- Email: [email protected]
[26] Conferring new specificities on restriction enzymes: Cleavage at any predetermined site by combining adapter oligodeoxynucleotide and class-IIS enzyme
[26]
CONFERRING SPECIFICITIES ON RESTRICTION ENZYMES
303
Strobel, S. A., Moser, H. E., and Dervan, P. B., J. Am. Chem. Soc. 110, 7927 (1988). Suwanto, A., and Kaplan, S., J. Bacteriol. 171, 5850 (1989). Taylor, J. D., Badcoe, I. G., Clarke, A. R., and Halford, S. E., Biochemistry 30, 8743 (1991). Waterbury, P. G., Rehfuss, R. P., Carroll, W. T., Smardon, A. M., Faldasz, B. D., Huckaby, C. S., and Lane, M. J., Nucleic Acids Res. 17, 9493 (1990). Weil, M. D., and McClelland, M., Proc. Natl. Acad. Sci. U.S.A. 86, 51 (1989). Wilson, G. G., Gene 74, 281 (1990). Wilson, G. G., Nucleic Acids Res. 19, 2539 (1991). Wilson, W. W., and Hoffman, R. M., Anal. Biochem. 191, 370 (1991). Winkler, F. K., D'Arcy, A., Blocker, H., Frank, R., and van Boom, J. H., J. Mol. Biol. 217, 235 (1991). Wong, K. K., and McClelland, M., J. Bacteriol. 174, 1656 (1992). Wu, J. C., and Santi, D. V., J. Biol. Chem. 262, 4786 (1987).
[26] Conferring New Specificities on Restriction Enzymes: Cleavage at Any Predetermined Site by Combining Adapter Oligodeoxynucleotide and Class-IIS Enzyme
By
A N N A J. PODHAJSKA, S U N C H A N G K I M , a n d W A C L A W SZYBALSKI
Introduction Class-II restriction endonucleases (ENases), the most widely used tools in genetic engineering today, cleave double-stranded (ds) DNA at preexisting recognition sites, which usually are 4-8 bp long. 1 Because the number of such recognition sites is quite limited, it would be convenient to have a means for cutting DNA at any predetermined site. Such a method, as adopted for single-stranded (ss) DNA, is described here and depends on the use of a specially designed oligodeoxyribonucleotide (oligo) adapter carrying the class-IIS ENase recognition site, which directs the cognant class-IIS ENase to a specific predetermined cleavage site. 2-4
Principle of Method The method exploits the mechanism of cleavage employed by the classIIS ENase, as exemplified here by the enzyme, FokI. FokI recognizes a I C. Kessler and V. Manta, Gene 92, 1 (1990); R. J. Roberts, Nucleic Acids Res. 17, r347 (1989). 2 W. Szybalski, Gene 40, 169 (1985). 3 A. J. Podhajska and W. Szybalski, Gene 40, 175 (1985). 4 S. C. Kim, A. J. Podhajska, and W. Szybalski, Science 240, 504 (1988).
METHODS IN ENZYMOLOGY,VOL. 216
Copyright © 1992by AcademicPress. Inc. All rightsof reproductionin any form reserved.
304
CLEAVING AND MANIPULATING D N A
(A)
recognition
[26]
cut site 9f10
~::~
~..,.::: 13114
(B)
hairpin ds domain Fokl
ss domain
C~A~C~?AAOGAGGGT
(C)
AGCA-3'
Adapter Fokl
Target DNA 1 3 3 0 ) M13mp7
~ +dNTPsPOlk (D)
Adapter-primer Fokl
"aroe'0'" M13mp7
~
(E) 5'
. . . . . . . . . . . .
3' - . . . . . . . . . . .
A A C G A G
TTGCTGOCAT
....J
Fokl
GGTA%C~~~--::::::t::: 3'2, +dNTPsPOlk
(F) 5'
- . . . . . . . . . . .
3' - . . . . . . . . . . .
AACGAGGGTA
TTGCTCCCAT
gg1%g'~g~gg2~g~f~gE:::-_-_:::t:-_:2,3'
[26]
CONFERRING SPECIFICITIESON RESTRICTION ENZYMES
305
5-bp sequence, 5'-GGATG, and cuts 9 and 13 nucleotides (nt) away C CTAC from this site (see Fig. 1A). In native DNA, both the 5-bp recognition site and the cleavage site are on the same molecule. However, it is possible to place the FokI recognition site on a special hairpin oligo adapter, shown in Fig. lB. 2-4 FokI binds to the ds domain of such an adapter, but cannot cleave the ss domain unless it is paired with a complementary region of the target ssDNA, as represented in Fig. IC. 3 By specifying the proper complementary sequence for the ss domain in the adapter, one could cut the target ssDNA at any predetermined site between any two bases (e.g., between T and C, as shown in Fig. 1C).
Experimental Procedures Solutions F o k I buffer: 20 m M Tris-HC1, p H 7.5, 10 m M MgCI2, a n d 1 m M dithiothreitol ( D T T ) T E : 10 m M Tris-HC1, 1 m M e t h y l e n e d i a m i n e t e t r a a c e t i c acid ( E D T A ) , p H 8.0/24 ° A l k a l i n e l o a d i n g b u f f e r ( A L B ) : 30 m M N a O H , 2 m M E D T A , 7% (w/v) Ficoll, 0.1% (w/v) s o d i u m d o d e c y l sulfate (SDS), 0.01% (w/v) b r o m phenol blue
FIG. 1. Design of an adapter oligodeoxynucleotide (oligo) for instructingthe FokI enzyme to cleave MI3 ssDNA at a predetermined point. (A) Both the FokI recognition and cutting domains for dsDNA are schematically outlined. The staggered cleavage points are represented by vertical arrows. (B) The 34-mer adapter oligo is designed to bind to FokI and to cut M13 ssDNA between nt 1341 and 1342 [P. M. G. F. Van Wezenbeek, T. J. M. Hulsebos, and J. G. G. Schoenmakers, Gene 11, 129 (1980)]. (C) The adapter is a 34-mer oligo with a 10-bp hairpin ds domain carrying the FokI recognition site (boxed) and a 14-nt ss domain complementary to nt 1339-1352 of phage M13mp7 ss target DNA. This 34-mer adapter is directing the FokI-mediated cleavages, as shown by the vertical arrows. (D) Alternatively, the 3' end of the 34-mer adapter-primer is elongated by PolIk and all four dNTPs, thus converting the Ml3mp7 ss target to dsDNA. (E) Addition of FokI results in staggered cleavages creating a predetermined end, the position of which depends solely on the design and the sequence of the adapter-primer. (F) Because of the presence of Pollk and four dNTPs, the cohesive ends are filled in. The bold letters represent the adapter and the outlined letters indicate the target ssDNA. Only one kind of adapter with the 5' hairpin ds domain and with the 3' ss target recognition domain is shown here. The high precision of cleavage was assessed by sequence analysis. 4
306
CLEAVINGAND MANIPULATINGDNA (A)
[26]
23-mer 6CCTAC
(CGQA--(B)
, - 5'
TGAGGGrAQC
t
32-mer (
(~.~.(~ACG ACGGATGTG
- 3'
ATGGGAG CAAG
- 5'
- 3'
FIG. 2. The 23- and 32-nt oligo adapters. The 5-bp FokI recognition sequences are marked by horizontal arrows within the ds hairpin domains. The additional base-paired nucleotide flanking both ends of the FokI recognition sequence in the ds domain are indicated by heavy dots. Vertical arrows indicate the predicted FokI cut sites in the adapters, after being annealed to the target ssDNA. The 23-mer adapter showed very poor binding to the FokI enzyme and did not direct any cleavage. 5
Design of Adapter The adapter shown in Fig. 1B will serve as an example. Its hairpin ds domain is 10 bp long and consists of a 5-bp FokI recognition site and 3and 2-bp ds flanking sequences. The ss domain is 14 nt long and its sequence, which is complementary to the target ssDNA, determines the exact point where the target will be cleaved (Fig. I C ) . 3'4
Notes on Adapter Design a. The hairpin ds domain shown in Fig. 1B (composed of a 5-bp FokI recognition site and two flanking sequences, 2 and 3 bp long) was found to be quite efficient both in binding to FokI and in subsequent cleavage. A ds domain, shorter by 1 bp and with flanking sequences of only 2 bp (see Fig. 2B), 3'5 was found to be as efficient as that shown in Fig. lB. However, a ds domain with each flanking sequence only 1 bp in length was a poor FokI binder and unsuitable as an adapter (see Fig. 2A). b. Adapters can dimerize by hybridization between two open hairpin domains, but they still remain active in the present procedure. Heating to 70 ° (5 min) and rapid cooling in ice produces predominantly monomeric adapters, as confirmed by 10% (w/v) polyacrylamide gel electrophoresis. 5 c. The ss domain of the adapter in Fig. 1B is 14 nt long, but a longer ss sequence would assure higher hybridization specificity for a given site on the target DNA. The design of the adapter could allow some mispairing
S. C, Kim and W. Szybalski, unpublished.
[26]
CONFERRING SPECIFICITIES ON RESTRICTION ENZYMES
307
in the region between the recognition site and cutting domain (around nt 1350 in Fig. 1C), while still directing rather efficient and precise cleavage, providing that 14 or more nucleotides remain base paired. 5 The latter property could be exploited when designing specific adapters. d. The 3' end in the ss domain of the adapter (shown in Fig. 1C) permits its use as a primer, as described below (and shown in Fig. 1D and E). 4 Adapters with a 5'-end ss domain were also constructed and found to direct precise cutting just as efficiently3'5; however, they cannot be used as primers. e. If required, the recognition specificity of FokI could be increased to 7 bp, 5'-CCGGATG, by double methylation. 6'7 GGCCTAC f. Analogous adapters could be designed for several other class-IIS ENases.8 Enzymes The class-IIS ENases must be free of endonucleolytic activity toward ssDNA. 3'4 FokI was selected, because of our previous experience with this enzyme, and because the M . F o k I methyltransferase has become commercially available, permitting protection of dsDNA from FokI digestion at preexisting restriction sites. The experimental approach we describe here is not limited to FokI. The procedure probably could be adapted to many of the over 35 other class-IIS ENases and their isoschizomers.8 Cleaving ssDNA to Produce Single-Stranded Products 1. Suspend M13mp7 ssDNA (2/zg) in 20/xl of FokI buffer, and add a 7.5-fold molar excess of the 34-met or 32-mer adapters shown in Figs. IB and 2 (see note a below). 2. Heat the sample for 10 min at 70 °, cool it rapidly in ice, and incubate for 30 min at 37° for an effective hybridization (see note c below). 3. Add FokI enzyme (12 units) and digest for 2 hr at 37 °. 4. Precipitate the sample with 4 vol of 95% ethanol in the presence of 0.3 M sodium acetate, place the tube in dry ice for 10 rain, spin in a microfuge for 10 min, and then wash with 70% ethanol. 5. Dry the sample under vacuum, and then resuspend in 20/~1 of ALB.
6 G. P6sfai and W. Szybalski, Nucleic Acids Res. 16, 6245 (1988). 7 S. C. Kim, G. P6sfai, and W. Szybalski, Gene 100, 45 (1991). 8 W. Szybalski, S. C. Kim, N. Hasan, and A. J. Podhajska, Gene 100, 13 (1991).
308
CLEAVING AND MANIPULATING D N A
[26]
6. Load the resuspended sample onto a I% (w/v) agarose gel, run for 2 hr at 150 V, and then stain the bands with ethidium bromide. Notes on Cleaving ssDNA
a. The M13mp7 ssDNA and adapter can be stored for several months at 4° in TE buffer. Any range between a 5- and 15-fold molar excess of adapter to M13mp7 ssDNA works well. b. Agarose gels (0.8-1%, w/v) are recommended for ssDNA of the M13 size. c. The annealing time can be reduced to 5 min with hardly any change in the digestion pattern. d. As the target, one could use dsDNA with a s s gap at the adapterannealing and -cutting region. 2'8 Cleaving ssDNA to Produce Double-Stranded Fragment(s)
1. Suspend M13mp7 ssDNA (2/xg) in 100/zl of FokI buffer, together with a 7.5-fold molar excess of the 34-mer adapter-primer (Fig. 1B). 2. Heat the sample for 10 min at 70 °, cool it rapidly in ice, and then anneal at 37° for 30 min (see note a below). 3. Add Escherichia coli polymerase I, Klenow fragment (Pollk, 10 units; New England BioLabs, Beverly, MA), and all four dNTPs (8/zl of each dNTP at 2.5 mM) to the reaction sample; add FokI (8 units) 5 min after the addition of the Pollk, and take samples after 15 min, 30 min, 1 hr, and 2 hr. 4. Stop the reaction in each sample by adding 100/~1 of 5 M ammonium acetate and 400/xl of 95% ethanol (see note b below). 5. Place the samples in dry ice, spin the sample in a microfuge for I0 min, wash twice with 70% ethanol, dry under vacuum, and then redissolve in FokI buffer. 6. Load the resuspended samples on a 1.5% (w/v) agarose gel (see note c below), run for 2 hr at 150 V, and then stain with ethidium bromide. Further details and photographs of the resulting gels are as in Ref. 4. Notes on Cleaving to Produce Double-Stranded Fragment(s)
a. To avoid the nonspecific binding of the adapter, a 37° hybridization temperature is recommended. (Also, see note c, above, in the Notes on Adapter Design section.) The annealing time can be reduced to 5 min with hardly any change in the digestion pattern. b. To remove the remaining dNTPs after the reaction, use ammonium acetate (final concentration, 2.5 M) instead of sodium acetate. c. A 1.5% (w/v) agarose gel is suitable for dsDNA fragments.
[27]
TRIPLE
HELIX-MEDIATED
ENZYMATIC
CLEAVAGE
309
Conclusions
A specially designed adapter-primer oligo permits one to produce DNA fragments with a predetermined end located between any specified two base pairs of the target DNA. This approach is especially useful for DNA fragments cloned in an ssDNA-generating vector, and it could also complement our other preprogrammed DNA-trimming method.9 The present experiment should also be important for dissecting the mechanisms of enzyme recognition, endonucleolytic cleavage, and methylation. Cleavage of ssDNA vectors is helpful for the alternative method of sequencing in the case of sequence ambiguities encountered during the dideoxy sequencing method. Only one or two hairpin adapters, complementary to the universal primer site(s), permit precise cleavage of ssDNA and its 32p end-labeling by Pollk (for the already prepared ss plasmids, which were used for dideoxy sequencing), for Maxam-Gilbert sequencing of the strand opposite to that already sequenced by the dideoxy technique. J0 9 N. Hasan, S. C. Kim, A. J. Podhajska, and W. Szybalski, Gene 50, 55 (1986). 10 B. Goszczynski and J. D. McGhee, Gene 104, 71 (1991).
[27] T r i p l e H e l i x - M e d i a t e d S i n g l e - S i t e E n z y m a t i c C l e a v a g e of M e g a b a s e G e n o m i c D N A
By SCOTT A. STROBELand PETER B. DERVAN
Oligonucleotide-directed triple helix formation is a generalizable chemical approach for the recognition and cleavage of a single target site within several megabase pairs of duplex genomic DNA. j-4 Pyrimidine oligodeoxyribonucleotides 15 to 25 bases in length form a highly specific triple helix structure with purine tracts in double-stranded DNA of high complexity. ~-4The pyrimidine oligonucleotide binds by Hoogsteen hydrogen bonding in the major groove of the DNA duplex parallel to the Watson-Crick purine strand. ~The recognition motif is generalizable to homopurine target sites utilizing thymine binding to adenine-thymine base pairs (T. A-T I H. E. 2 S. A. 3 S. A. 4 S. A.
Moser and P. B. Dervan, Science 238, 645 (1987). Strobel, H. E. Moser, and P. B. Dervan, J. Am. Chem. Soc. 110, 7927 (1988). Strobel and P. B. Dervan, Science 249, 73 (1990). Strobel and P. B. Dervan, Nature (London) 350, 172 (1991).
METHODS IN ENZYMOLOGY, VOL. 216
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
CONFERRING SPECIFICITIES ON RESTRICTION ENZYMES
303
Strobel, S. A., Moser, H. E., and Dervan, P. B., J. Am. Chem. Soc. 110, 7927 (1988). Suwanto, A., and Kaplan, S., J. Bacteriol. 171, 5850 (1989). Taylor, J. D., Badcoe, I. G., Clarke, A. R., and Halford, S. E., Biochemistry 30, 8743 (1991). Waterbury, P. G., Rehfuss, R. P., Carroll, W. T., Smardon, A. M., Faldasz, B. D., Huckaby, C. S., and Lane, M. J., Nucleic Acids Res. 17, 9493 (1990). Weil, M. D., and McClelland, M., Proc. Natl. Acad. Sci. U.S.A. 86, 51 (1989). Wilson, G. G., Gene 74, 281 (1990). Wilson, G. G., Nucleic Acids Res. 19, 2539 (1991). Wilson, W. W., and Hoffman, R. M., Anal. Biochem. 191, 370 (1991). Winkler, F. K., D'Arcy, A., Blocker, H., Frank, R., and van Boom, J. H., J. Mol. Biol. 217, 235 (1991). Wong, K. K., and McClelland, M., J. Bacteriol. 174, 1656 (1992). Wu, J. C., and Santi, D. V., J. Biol. Chem. 262, 4786 (1987).
[26] Conferring New Specificities on Restriction Enzymes: Cleavage at Any Predetermined Site by Combining Adapter Oligodeoxynucleotide and Class-IIS Enzyme
By
A N N A J. PODHAJSKA, S U N C H A N G K I M , a n d W A C L A W SZYBALSKI
Introduction Class-II restriction endonucleases (ENases), the most widely used tools in genetic engineering today, cleave double-stranded (ds) DNA at preexisting recognition sites, which usually are 4-8 bp long. 1 Because the number of such recognition sites is quite limited, it would be convenient to have a means for cutting DNA at any predetermined site. Such a method, as adopted for single-stranded (ss) DNA, is described here and depends on the use of a specially designed oligodeoxyribonucleotide (oligo) adapter carrying the class-IIS ENase recognition site, which directs the cognant class-IIS ENase to a specific predetermined cleavage site. 2-4
Principle of Method The method exploits the mechanism of cleavage employed by the classIIS ENase, as exemplified here by the enzyme, FokI. FokI recognizes a I C. Kessler and V. Manta, Gene 92, 1 (1990); R. J. Roberts, Nucleic Acids Res. 17, r347 (1989). 2 W. Szybalski, Gene 40, 169 (1985). 3 A. J. Podhajska and W. Szybalski, Gene 40, 175 (1985). 4 S. C. Kim, A. J. Podhajska, and W. Szybalski, Science 240, 504 (1988).
METHODS IN ENZYMOLOGY,VOL. 216
Copyright © 1992by AcademicPress. Inc. All rightsof reproductionin any form reserved.
304
CLEAVING AND MANIPULATING D N A
(A)
recognition
[26]
cut site 9f10
~::~
~..,.::: 13114
(B)
hairpin ds domain Fokl
ss domain
C~A~C~?AAOGAGGGT
(C)
AGCA-3'
Adapter Fokl
Target DNA 1 3 3 0 ) M13mp7
~ +dNTPsPOlk (D)
Adapter-primer Fokl
"aroe'0'" M13mp7
~
(E) 5'
. . . . . . . . . . . .
3' - . . . . . . . . . . .
A A C G A G
TTGCTGOCAT
....J
Fokl
GGTA%C~~~--::::::t::: 3'2, +dNTPsPOlk
(F) 5'
- . . . . . . . . . . .
3' - . . . . . . . . . . .
AACGAGGGTA
TTGCTCCCAT
gg1%g'~g~gg2~g~f~gE:::-_-_:::t:-_:2,3'
[26]
CONFERRING SPECIFICITIESON RESTRICTION ENZYMES
305
5-bp sequence, 5'-GGATG, and cuts 9 and 13 nucleotides (nt) away C CTAC from this site (see Fig. 1A). In native DNA, both the 5-bp recognition site and the cleavage site are on the same molecule. However, it is possible to place the FokI recognition site on a special hairpin oligo adapter, shown in Fig. lB. 2-4 FokI binds to the ds domain of such an adapter, but cannot cleave the ss domain unless it is paired with a complementary region of the target ssDNA, as represented in Fig. IC. 3 By specifying the proper complementary sequence for the ss domain in the adapter, one could cut the target ssDNA at any predetermined site between any two bases (e.g., between T and C, as shown in Fig. 1C).
Experimental Procedures Solutions F o k I buffer: 20 m M Tris-HC1, p H 7.5, 10 m M MgCI2, a n d 1 m M dithiothreitol ( D T T ) T E : 10 m M Tris-HC1, 1 m M e t h y l e n e d i a m i n e t e t r a a c e t i c acid ( E D T A ) , p H 8.0/24 ° A l k a l i n e l o a d i n g b u f f e r ( A L B ) : 30 m M N a O H , 2 m M E D T A , 7% (w/v) Ficoll, 0.1% (w/v) s o d i u m d o d e c y l sulfate (SDS), 0.01% (w/v) b r o m phenol blue
FIG. 1. Design of an adapter oligodeoxynucleotide (oligo) for instructingthe FokI enzyme to cleave MI3 ssDNA at a predetermined point. (A) Both the FokI recognition and cutting domains for dsDNA are schematically outlined. The staggered cleavage points are represented by vertical arrows. (B) The 34-mer adapter oligo is designed to bind to FokI and to cut M13 ssDNA between nt 1341 and 1342 [P. M. G. F. Van Wezenbeek, T. J. M. Hulsebos, and J. G. G. Schoenmakers, Gene 11, 129 (1980)]. (C) The adapter is a 34-mer oligo with a 10-bp hairpin ds domain carrying the FokI recognition site (boxed) and a 14-nt ss domain complementary to nt 1339-1352 of phage M13mp7 ss target DNA. This 34-mer adapter is directing the FokI-mediated cleavages, as shown by the vertical arrows. (D) Alternatively, the 3' end of the 34-mer adapter-primer is elongated by PolIk and all four dNTPs, thus converting the Ml3mp7 ss target to dsDNA. (E) Addition of FokI results in staggered cleavages creating a predetermined end, the position of which depends solely on the design and the sequence of the adapter-primer. (F) Because of the presence of Pollk and four dNTPs, the cohesive ends are filled in. The bold letters represent the adapter and the outlined letters indicate the target ssDNA. Only one kind of adapter with the 5' hairpin ds domain and with the 3' ss target recognition domain is shown here. The high precision of cleavage was assessed by sequence analysis. 4
306
CLEAVINGAND MANIPULATINGDNA (A)
[26]
23-mer 6CCTAC
(CGQA--(B)
, - 5'
TGAGGGrAQC
t
32-mer (
(~.~.(~ACG ACGGATGTG
- 3'
ATGGGAG CAAG
- 5'
- 3'
FIG. 2. The 23- and 32-nt oligo adapters. The 5-bp FokI recognition sequences are marked by horizontal arrows within the ds hairpin domains. The additional base-paired nucleotide flanking both ends of the FokI recognition sequence in the ds domain are indicated by heavy dots. Vertical arrows indicate the predicted FokI cut sites in the adapters, after being annealed to the target ssDNA. The 23-mer adapter showed very poor binding to the FokI enzyme and did not direct any cleavage. 5
Design of Adapter The adapter shown in Fig. 1B will serve as an example. Its hairpin ds domain is 10 bp long and consists of a 5-bp FokI recognition site and 3and 2-bp ds flanking sequences. The ss domain is 14 nt long and its sequence, which is complementary to the target ssDNA, determines the exact point where the target will be cleaved (Fig. I C ) . 3'4
Notes on Adapter Design a. The hairpin ds domain shown in Fig. 1B (composed of a 5-bp FokI recognition site and two flanking sequences, 2 and 3 bp long) was found to be quite efficient both in binding to FokI and in subsequent cleavage. A ds domain, shorter by 1 bp and with flanking sequences of only 2 bp (see Fig. 2B), 3'5 was found to be as efficient as that shown in Fig. lB. However, a ds domain with each flanking sequence only 1 bp in length was a poor FokI binder and unsuitable as an adapter (see Fig. 2A). b. Adapters can dimerize by hybridization between two open hairpin domains, but they still remain active in the present procedure. Heating to 70 ° (5 min) and rapid cooling in ice produces predominantly monomeric adapters, as confirmed by 10% (w/v) polyacrylamide gel electrophoresis. 5 c. The ss domain of the adapter in Fig. 1B is 14 nt long, but a longer ss sequence would assure higher hybridization specificity for a given site on the target DNA. The design of the adapter could allow some mispairing
S. C, Kim and W. Szybalski, unpublished.
[26]
CONFERRING SPECIFICITIES ON RESTRICTION ENZYMES
307
in the region between the recognition site and cutting domain (around nt 1350 in Fig. 1C), while still directing rather efficient and precise cleavage, providing that 14 or more nucleotides remain base paired. 5 The latter property could be exploited when designing specific adapters. d. The 3' end in the ss domain of the adapter (shown in Fig. 1C) permits its use as a primer, as described below (and shown in Fig. 1D and E). 4 Adapters with a 5'-end ss domain were also constructed and found to direct precise cutting just as efficiently3'5; however, they cannot be used as primers. e. If required, the recognition specificity of FokI could be increased to 7 bp, 5'-CCGGATG, by double methylation. 6'7 GGCCTAC f. Analogous adapters could be designed for several other class-IIS ENases.8 Enzymes The class-IIS ENases must be free of endonucleolytic activity toward ssDNA. 3'4 FokI was selected, because of our previous experience with this enzyme, and because the M . F o k I methyltransferase has become commercially available, permitting protection of dsDNA from FokI digestion at preexisting restriction sites. The experimental approach we describe here is not limited to FokI. The procedure probably could be adapted to many of the over 35 other class-IIS ENases and their isoschizomers.8 Cleaving ssDNA to Produce Single-Stranded Products 1. Suspend M13mp7 ssDNA (2/zg) in 20/xl of FokI buffer, and add a 7.5-fold molar excess of the 34-met or 32-mer adapters shown in Figs. IB and 2 (see note a below). 2. Heat the sample for 10 min at 70 °, cool it rapidly in ice, and incubate for 30 min at 37° for an effective hybridization (see note c below). 3. Add FokI enzyme (12 units) and digest for 2 hr at 37 °. 4. Precipitate the sample with 4 vol of 95% ethanol in the presence of 0.3 M sodium acetate, place the tube in dry ice for 10 rain, spin in a microfuge for 10 min, and then wash with 70% ethanol. 5. Dry the sample under vacuum, and then resuspend in 20/~1 of ALB.
6 G. P6sfai and W. Szybalski, Nucleic Acids Res. 16, 6245 (1988). 7 S. C. Kim, G. P6sfai, and W. Szybalski, Gene 100, 45 (1991). 8 W. Szybalski, S. C. Kim, N. Hasan, and A. J. Podhajska, Gene 100, 13 (1991).
308
CLEAVING AND MANIPULATING D N A
[26]
6. Load the resuspended sample onto a I% (w/v) agarose gel, run for 2 hr at 150 V, and then stain the bands with ethidium bromide. Notes on Cleaving ssDNA
a. The M13mp7 ssDNA and adapter can be stored for several months at 4° in TE buffer. Any range between a 5- and 15-fold molar excess of adapter to M13mp7 ssDNA works well. b. Agarose gels (0.8-1%, w/v) are recommended for ssDNA of the M13 size. c. The annealing time can be reduced to 5 min with hardly any change in the digestion pattern. d. As the target, one could use dsDNA with a s s gap at the adapterannealing and -cutting region. 2'8 Cleaving ssDNA to Produce Double-Stranded Fragment(s)
1. Suspend M13mp7 ssDNA (2/xg) in 100/zl of FokI buffer, together with a 7.5-fold molar excess of the 34-mer adapter-primer (Fig. 1B). 2. Heat the sample for 10 min at 70 °, cool it rapidly in ice, and then anneal at 37° for 30 min (see note a below). 3. Add Escherichia coli polymerase I, Klenow fragment (Pollk, 10 units; New England BioLabs, Beverly, MA), and all four dNTPs (8/zl of each dNTP at 2.5 mM) to the reaction sample; add FokI (8 units) 5 min after the addition of the Pollk, and take samples after 15 min, 30 min, 1 hr, and 2 hr. 4. Stop the reaction in each sample by adding 100/~1 of 5 M ammonium acetate and 400/xl of 95% ethanol (see note b below). 5. Place the samples in dry ice, spin the sample in a microfuge for I0 min, wash twice with 70% ethanol, dry under vacuum, and then redissolve in FokI buffer. 6. Load the resuspended samples on a 1.5% (w/v) agarose gel (see note c below), run for 2 hr at 150 V, and then stain with ethidium bromide. Further details and photographs of the resulting gels are as in Ref. 4. Notes on Cleaving to Produce Double-Stranded Fragment(s)
a. To avoid the nonspecific binding of the adapter, a 37° hybridization temperature is recommended. (Also, see note c, above, in the Notes on Adapter Design section.) The annealing time can be reduced to 5 min with hardly any change in the digestion pattern. b. To remove the remaining dNTPs after the reaction, use ammonium acetate (final concentration, 2.5 M) instead of sodium acetate. c. A 1.5% (w/v) agarose gel is suitable for dsDNA fragments.
[27]
TRIPLE
HELIX-MEDIATED
ENZYMATIC
CLEAVAGE
309
Conclusions
A specially designed adapter-primer oligo permits one to produce DNA fragments with a predetermined end located between any specified two base pairs of the target DNA. This approach is especially useful for DNA fragments cloned in an ssDNA-generating vector, and it could also complement our other preprogrammed DNA-trimming method.9 The present experiment should also be important for dissecting the mechanisms of enzyme recognition, endonucleolytic cleavage, and methylation. Cleavage of ssDNA vectors is helpful for the alternative method of sequencing in the case of sequence ambiguities encountered during the dideoxy sequencing method. Only one or two hairpin adapters, complementary to the universal primer site(s), permit precise cleavage of ssDNA and its 32p end-labeling by Pollk (for the already prepared ss plasmids, which were used for dideoxy sequencing), for Maxam-Gilbert sequencing of the strand opposite to that already sequenced by the dideoxy technique. J0 9 N. Hasan, S. C. Kim, A. J. Podhajska, and W. Szybalski, Gene 50, 55 (1986). 10 B. Goszczynski and J. D. McGhee, Gene 104, 71 (1991).
[27] T r i p l e H e l i x - M e d i a t e d S i n g l e - S i t e E n z y m a t i c C l e a v a g e of M e g a b a s e G e n o m i c D N A
By SCOTT A. STROBELand PETER B. DERVAN
Oligonucleotide-directed triple helix formation is a generalizable chemical approach for the recognition and cleavage of a single target site within several megabase pairs of duplex genomic DNA. j-4 Pyrimidine oligodeoxyribonucleotides 15 to 25 bases in length form a highly specific triple helix structure with purine tracts in double-stranded DNA of high complexity. ~-4The pyrimidine oligonucleotide binds by Hoogsteen hydrogen bonding in the major groove of the DNA duplex parallel to the Watson-Crick purine strand. ~The recognition motif is generalizable to homopurine target sites utilizing thymine binding to adenine-thymine base pairs (T. A-T I H. E. 2 S. A. 3 S. A. 4 S. A.
Moser and P. B. Dervan, Science 238, 645 (1987). Strobel, H. E. Moser, and P. B. Dervan, J. Am. Chem. Soc. 110, 7927 (1988). Strobel and P. B. Dervan, Science 249, 73 (1990). Strobel and P. B. Dervan, Nature (London) 350, 172 (1991).
METHODS IN ENZYMOLOGY, VOL. 216
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.