Sequencing Kit (Amersham) using EcoRI-digested PBR322 and fluorescein-labeled primer.
Enzymatic ligation of scission fragments and sequencing of ligation products
After the site-selective scission, the longer products in the gel were purified by agarose gel electrophoresis and extracted from the gel by CONCERT Rapid Gel Extraction System (GibcoBRL). The DNA(1)/DNA(2) mixture was annealed in pH 7.0 solution containing 25 mM NaCl, and then added to the solution of longer scission products (final concentration of foreign DNA was 0.1 μM). By treating the mixture with T4 DNA ligase in the ligation buffer (DNA Ligation Kit of Takara) at 16°C for 30 min, recombinant DNA was obtained and then purified by QIAquick PCR Purification Kit (Qiagen). In order to analyze the ligation product in gel electrophoresis, a foreign DNA, which was 1077 bp longer than DNA(1)/DNA(2) duplex and had the same sticky end, was used.
The purified recombinant DNA was amplified by hot start PCR using primer(1) and primer(2). The PCR cycle is as follows: 95°C for 30 s to 68°C for 60 s, 25 cycles. The PCR product was cloned using pGEM-T Easy Vector (Promega) and JM109 competent cell (Toyobo), and then its sequence was determined on an ABI PRISM 310 Genetic Analyzer.
Digestion of double-stranded DNA by nuclease S1 in the presence of two pcPNAs
Invasion complex composed of linearized PBR322, pcPNA(1) and pcPNA(2) was prepared as described above. Then, nuclease S1 reaction buffer containing sodium acetate, zinc acetate and NaCl (adjusted to pH 4.6) was added. The enzymatic reaction was started by the addition of nuclease S1. Digestion conditions: = 4 nM, = 100 nM, = 5 U/μl, = 100 mM, = 1 mM, = 30 mM, = 5 mM at 25°C and pH 4.6. After a predetermined time, the mixture was analyzed by 0.8% agarose gel electrophoresis, exactly as described for the site-selective scission by Ce(IV)/EDTA.
RESULTS AND DISCUSSION
Site-selective two-strand scission of double-stranded DNA by combining Ce(IV)/EDTA with two pcPNAs
Site-selective scission of linear double-stranded DNA
Outline of the manipulation of linear double-stranded DNA is shown in Figure 1. First, PBR322 plasmid DNA (supercoiled form I) was cut at one site by restriction enzyme EcoRI, and converted to its linearized form (form III). Supercoiled DNA can also be site-selectively hydrolyzed by the present method: vide infra. This linearized plasmid DNA (4361 bp) was treated with Ce(IV)/EDTA at 37°C and pH 7.0 in the presence of pcPNA(1) and pcPNA(2) (step 1 in Figure 1). These two pcPNA additives are complementary with C1826-A1840 in the upper strand of the linearized DNA and A1821-G1835 in its lower strand, respectively. Accordingly, almost all the nucleobases in the substrate DNA are pairing with the counterpart nucleobase in either the DNA or the pcPNA, and only T1821-T1825 in the upper strand and G1836-T1840 in the lower strand are kept single-stranded (underlined nucleotides in Figure 2a). These gap-like sites are the targets for the scission. Formation of pcPNA(1)/pcPNA(2) duplex is inefficient because of the steric repulsion between 2'-S in U and 2'-NH2 in D, although 10 nt in them are complementary with each other (16).
Figure 1. Outline of the present DNA manipulation.
Figure 2. (a) DNA substrate and pcPNA additives used for site-selective scission by Ce(IV)/EDTA. In the invasion complex composed of linearized PBR322, pcPNA(1) and pcPNA(2), the underlined nucleotides remain unpaired. In place of conventional bases, U and D in pcPNA bear 2-thiouracil and 2,6-diaminopurine residues, respectively. (b) DNA fragments used for enzymatic ligation. The sticky end of foreign DNA is complementary to that of the scission fragment . (c) Primers used for PCR amplification of recombinant DNA.
Upon the treatment of this invasion complex with Ce(IV)/EDTA, two bands were clearly observed by agarose gel electrophoresis (lane 3 in Figure 3). No other bands were detectable, indicating high site-selectivity of the present DNA scission. One of the products in the gel is slightly smaller than 2000mer, and the other is between 2000 and 3000mer. These values are consistent with the scission of the two strands in the substrate DNA (4361 bp) at the corresponding gap-like site (approximately 1830 and 2530mer products should be formed from the left-hand side of the substrate DNA and its right-hand side, respectively). The structures of these products were further confirmed by the ligation experiments described below. Under the conditions employed here, the conversion for the site-selective scission of the 4361mer DNA was 30%. The site-selective scission of double-stranded DNA could also be accomplished at higher temperatures (45 and 50°C) to shorten the reaction times (lanes 5 and 6 in Figure 3). The selectivity remained satisfactorily high even under these conditions.
Figure 3. Agarose gel electrophoresis patterns for the site-selective two-strand hydrolysis of the linearized PBR322 DNA by Ce(IV)/EDTA in the presence of pcPNA additives. Lane 1, pcPNA(1)/pcPNA(2) only ; lane 2, Ce(IV)/EDTA only ; lane 3, pcPNA(1)/pcPNA(2) + Ce(IV)/EDTA; lane 4, pcPNA(3)/pcPNA(4) + Ce(IV)/EDTA; M, 1000 bp ladder. Scission conditions: = 4 nM, = 100 nM, = 20 μM, = 100 mM, and = 5 mM at 37°C and pH 7.0 for 64 h. In lane 5, the reaction in lane 3 was achieved at 45°C for 22 h, and in lane 6 at 50°C for 11.5 h. The bands were detected by staining with GelStar.
Site-selective scission of supercoiled double-stranded DNA
By combining Ce(IV)/EDTA and pcPNA additives, supercoiled DNA (form I DNA) can also be hydrolyzed selectively at the desired site (Figure 4). In lane 2, PBR322 DNA was directly treated with Ce(IV)/EDTA in the presence of pcPNA(1) and pcPNA(2). In this treatment, the supercoiled DNA was converted to a linear form III DNA. Then, the product was digested by EcoRI. As shown in lane 3, two fragments of expected sizes (the same ones as obtained in Figure 3) were formed. The two-strand scission of the supercoiled DNA at the target site by Ce(IV)/EDTA has been substantiated.
Figure 4. Direct site-selective hydrolysis of supercoiled PBR322 DNA by Ce(IV)/EDTA in the presence of pcPNA additives. Lane 1, pcPNA(1)/pcPNA(2) only ; lane 2, pcPNA(1)/pcPNA(2) + Ce(IV)/EDTA; lane 3, EcoRI digests of the products in lane 2; M, 1000 bp ladder. Reaction conditions are the same as described for Figure 3.
Gel-shift assay for the invasion of two pcPNAs to double-stranded DNA
The efficiency of the formation of invasion complex was investigated by gel-shift assay using 5% non-denaturing PAGE (Figure 5). In order to make the assay still more clear-cut, 408mer DNA (T1651-T2058 of the linearized PBR322 DNA) involving the target scission-site was used in place of the whole DNA. Otherwise, the conditions were the same as those employed for the site-selective DNA scission (at 37°C and pH 7.0). Upon the addition of 1:1 mixture of pcPNA(1) and pcPNA(2), a new band for the invasion complex appeared at smaller mobility than the band for the 408mer DNA (lane 2). More than 80% of the DNA forms the invasion complex with pcPNA(1)/pcPNA(2) under these conditions. Furthermore, this invasion complex remained satisfactorily intact when it was incubated under the reaction conditions for a long time (lane 3).
Figure 5. Gel-shift assay for the formation of invasion complex from the 408mer double-stranded DNA, pcPNA(1), and pcPNA(2). The DNA is T1651-T2058 of PBR322 and involves the target scission-site. Lane 1, DNA only; lane 2, in the presence of 1:1 mixture of pcPNA(1) and pcPNA(2). Invasion conditions: = 20 nM, = 200 nM, and = 5 mM at 37°C and pH 7.0 for 3 h (no NaCl was added to the mixture). In lane 3, the stability of the invasion complex under the conditions for site-selective scission (in Figures 3 and 4) was investigated by adding 100 mM NaCl to the mixture and incubating it at 37°C for 66 h.
Detailed analysis of the scission sites
In order to investigate the scission sites more in detail, each strand of the 408mer double-stranded DNA (T1651-T2058 of the linearized PBR322 DNA) was labeled with fluorescein at the 5'-end, and the scission fragments were analyzed by denaturing PAGE (Figure 6). As shown in Figure 6a, the upper strand was mostly hydrolyzed at G1820-A1830. The scission at C1823-A1827 is the most prevailing. Apparently, the scission by Ce(IV)/EDTA occurred at the gap-like site formed through invasion of the pcPNA additives, although 3'-side of this strand was also hydrolyzed to some extent. Virtually the same result was obtained for the scission of the lower strand (see Figure 6b). Complete separation of the bands was not successful partially because the scission provided both the fragments having OH termini and the ones having phosphate termini.
Figure 6. PAGE patterns for site-selective hydrolysis of the 408mer double-stranded DNA (T1651-T2058). In (a), the upper strand was labeled with fluorescein at the 5'-end, whereas the lower strand was labeled in (b). Lanes A, C, G and T are the Sanger standard lanes. Reaction conditions: = 20 nM, = 200 nM, = 100 μM, = 100 mM, and = 5 mM at 37°C and pH 7.0 for 69 h. The bands designated by asterisk are associated with incompletely denatured substrate DNA. The PNA additives show no significant effects, as confirmed by the control lane ‘pcPNA(1)/pcPNA(2) only’ where simple mixture of these additives and the DNA substrate was charged to the gel.
Requirements for the present site-selective scission of double-stranded DNA by Ce(IV)/EDTA
The pcPNA additives must have flanking portions and provide gap-like structures in the double-stranded DNA. Accordingly, the scission was marginal when pcPNA(3) and pcPNA(4) were combined (see lane 4 in Figure 3). These two pcPNAs are completely complementary to each other (although their duplex is not much formed due to mutual steric repulsion), and thus no gap-like structures are formed when they invade into the linearized plasmid DNA (see the structure in the bottom of Figure 2a). In the absence of pcPNA additives, no scission occurred as expected (lane 2 in Figure 3).
In the present system, invasion complex composed of PBR322 plasmid DNA and pcPNA(1)/pcPNA(2) was first formed in the absence of NaCl, since low salt concentrations are more favorable for the formation of invasion complex (19). Then, NaCl was added to the mixture to a final concentration of 100 mM, and the DNA hydrolysis was started (see Materials and Methods for details). This subsequent addition of NaCl is necessary to stabilize the DNA/DNA duplexes in the solution and achieve high site-selectivity (data not shown). Under the conditions for site-selective scission, the invasion complex was sufficiently stable as was confirmed by the gel-shift assay in lane 3 of Figure 5.
Enzymatic ligation of the scission fragments with foreign DNA using DNA ligase
Since DNA scission by Ce(IV)/EDTA complex proceeds via hydrolytic pathway (20), the scission fragments can be recombined with foreign DNA by ligase to provide desired recombinant DNA. The longer scission products from the linearized plasmid DNA (observed between 2000 and 3000mer in the gel in Figure 3) were mixed with DNA(1)/DNA(2) duplex, and the mixture was treated with T4 DNA ligase (step 2 in Figure 1). The sticky end of this foreign DNA is complementary to the end of the fragment(L) that is formed by cutting the upper strand of the linearized DNA at the 5'-side of A1824 and the lower strand at the 5'-side of G1837 (see the right-hand side of Figure 2b). Successful ligation of these two double-stranded DNAs was confirmed by the following experiments. The ligation product was first amplified by PCR. Of two primers used, primer(1) is complementary to DNA(2), whereas primer(2) is complementary to G2271-C2292 of the upper strand of the fragment(L) (Figure 2c). As shown by the gel electrophoresis in lane 1 of Figure 7, PCR product of 500mer size was efficiently formed . Consistently, no PCR products were formed when a mixture of the longer scission products in Figure 3 and DNA(1)/DNA(2) duplex was directly (without the treatment with T4 ligase) used for PCR (see lane 2 in Figure 7). Furthermore, the sequence of the ligation product was completely characterized by the sequencing experiment in Figure 8 (detailed procedures are described in Materials and Methods).
Figure 7. Agarose gel electrophoresis of the PCR product using the recombinant DNA obtained from the scission fragment(L) and DNA(1)/DNA(2) duplex. Lane 1, PCR product from the recombinant DNA; lane 2, the product of PCR reaction using the mixture of fragment(L) and DNA(1)/DNA(2) without the enzymatic ligation; M, 200 bp ladder. The primer(1) is complementary to DNA(2), whereas primer(2) is complementary to G2271-C2292 of the upper strand of the fragment(L). The bands were detected by staining with GelStar.
Figure 8. Sequencing analysis of the recombinant DNA obtained by the ligation of fragment(L) with DNA(1)/DNA(2) duplex. The conjunction between DNA(1) and the 5' side portion of upper strand of the fragment(L) is shown.
The ligation was also successful when foreign DNA was much (1077 bp) longer than DNA(1)/DNA(2) duplex and had the same sticky end as this duplex. The ligation product was clearly separated from the other bands in agarose gel electrophoresis, and its conversion was 20%. This value probably reflects the amount of fragment(L) obtained by the present site-selective scission. Of several fragments formed by the scission, this fragment was selectively incorporated into the recombinant DNA, since its sticky end completely fits that of DNA(1)/DNA(2) duplex (and of the longer foreign DNA).
Use of nuclease S1 as the scissors in place of Ce(IV)/EDTA
The invasion complex composed of the linearized plasmid DNA, pcPNA(1), and pcPNA(2) was treated with nuclease S1 (single-strand specific nuclease) at 25°C and pH 4.6 (see Supplemental Figure). In the very early stage of the reaction, two bands were detected in agarose gel electrophoresis, as was observed in Figure 3. In prolonged reactions, however, these bands gradually weakened and became smeary. Apparently, these double-stranded DNA products were further digested by nuclease S1. The substrate (the linearized plasmid DNA) was also notably digested by this single-strand specific nuclease, providing a smeary band. Probably, the binding of nuclease S1 toward single-stranded DNA is so strong that either strand in double-stranded DNA is partially removed from its complementary strand by this enzyme and is digested. In contrast, Ce(IV)/EDTA binds single-stranded DNA less efficiently, and thus similar process hardly occurs in the non-enzymatic reactions (13,14). Furthermore, double-stranded DNA is hardly hydrolyzed by this complex. As the result, the double-stranded DNA products, formed by the primary scission by this complex, remain satisfactorily intact even for prolonged reactions. Attempts to find appropriate conditions for enzymatic reactions where the primary scission products are obtained in high yields were unsuccessful.
CONCLUSION
By using two pcPNAs, invasion structures are formed in double-stranded DNA, and designated sites in both strands are made single-stranded. These sites are selectively hydrolyzed by Ce(IV)/EDTA, because of the substrate-specificity of this complex (single-stranded DNA is hydrolyzed much faster than double-stranded DNA). As the substrates for the present site-selective two-strand scission, both linear DNA and supercoiled DNA are usable. The lengths of pcPNAs and their sequences can be versatile, and thus these systems are potent for site-selective scission of huge DNAs. Furthermore, the products of the present site-selective scission can be connected with foreign DNA using DNA ligase. Accordingly, fundamental processes of DNA manipulation (site-selective two-strand scission of double-stranded DNA at designated site, followed by enzymatic ligation) have been successfully accomplished.
Quite recently, gap-selective scission of single-stranded DNA by Ce(IV)/EDTA has been promoted by introducing monophosphate groups to the gap-site (21,22). It was proposed that these groups recruit the complex to the gap-site and accelerate the reaction there. A similar strategy could promote the present site-selective scission of double-stranded DNA. Appropriate chemical modification of pcPNA additives is also expected to be useful for further increase in the site-selectivity and scission-efficiency. These attempts, as well as the application of the present findings to DNA manipulation, are currently under way in our laboratory.
SUPPLEMENTARY MATERIAL
Supplementary Material is available at NAR Online.
ACKNOWLEDGEMENTS
We would like to thank Prof. Teruyuki Nagamune for his valuable comments on the analysis of scission fragments and Dr J.-M. Zhou for the assistance in sequencing the recombinant DNA. This work was partially supported by PROBRAIN. The support by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, Culture and Technology, Japan is also acknowledged.
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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
ABSTRACT
By combining Ce(IV)/EDTA with two pseudo-complementary peptide nucleic acids (pcPNAs), both strands in double-stranded DNA were site-selectively hydrolyzed at the target site. Either plasmid DNA (4361 bp) or its linearized form was used as the substrate. When two pcPNAs invaded into the double-stranded DNA, only the designated portion in each of the two strands was free from Watson–Crick base pairing with the counterpart DNA or the pcPNA. Upon the treatment of this invasion complex with Ce(IV)/EDTA at 37°C and pH 7.0, both of these single-stranded portions were selectively hydrolyzed at the designated site, resulting in the site-selective two-strand scission of the double-stranded DNA. Furthermore, the hydrolytic scission products were successfully connected with foreign double-stranded DNA by using ligase. The potential of these artificial systems for manipulation of huge DNA has been indicated.
INTRODUCTION
Preparation of artificial restriction enzymes has been attracting significant interests of chemists and biochemists, mainly because they are necessary to manipulate huge DNAs of animals and plants (1–7). Site-specificity of naturally occurring restriction enzymes is too low for this manipulation. Thus, artificial enzymes, which have much higher site-specificity and can selectively hydrolyze huge DNA at predetermined position, are crucially important for further developments. To date, a number of challenging attempts for the development of artificial restriction enzymes have been already made. Single-stranded DNA was selectively cleaved at the target site by conjugating catalysts for DNA hydrolysis with sequence-recognizing oligonucleotides or their equivalent (8–10). However, only little has been known on site-selective scission of double-stranded DNA that is more widely spread in nature. Conjugation of metal complexes with DNA-binding protein has been proposed as one of the solutions (11). Alternatively, the target site in double-stranded DNA (homopurine–homopyrimidine sequences) was made single-stranded through triple-helix formation by peptide nucleic acid, and selectively digested by single-strand specific nuclease S1 (12).
Recently, it was found that Ce(IV)/EDTA complex preferentially hydrolyzes the phosphodiester linkages in gap-sites in single-stranded DNA substrates (13–15). This scission is selective because gap-sites are more susceptible to the catalysis by the complex. Thus, no covalent fixation of the complex near the target site is necessary. These results have indicated that site-selective two-strand scission of double-stranded DNA should also be possible if one can form gap-like structure in both strands of double-stranded DNA. In this paper, gap-like structures are formed at the designated sites in double-stranded DNA substrate by using invasion of two pseudo-complementary peptide nucleic acids (pcPNAs) additives (16). Upon treatment of these systems with Ce(IV)/EDTA, each of the strands in the substrate DNA is selectively hydrolyzed at the target site, and thus the substrate is divided into desired two fragments. This site-selective two-strand scission is also applicable to supercoiled plasmid DNA. Furthermore, the double-stranded fragments obtained by the scission are connected with foreign double-stranded DNA by using DNA ligase to provide recombinant DNA. A promising applicability of the present DNA scission to molecular biology is indicated.
MATERIALS AND METHODS
Materials
PBR322 plasmid DNA was linearized by EcoRI (Takara). The 408mer double-stranded DNA (T1651-T2058 in the linearized DNA) was prepared by PCR using the following two primers; 5'-TGCACCATTATGTTCCGGATCTG-3' and 5'-AAGCTCATCAGCGTGGTCGTG-3', and purified by QIAquick PCR Purification Kit (Qiagen). Fluorescein-labeled 408mer double-stranded DNA of the same sequence was also prepared by PCR using the corresponding fluorescein-labeled primers. For the preparation of pcPNAs, Boc-protected 2-thiouracil and 2,6-diaminopurine were used together with commercially available Boc-protected monomers and (4-methylbenzhydryl)amine resin (both from ABI) (18). The DNA oligomers (primers and foreign DNAs) were prepared on an ABI 394 DNA/RNA synthesizer using the phosphoroamidite monomers (Glen Research). All these PNAs and DNAs were purified by reversed-phase high-performance liquid chromatography and characterized by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) (Shimadzu, KOMPACT MALDI II). The Ce(IV)/EDTA complex was prepared by mixing 20 mM solution of Ce(NH4)2(NO3)6 (Nacalai Tesque) in water and 20 mM EDTA·4Na (Tokyo Kasei Kogyo) in HEPES buffer, and then the pH was adjusted to 7.0 with small amount of NaOH.
Site-selective scission of double-stranded DNA by Ce(IV)/EDTA
At pH 7.0 (5 mM HEPES buffer containing no NaCl), the linearized PBR322 was incubated with 1:1 mixture of pcPNA(1) and pcPNA(2) (100 nM each) or pcPNA(3)/pcPNA(4) mixture at 37°C for 3 h. Then, NaCl was added to a final concentration of 100 mM. The DNA hydrolysis was started by adding aqueous solution of Ce(IV)/EDTA. Typical cleavage conditions: = 4 nM, = 100 nM, = 20 μM, = 100 mM, = 5 mM and pH 7.0. After a predetermined time, loading buffer (adjusted to pH 7.0) containing bromophenol blue (0.05%), glycerol (30%) and ethylenediaminetetramethylenephosphonic acid (5 mM) in 0.5x TBE buffer was added. The mixture was further incubated for 2 h at 37°C, and then subjected to 0.8% agarose gel electrophoresis. The bands were detected by staining with GelStar (Cambrex). The direct site-selective hydrolysis of supercoiled plasmid DNA was accomplished under exactly the same conditions.
Gel-shift assay for the formation of invasion complex
Invasion complex composed of the 408mer double-stranded DNA (T1651-T2058 in the linearized DNA), pcPNA(1) and pcPNA(2) was prepared as described in the above section. Invasion conditions: = 20 nM, = 200 nM, and = 5 mM at 37°C and pH 7.0 for 3 h. Loading buffer containing bromophenol blue (0.05%) and glycerol (30%) in 0.5x TBE buffer was added to the mixture and then subjected to 5% non-denaturing PAGE. In order to investigate the stability of the invasion complex under the conditions for site-selective DNA scission, NaCl was added to the invasion mixture to a final concentration of 100 mM, incubated at 37°C for 66 h and then subjected to PAGE. The bands were detected by staining with GelStar.
Detailed analysis of the scission sites
Either strand of the 408mer double-stranded DNA (T1651-T2058) was labeled with fluorescein at the 5'-end, and treated with Ce(IV)/EDTA in the presence of 1:1 mixture of pcPNA(1) and pcPNA(2). The reaction conditions were the same as described above. After the reaction, ethylenediaminetetramethylenephosphonic acid was added to a final concentration of 500 μM and the mixture was purified by ethanol precipitation. The resultant pellet was dissolved in loading buffer (Amersham), heated for 10 min at 95°C, cooled on ice and then subjected to 6% denaturing PAGE at 50°C. The Sanger standard lanes were prepared by Thermo Sequenase Primer Cycle Sequencing Kit (Amersham) using EcoRI-digested PBR322 and fluorescein-labeled primer.
Enzymatic ligation of scission fragments and sequencing of ligation products
After the site-selective scission, the longer products in the gel were purified by agarose gel electrophoresis and extracted from the gel by CONCERT Rapid Gel Extraction System (GibcoBRL). The DNA(1)/DNA(2) mixture was annealed in pH 7.0 solution containing 25 mM NaCl, and then added to the solution of longer scission products (final concentration of foreign DNA was 0.1 μM). By treating the mixture with T4 DNA ligase in the ligation buffer (DNA Ligation Kit of Takara) at 16°C for 30 min, recombinant DNA was obtained and then purified by QIAquick PCR Purification Kit (Qiagen). In order to analyze the ligation product in gel electrophoresis, a foreign DNA, which was 1077 bp longer than DNA(1)/DNA(2) duplex and had the same sticky end, was used.
The purified recombinant DNA was amplified by hot start PCR using primer(1) and primer(2). The PCR cycle is as follows: 95°C for 30 s to 68°C for 60 s, 25 cycles. The PCR product was cloned using pGEM-T Easy Vector (Promega) and JM109 competent cell (Toyobo), and then its sequence was determined on an ABI PRISM 310 Genetic Analyzer.
Digestion of double-stranded DNA by nuclease S1 in the presence of two pcPNAs
Invasion complex composed of linearized PBR322, pcPNA(1) and pcPNA(2) was prepared as described above. Then, nuclease S1 reaction buffer containing sodium acetate, zinc acetate and NaCl (adjusted to pH 4.6) was added. The enzymatic reaction was started by the addition of nuclease S1. Digestion conditions: = 4 nM, = 100 nM, = 5 U/μl, = 100 mM, = 1 mM, = 30 mM, = 5 mM at 25°C and pH 4.6. After a predetermined time, the mixture was analyzed by 0.8% agarose gel electrophoresis, exactly as described for the site-selective scission by Ce(IV)/EDTA.
RESULTS AND DISCUSSION
Site-selective two-strand scission of double-stranded DNA by combining Ce(IV)/EDTA with two pcPNAs
Site-selective scission of linear double-stranded DNA
Outline of the manipulation of linear double-stranded DNA is shown in Figure 1. First, PBR322 plasmid DNA (supercoiled form I) was cut at one site by restriction enzyme EcoRI, and converted to its linearized form (form III). Supercoiled DNA can also be site-selectively hydrolyzed by the present method: vide infra. This linearized plasmid DNA (4361 bp) was treated with Ce(IV)/EDTA at 37°C and pH 7.0 in the presence of pcPNA(1) and pcPNA(2) (step 1 in Figure 1). These two pcPNA additives are complementary with C1826-A1840 in the upper strand of the linearized DNA and A1821-G1835 in its lower strand, respectively. Accordingly, almost all the nucleobases in the substrate DNA are pairing with the counterpart nucleobase in either the DNA or the pcPNA, and only T1821-T1825 in the upper strand and G1836-T1840 in the lower strand are kept single-stranded (underlined nucleotides in Figure 2a). These gap-like sites are the targets for the scission. Formation of pcPNA(1)/pcPNA(2) duplex is inefficient because of the steric repulsion between 2'-S in U and 2'-NH2 in D, although 10 nt in them are complementary with each other (16).
Figure 1. Outline of the present DNA manipulation.
Figure 2. (a) DNA substrate and pcPNA additives used for site-selective scission by Ce(IV)/EDTA. In the invasion complex composed of linearized PBR322, pcPNA(1) and pcPNA(2), the underlined nucleotides remain unpaired. In place of conventional bases, U and D in pcPNA bear 2-thiouracil and 2,6-diaminopurine residues, respectively. (b) DNA fragments used for enzymatic ligation. The sticky end of foreign DNA is complementary to that of the scission fragment . (c) Primers used for PCR amplification of recombinant DNA.
Upon the treatment of this invasion complex with Ce(IV)/EDTA, two bands were clearly observed by agarose gel electrophoresis (lane 3 in Figure 3). No other bands were detectable, indicating high site-selectivity of the present DNA scission. One of the products in the gel is slightly smaller than 2000mer, and the other is between 2000 and 3000mer. These values are consistent with the scission of the two strands in the substrate DNA (4361 bp) at the corresponding gap-like site (approximately 1830 and 2530mer products should be formed from the left-hand side of the substrate DNA and its right-hand side, respectively). The structures of these products were further confirmed by the ligation experiments described below. Under the conditions employed here, the conversion for the site-selective scission of the 4361mer DNA was 30%. The site-selective scission of double-stranded DNA could also be accomplished at higher temperatures (45 and 50°C) to shorten the reaction times (lanes 5 and 6 in Figure 3). The selectivity remained satisfactorily high even under these conditions.
Figure 3. Agarose gel electrophoresis patterns for the site-selective two-strand hydrolysis of the linearized PBR322 DNA by Ce(IV)/EDTA in the presence of pcPNA additives. Lane 1, pcPNA(1)/pcPNA(2) only ; lane 2, Ce(IV)/EDTA only ; lane 3, pcPNA(1)/pcPNA(2) + Ce(IV)/EDTA; lane 4, pcPNA(3)/pcPNA(4) + Ce(IV)/EDTA; M, 1000 bp ladder. Scission conditions: = 4 nM, = 100 nM, = 20 μM, = 100 mM, and = 5 mM at 37°C and pH 7.0 for 64 h. In lane 5, the reaction in lane 3 was achieved at 45°C for 22 h, and in lane 6 at 50°C for 11.5 h. The bands were detected by staining with GelStar.
Site-selective scission of supercoiled double-stranded DNA
By combining Ce(IV)/EDTA and pcPNA additives, supercoiled DNA (form I DNA) can also be hydrolyzed selectively at the desired site (Figure 4). In lane 2, PBR322 DNA was directly treated with Ce(IV)/EDTA in the presence of pcPNA(1) and pcPNA(2). In this treatment, the supercoiled DNA was converted to a linear form III DNA. Then, the product was digested by EcoRI. As shown in lane 3, two fragments of expected sizes (the same ones as obtained in Figure 3) were formed. The two-strand scission of the supercoiled DNA at the target site by Ce(IV)/EDTA has been substantiated.
Figure 4. Direct site-selective hydrolysis of supercoiled PBR322 DNA by Ce(IV)/EDTA in the presence of pcPNA additives. Lane 1, pcPNA(1)/pcPNA(2) only ; lane 2, pcPNA(1)/pcPNA(2) + Ce(IV)/EDTA; lane 3, EcoRI digests of the products in lane 2; M, 1000 bp ladder. Reaction conditions are the same as described for Figure 3.
Gel-shift assay for the invasion of two pcPNAs to double-stranded DNA
The efficiency of the formation of invasion complex was investigated by gel-shift assay using 5% non-denaturing PAGE (Figure 5). In order to make the assay still more clear-cut, 408mer DNA (T1651-T2058 of the linearized PBR322 DNA) involving the target scission-site was used in place of the whole DNA. Otherwise, the conditions were the same as those employed for the site-selective DNA scission (at 37°C and pH 7.0). Upon the addition of 1:1 mixture of pcPNA(1) and pcPNA(2), a new band for the invasion complex appeared at smaller mobility than the band for the 408mer DNA (lane 2). More than 80% of the DNA forms the invasion complex with pcPNA(1)/pcPNA(2) under these conditions. Furthermore, this invasion complex remained satisfactorily intact when it was incubated under the reaction conditions for a long time (lane 3).
Figure 5. Gel-shift assay for the formation of invasion complex from the 408mer double-stranded DNA, pcPNA(1), and pcPNA(2). The DNA is T1651-T2058 of PBR322 and involves the target scission-site. Lane 1, DNA only; lane 2, in the presence of 1:1 mixture of pcPNA(1) and pcPNA(2). Invasion conditions: = 20 nM, = 200 nM, and = 5 mM at 37°C and pH 7.0 for 3 h (no NaCl was added to the mixture). In lane 3, the stability of the invasion complex under the conditions for site-selective scission (in Figures 3 and 4) was investigated by adding 100 mM NaCl to the mixture and incubating it at 37°C for 66 h.
Detailed analysis of the scission sites
In order to investigate the scission sites more in detail, each strand of the 408mer double-stranded DNA (T1651-T2058 of the linearized PBR322 DNA) was labeled with fluorescein at the 5'-end, and the scission fragments were analyzed by denaturing PAGE (Figure 6). As shown in Figure 6a, the upper strand was mostly hydrolyzed at G1820-A1830. The scission at C1823-A1827 is the most prevailing. Apparently, the scission by Ce(IV)/EDTA occurred at the gap-like site formed through invasion of the pcPNA additives, although 3'-side of this strand was also hydrolyzed to some extent. Virtually the same result was obtained for the scission of the lower strand (see Figure 6b). Complete separation of the bands was not successful partially because the scission provided both the fragments having OH termini and the ones having phosphate termini.
Figure 6. PAGE patterns for site-selective hydrolysis of the 408mer double-stranded DNA (T1651-T2058). In (a), the upper strand was labeled with fluorescein at the 5'-end, whereas the lower strand was labeled in (b). Lanes A, C, G and T are the Sanger standard lanes. Reaction conditions: = 20 nM, = 200 nM, = 100 μM, = 100 mM, and = 5 mM at 37°C and pH 7.0 for 69 h. The bands designated by asterisk are associated with incompletely denatured substrate DNA. The PNA additives show no significant effects, as confirmed by the control lane ‘pcPNA(1)/pcPNA(2) only’ where simple mixture of these additives and the DNA substrate was charged to the gel.
Requirements for the present site-selective scission of double-stranded DNA by Ce(IV)/EDTA
The pcPNA additives must have flanking portions and provide gap-like structures in the double-stranded DNA. Accordingly, the scission was marginal when pcPNA(3) and pcPNA(4) were combined (see lane 4 in Figure 3). These two pcPNAs are completely complementary to each other (although their duplex is not much formed due to mutual steric repulsion), and thus no gap-like structures are formed when they invade into the linearized plasmid DNA (see the structure in the bottom of Figure 2a). In the absence of pcPNA additives, no scission occurred as expected (lane 2 in Figure 3).
In the present system, invasion complex composed of PBR322 plasmid DNA and pcPNA(1)/pcPNA(2) was first formed in the absence of NaCl, since low salt concentrations are more favorable for the formation of invasion complex (19). Then, NaCl was added to the mixture to a final concentration of 100 mM, and the DNA hydrolysis was started (see Materials and Methods for details). This subsequent addition of NaCl is necessary to stabilize the DNA/DNA duplexes in the solution and achieve high site-selectivity (data not shown). Under the conditions for site-selective scission, the invasion complex was sufficiently stable as was confirmed by the gel-shift assay in lane 3 of Figure 5.
Enzymatic ligation of the scission fragments with foreign DNA using DNA ligase
Since DNA scission by Ce(IV)/EDTA complex proceeds via hydrolytic pathway (20), the scission fragments can be recombined with foreign DNA by ligase to provide desired recombinant DNA. The longer scission products from the linearized plasmid DNA (observed between 2000 and 3000mer in the gel in Figure 3) were mixed with DNA(1)/DNA(2) duplex, and the mixture was treated with T4 DNA ligase (step 2 in Figure 1). The sticky end of this foreign DNA is complementary to the end of the fragment(L) that is formed by cutting the upper strand of the linearized DNA at the 5'-side of A1824 and the lower strand at the 5'-side of G1837 (see the right-hand side of Figure 2b). Successful ligation of these two double-stranded DNAs was confirmed by the following experiments. The ligation product was first amplified by PCR. Of two primers used, primer(1) is complementary to DNA(2), whereas primer(2) is complementary to G2271-C2292 of the upper strand of the fragment(L) (Figure 2c). As shown by the gel electrophoresis in lane 1 of Figure 7, PCR product of 500mer size was efficiently formed . Consistently, no PCR products were formed when a mixture of the longer scission products in Figure 3 and DNA(1)/DNA(2) duplex was directly (without the treatment with T4 ligase) used for PCR (see lane 2 in Figure 7). Furthermore, the sequence of the ligation product was completely characterized by the sequencing experiment in Figure 8 (detailed procedures are described in Materials and Methods).
Figure 7. Agarose gel electrophoresis of the PCR product using the recombinant DNA obtained from the scission fragment(L) and DNA(1)/DNA(2) duplex. Lane 1, PCR product from the recombinant DNA; lane 2, the product of PCR reaction using the mixture of fragment(L) and DNA(1)/DNA(2) without the enzymatic ligation; M, 200 bp ladder. The primer(1) is complementary to DNA(2), whereas primer(2) is complementary to G2271-C2292 of the upper strand of the fragment(L). The bands were detected by staining with GelStar.
Figure 8. Sequencing analysis of the recombinant DNA obtained by the ligation of fragment(L) with DNA(1)/DNA(2) duplex. The conjunction between DNA(1) and the 5' side portion of upper strand of the fragment(L) is shown.
The ligation was also successful when foreign DNA was much (1077 bp) longer than DNA(1)/DNA(2) duplex and had the same sticky end as this duplex. The ligation product was clearly separated from the other bands in agarose gel electrophoresis, and its conversion was 20%. This value probably reflects the amount of fragment(L) obtained by the present site-selective scission. Of several fragments formed by the scission, this fragment was selectively incorporated into the recombinant DNA, since its sticky end completely fits that of DNA(1)/DNA(2) duplex (and of the longer foreign DNA).
Use of nuclease S1 as the scissors in place of Ce(IV)/EDTA
The invasion complex composed of the linearized plasmid DNA, pcPNA(1), and pcPNA(2) was treated with nuclease S1 (single-strand specific nuclease) at 25°C and pH 4.6 (see Supplemental Figure). In the very early stage of the reaction, two bands were detected in agarose gel electrophoresis, as was observed in Figure 3. In prolonged reactions, however, these bands gradually weakened and became smeary. Apparently, these double-stranded DNA products were further digested by nuclease S1. The substrate (the linearized plasmid DNA) was also notably digested by this single-strand specific nuclease, providing a smeary band. Probably, the binding of nuclease S1 toward single-stranded DNA is so strong that either strand in double-stranded DNA is partially removed from its complementary strand by this enzyme and is digested. In contrast, Ce(IV)/EDTA binds single-stranded DNA less efficiently, and thus similar process hardly occurs in the non-enzymatic reactions (13,14). Furthermore, double-stranded DNA is hardly hydrolyzed by this complex. As the result, the double-stranded DNA products, formed by the primary scission by this complex, remain satisfactorily intact even for prolonged reactions. Attempts to find appropriate conditions for enzymatic reactions where the primary scission products are obtained in high yields were unsuccessful.
CONCLUSION
By using two pcPNAs, invasion structures are formed in double-stranded DNA, and designated sites in both strands are made single-stranded. These sites are selectively hydrolyzed by Ce(IV)/EDTA, because of the substrate-specificity of this complex (single-stranded DNA is hydrolyzed much faster than double-stranded DNA). As the substrates for the present site-selective two-strand scission, both linear DNA and supercoiled DNA are usable. The lengths of pcPNAs and their sequences can be versatile, and thus these systems are potent for site-selective scission of huge DNAs. Furthermore, the products of the present site-selective scission can be connected with foreign DNA using DNA ligase. Accordingly, fundamental processes of DNA manipulation (site-selective two-strand scission of double-stranded DNA at designated site, followed by enzymatic ligation) have been successfully accomplished.
Quite recently, gap-selective scission of single-stranded DNA by Ce(IV)/EDTA has been promoted by introducing monophosphate groups to the gap-site (21,22). It was proposed that these groups recruit the complex to the gap-site and accelerate the reaction there. A similar strategy could promote the present site-selective scission of double-stranded DNA. Appropriate chemical modification of pcPNA additives is also expected to be useful for further increase in the site-selectivity and scission-efficiency. These attempts, as well as the application of the present findings to DNA manipulation, are currently under way in our laboratory.
SUPPLEMENTARY MATERIAL
Supplementary Material is available at NAR Online.
ACKNOWLEDGEMENTS
We would like to thank Prof. Teruyuki Nagamune for his valuable comments on the analysis of scission fragments and Dr J.-M. Zhou for the assistance in sequencing the recombinant DNA. This work was partially supported by PROBRAIN. The support by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, Culture and Technology, Japan is also acknowledged.
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