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TIBTECH
21 Hilvert, D. and Nared, K. D. (1988) J. A m . Chem. Soc. 110, 5593 22 Shokat, K. M., Leumann, C. J., Sugasawara, R. and Schultz, P.G. (1989) Nature 338, 269-271 23 Leatherbarrow, R. J. (1989) Nature 338, 206-207 24 Balan, A., Doctor, B. P., Green, B. S., Torten, M. and Ziffer, H. (1988) J. C. S. Chem. Comm. 106-108 []
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25 Cochran, A. G., Sugasawara, R. and Schultz, P.G. (1988) J. A m . Chem. Soc. 110, 7888-7890 26 Pollack, S. J. and Schultz, P. G. (1989) J. Am. Chem. Soc. 111, 1929-1931 27 Iverson, B. L. and Lerner, R. A. (1989) Science 243, 1184-1188 28 Williams, G. (1989) Trends Biotechnol. 6, 36-41 29 Kamely, D. (1989) Bio/Technology 7, []
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Sequence-specific artificial endonucleases Claude H61~ne, Nguyen T. Thuong, Tula Saison-Behmoaras and Jean-Christophe Fran£~ois Artificial sequence-specific nucleic acid cleaving reagents provide new opportunities in nucleic acid chemistry and biology. This article reviews the different strategies used to attach nucleic acid cleaving reagents to sequence-specific ligands. Metal chelates, photoactive groups and nonspecific nucleases have been used as cleaving reagents. Sequence-specific recognition was achieved by oligonucleotides, oligopeptides, proteins and DNA minor-groove ligands. Sequence-specific cleavage can be targeted to single-stranded nucleic acids, such as messenger and viral RNAs, and to double-stranded DNA. Such artificial nucleases might be used as tools in molecular and cellular biology. They also provide a rational basis for the design of specific gene expression inhibitors. During recent years there has been a growing interest in the use of synthetic oligodeoxynucleotides [oligo(dN)] as specific inhibitors of gene expression 1. It was originally assumed that upon binding to a messenger RNA, an oligo(dN) would prevent protein synthesis by physically blocking translation of the message by ribosomes. However, the situation was much more complex than expected. Even though an oligo(dN) bound to the 5'-untranslated region of a mRNA might interfere with ribosome assembly, ribosomes have an unwinding acC. H6Jbne, 71. Saison-Behmoaras and ]-C. Francois are at the Laboratoire de Biophysique, Mus6um National d'Histoire Naturelle, INSERM U.2OICNRS UA.481, 4 3 r u e Cuvier, 75231 Paris Cedex 05, France. N. T. Thuong is at the Centre de Biophysique Mol~culaire, CNRS, 45071 Orleans Cedex 02, France. ~) 1989, Elsevier Science Publishers Ltd (UK)
tivity that releases any oligo(dN) hybridized to the coding region (i.e., downstream of the translation initiation site). Therefore, oligo(dN) should not be able to inhibit protein synthesis when targeted to coding regions of mRNAs. However, they were shown to be active in i n vitro translation systems, in microinjected oocytes and in cells in culture. In the first two systems, the efficiency of oligo(dN) was due to the activity of ribonuclease H (RNase H), a cellular enzyme that cleaves the mRNA moiety in an oligo(dN)-mRNA hybrid at the site of oligo(dN) binding 2. As a consequence of mRNA cleavage, protein synthesis is obviously inhibited. The use of oligo(dN) i n v i v o is limited by sensitivity to both endoand exonucleases. Several attempts 1 have been made to overcome this difficulty by chemical modification
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NOVEMBER 1989 [Vol. 7]
447-451 30 Wong, C-H. (1989) Science 244, 11451152 31 Paul, S.,Volle, D.J.,Beach, C. M. eta]. (1989) Science 244, 182-188 32 Ekberg, B. and Mosbach, K. (1989) Trends Biotechnol. 7, 92-96 33 Pollack, S. I., Hsiun, P. and Schultz, P. G. (1989) J. A m . Chem. Soc. 111, 5961-5962 []
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of the oligo(dN). Oligophosphonates, oligo-[e~]-deoxynucleotides [oc-oligo(dN), which make use of the synthetic ~-anomers of the nucleotide units instead of the natural [3-anomers] and oligophosphorothioates have been proven resistant to nucleases. However, oligophosphonates and ~oligo(dN) hybrids with mRNA are refractory to RNase H action and, consequently, these two modified oligonucleotides are weak inhibitors of mRNA translation. In contrast, oligophosphorothioates inhibit protein synthesis efficiently because they form RNase H-sensitive hybrids with RNAs 1. However, they exhibit nonspecific effects which limit their potential use in vivo. Nucleaseresistant, efficient inhibitors of mRNA translation are, therefore, still needed. Artificial control of gene expression can be achieved at levels other than mRNA translation. DNA itself can be chosen as a target for sequence-specific ligands that could inhibit transcription, e.g., via triple helix formation. Splicing of premRNA and export of mature mRNA from the nucleus to the cytoplasm are other potential targets for sequencespecific inhibitors based upon oligonucleotides. Both reversible binding and irreversible site-directed modifications of the target nucleic acid can be contemplated as ways of effecting these biological processes. Here, we review the approaches which have recently been developed to induce sequence-specific cleavage of nucleic acids. Development of sequencespecific artificial nucleases might permit the use of these approaches for a range of applications, from regulation of gene expression to gene mapping on long DNA fragments,
TIBTECH --Fig.
NOVEMBER1989[Vol.7]
1
Target seq~nce
Oligonucleotide¢
Intercalation
Cleaving reagent An artificial endonuclease. An oligonucleotide (black ribbon) hydrogenbonded to a complementary sequence on a target nucleic acid carries an intercalating agent at one e n d to stabilize the complex 13and a reactive group (scissors) that cleaves close to the target sequence.
site-directed mutagenesis and accessibility studies, e.g. in nucleoprotein complexes.
Single-stranded nucleic acids as targets for sequence-specific artificial nucleases Any nucleic acid ligand can potentially be converted into a nuclease by attaching to it a cleaving reagent. For single-stranded substrates, the simplest way to recognize a specific sequence consists of synthesizing an oligo(dN) with a complementary sequence. The oligo(dN) can then be attached to nucleic acid cleaving reagents (Fig. 1). Three types of cleaving agents have been developed: • Metal chelates. Fe-EDTA 3-6, Cuphenanthroline 7-1° or Fe-porphyrin ~'~2 can be used to generate O H radicals or metal-oxo derivatives that attack deoxyribose or ribose and lead to cleavage of phosphodiester bonds. The mechanism involves oxidative attack of the deoxyribose at the C1' and/or C4' position. A reducing agent is required. Metal chelate derivatives of oligonucleotides can also be used to cleave RNAs at specific sites. Again, the mechanism involves oxidative attack at the C1' and C4' position (of ribose) 8. To stabilize the complex formed between the oligonucleotide moiety of the artificial nuclease and its complementary target sequence, an intercalating agent may be attached to the end of the oligonucleotide not linked to the cleaving agent (Fig. 1). The inter-
calating agent provides additional binding energy6'1°'13; stabilization increases the residence time of the oligonucleotide-nuclease on its target and thereby favors the cleavage reaction. • Photoactive groups. Groups such as azidophenacyl or azidoproflavine derivatives, proflavine and porphyrins can be attached to the oligonucleotide. Treatment of the nuclease-target sequence with UV or visible light leads to photocrosslinking of the oligonucleotide to its target s equ ence 13--17.The photocrosslinked sample can then be cleaved at the crosslinking site by treatent with piperidine. The crosslinking reaction involves the bases and shows some basespecificity. Guanines are the most reactive bases when proflavine and porphyrin are used as photosensitizers; thymines are highly reactive towards azido-derivatives. Direct cleavage induced by irradiation at neutral pH occurs with ellipticine and diazapyrene derivatives 17. These derivatives are the first example of sequencespecific photoendonucleases. • Nonspecific enzymes. RNases or DNases can be tethered to the targeting oligonucleotide ~8,~9. The nonspecific cleavage observed when the nuclease is used alone may be directed to a specific sequence by the attached oligonucleotide binding to its complementary sequence on a singlestranded nucleic acid 19. These oligonucleotide-nuclease chimeras
induce site-specific cleavage after short incubation times, but product yields are low 19. After longer times, however, there is a significant amount of nonspecific cleavage. This could be due to the degradation of the targeting oligonucleotide by the enzyme; a problem which might be solved by using nuclease-resistant oligo(dN) [such as the chemically-modified oligo(dN)]. Oligonucleotides carrying a cleaving reagent or a photoactive group are also inactivated, at least partially, during their reactions with target sequences. The OH' radicals and other oxidizing species react both with the metal-chelating ligands (EDTA, porphyrin, phenanthroline) and the targeting oligonucleotide (deoxyribose and base moieties) leading to an inactivation of the artificial nuclease. Most of the photoactive groups become photooxidized during the course of their reactions and this leads to the loss of photocrosslinking or photocleavage properties. Research into the development of photoactive groups that would, for instance, remove hydrogen atoms from the ribose or deoxyribose moieties without undergoing photochemical degradation in the absence of substrate, are in progress.
Ribozymes Single-stranded RNA can also be cleaved by short RNAs without relying u p o n attachment of a cleaving reagent. Several RNAs undergo selfsplicing 2°. The definition of the minimal structural requirements for such a reaction have led to the synthesis of ribozymes 21. These are short RNA molecules capable of inducing a site-specific hydrolytic reaction in the target RNA, a reaction which is highly dependent on the tertiary structure of the RNA-RNA complex. Until now, only RNAs have been shown to induce such hydrolyric reactions. The advantage of ribozymes compared with the artificial nucleases built from oligo= deoxynucleotides that we have already discussed, is that they are true catalysts: the ribozyme is not inactivated during the hydrolytic reaction. However, short RNAs are very
TIBTECH- NOVEMBER 1989 [Vol. 7]
--Fig. 1
O
a
sensitive to ribonucleases and thus limits the potential use of ribozymes in vivo. Further studies of the tertiary structures of ribozyme-RNA complexes are clearly needed to design nuclease-resistant analogues of ribozymes.
Nuclease-resistant artificial nucleases In order to use artificial nucleases in vivo, the targeting moiety must be resistant to endogenous nucleases. In the case of oligodeoxynucleotides, this can be achieved by modifying the backbone (having, for instance, methylphosphonate, phosphorothioate, or phosphotriester groups linking the sugars) or by changing the anomeric configuration of the nucleoside units. All natural nucleic acids are made of [3-anomers (Fig. 2). If synthetic e~-anomers are used instead, synthetic oligo-[o~]-deoxynucleotides are produced and these are resistant to nucleases 22'23. The e~oligo(dN) bind to complementary nucleic acid sequences, forming double helices with parallel strands (as opposed to antiparallel strands in natural double-stranded DNA) 24. [However, an o~-(dT)8 (octamer of o~-deoxythymidine) binds in an antiparallel orientation with oligo-r A 25. This is the only known exception to the parallel orientation of the two strands in an o~-~ hybrid.] By attaching a cleaving reagent (e.g., Cuphenanthroline) at one end of these o~-oligo(dN), nuclease-resistant artificial nucleases can be produced ( F i g . 2) 10'24'25 .
O
Me~
NH
CH2
N,'~O J
Me~
NH
5 p
I
O~
3'%,O ~N,~O
CH2 0 3'
0/
/
i
5'
p-nucleoside
0c-nucleoside
b 3'
5'
(OP)-TTTT TT TT
[P]
,llll, [P]
TG^GTGAGTi
, [=]
X
X
ITGAGTGCCAA 3'
-,iT t ( O P ) - T T T T TT T T
5'
3'
The/3 and ~ anomers of nucleosides and binding o f oligomers. (a) In the ~-anomers, the base and the 5'-OH group are on the same side of the deoxyribose plane. In the o~anomers, the 3'-OH group is on the same side as the base. (b) Cleavage of a target single-stranded DNA sequence by [J- and ~-(dT)8 covalently linked to a copper-phenanthroline chelate(OP). The phenanthroline ring is attached to the 3'-end of ~-(dT)8 and to the 5'-end of o~(dT)8. The arrows indicate the sites and relative frequency of cleavage, t~-(dT)8 (top) forms an antiparallel double helix with its complementary sequence whereas ~-(dT)8 (bottom) forms a parallel-stranded double helix w
D o u b l e - s t r a n d e d D N A as a target for s e q u e n c e - s p e c i f i c artificial nucleases
Transcription of DNA is under the control of regulatory proteins that bind to specific sequences, thereby activating or repressing the RNA polymerase complex. Regulatory proteins or their DNA-binding domains can be converted to DNAcleaving reagents. Phenanthroline has been covalently linked to the trp repressor 26, for instance: in the presence of copper ions and a reducing agent, DNA cleavage occurs at operator sequences to which the trp repressor binds selectively. Another example is a synthetic
peptide of 52 amino acid residues from the Hin recombinase which binds selectively to the recognition site of this enzyme but is normally devoid of any DNA-cleaving activity. However, when the peptide is covalently linked to the EDTA-Fe chelate 27 or to a copper-chelating peptide (Gly-Gly-His 28) in the presence of a reducing agent, both complexes cleave DNA specifically at the sites recognized by the peptide. However, several cleavage sites were revealed on a plasmid DNA whereas only one was expected. The expla-
nation is that only a few of the base pairs that fall within the binding site of the peptide or protein are selectively recognized. Regulatory proteins very frequently bind as dimers and recognize pseudosymmetrical sequences: when the peptide fragment responsible for recognition is separated from the rest of the protein, therefore, dimers can no longer form and both selectivity and strength of binding of the protein are reduced. Many DNA-binding drugs can also be converted into cleaving reagents. For example, intercalating agents
TIBTECH- NOVEMBER1989[Vol. 7]
--Fig. 3 a
5'
3'
b
MAJOR GROOVE
/ ~H o' o g s '~H t e e ,i,n ~ / H ½N-H /l'~--H-~--....O.:~ _/,N'~.~./H~ '~C c--c c--GX I H--C C N----H--N G u / &=./
/
N----Ckk
R (5'- 3')
/
o--:-.,-, t H Watson-Crick /
'-. '
'/C_-o
Hoogsteen Hoogsteen ~._pv~-N,~ Watson-Crick H: ~Hoogsteen _ . ......... ~...... H:
II
/ CHo3 n O .)__.w--~ . . . . . M - - I X I{ / L~ I~l~r.,/' H C--O# ~ -- C/ L,I
/i

/1
H--C T N--H---N P, C N - - C/ ! C ~ N /
'~ R (3' -5)'
/
R (5' - 3')
/
-
Watson-Crick/ /
Oligonucleotide targeting to double-stranded DNA. (a) A homopyrimidine oligonucleotide bound to the major groove of double helical DNA at a homopurine.homopyrimidine sequence. The oligonucleotide carries a reactive group (star) that can be used to induce irreversible reactions on both strands of the double helix. The pyrimidine oligonucleotide binds by virtue of Hoogsteen hydrogen bonding of thymidine and protonated cytosine to Watson-Crick A T and GC basepairs, respectively (b).
attached to a cleaving reagent such as Fe-EDTA 29 or to a photoactive group such as an azido or diazo derivative 3° can cleave DNA, although the base sequence selectivity is low, reflecting the poor specificity of binding of the intercalating agent. Minor groove ligands such as netropsin have also been attached to Fe-EDTA and the complex shown to cleave at the sequences recognized by netropsin 29. However, the specificity is still limited to that conferred by sequences of a few base pairs, the size of the ligand recognition site. Recognition of DNA grooves Homopurine.homopyrimidine sequences in DNA can be recognized by homopyrimidine oligonucleotides. Binding occurs in the major groove where thymine and protonated cytosine can form Hoogsteen hydrogen bonds with Watson-Crick A-T and G.C base pairs, respectively (Fig. 3). If active groups are attached at the end of the oligonucleotide, reactions can be induced on both strands of duplex DNA. Azido derivatives (azidophenacyl, azidoproflavine) photocrosslink the oligonucleotide to each strand of DNA and cleavage of DNA
~Fig. 4,
H a e l ~
5'TCCTGAT 3. A G G A C T A 11.
r ...................................
1
AAAGGAGGAGA, TGA AGAoTGA 3' TTTCC TCCTCTIAC T TCToAC~5.
• ...................................
Op~sT. T T C C T C C
i
TCT
3'
Cleavage of simian virus SV40 DNA at a single site by an oligonucleotide d5'(TTTCCTCCTCT) 3' carrying a copper-phenanthroline complex attached to its 5'-phosphate. The bars indicate the sites and relative frequency of cleavage. The skewed distribution of cleavage sites indicates reactions in the minor groove.
TIBTECH- NOVEMBER1989 [Vol. 7]
follows piperidine treatment 14'15. Metal chelates, such as Fe-EDTA 31 or Cu-phenanthroline 1°'32, covalently attached to homopyrimidine oligonucleotides induce double-strand cleavage. The positions of the photocrosslinking and cleavage sites clearly show that a homopyrimidine oligo(dN) binds to the major groove in a parallel orientation to the homopurine-containing strand. When the homopyrimidine oligo(dN) is synthesized with the o:anomers of nucleoside units instead of the natural [3-anomers, the orientation within the major groove depends on the sequence: Ion] (dT)8 binds parallel to the (dA)8-containing strand of duplex DNA as does the corresponding [[3] (dT)814.15; in contrast an 11-mer containing both C and T (5'-TTTCCTCCTCT-3') binds antiparallel when synthesized with ec anomers but parallel when synthesized with the ~ anomers (J. S. Sun and J-C. Fran§ois, unpublished data). It is not known yet h o w many o~-C have to be inserted in an e~ (dT)n to reverse the orientation of the oligonucleotide within the major groove.
Cleavage specificity Cleavage reactions induced by homopyrimidine oligo(dN) bound to the major groove of DNA are highly sequence-specific. A copper-phenanthroline-substituted 11-mer cleaved supercoiled and linear SV40 DNA at a single site 32 (Fig. 4). A 17-mer oligonucleotide with Fe-EDTA attached induced a single doublestrand cut in )~ phage DNA 33. The specificity is temperature-dependent: lowering the temperature favors binding of the oligonucleotide to mismatched sequences. Specificity can also depend on the pH: increasing cytosine protonation as pH is lowered encourages binding (and hence cleavage) at mismatched sequences. The yield of the cleavage reactions (percentage of available sites cleaved) is 15-25% with Fe-EDTA 33 and 5070% with Cu-phenanthroline 32. In order for these sequence-specific artificial endonucleases to be useful in gene mapping of long DNA fragments, however, yields close to 100% are required: otherwise, incom-
~Fig. 5
Minor - ' ] groove I--~ reactio3
Intercalation m
Major groove recognition
I Attachment to oligonucleotide
Mechanism for the action of a Cu-phenanthroline artificial nuclease. A homopyrimidine oligonucleotide recognizes the major groove of DNA at a homopurine.homopyrimidine sequence. A phenanthroline ring attached at its 5'-end (see bottom right of figure) intercalates at the junction between the triplex and duplex structures, bringing the copper-chelating nitrogen atoms into the minor groove where the cleavage reaction takes place.
plete cleavage will create too many intermediate fragments. The pH and/ or temperature dependence of each potential endonuclease will need to be studied in order to define the best conditions for efficient site-specific cleavage.
Cleavage mechanism The ways in which duplex DNA is cut by artificial endonucleases and restriction endonucleases differ. Whereas nearly all restriction endonucleases cut precisely at a single bond, the artificial endonuclease cut is less clean: when the chemical species responsible for the cleavage is diffusible, a distribution of cut sites is seen. This is observed with the OH' radicals generated by Fe-EDTA and by Cu-phenanthroline (Fig. 4). The distribution of cleavage sites is narrower with Cu-phenanthroline
than with Fe-EDTA, probably because oxidative species are generated closer to the reactive sites. Some interesting results have been obtained concerning the action of Cuphenanthroline tethered to a homo= pyrimidine oligo(dN) targeted to SV40 DNA (Fig. 4). On both strands, the distribution of the cuts is skewed towards the 3' end and this is indicative of reactions taking place in the minor groove. However, it is the major groove to which the oligo(dN) binds. The explanation of this is that phenanthroline intercalates at the triplex-duplex junction (Fig. 5). The oligonucleotide is attached via C-5 on the phenanthroline ring. Therefore, intercalation brings the two copper-chelating nitrogen atoms into the minor groove where the cleavage reaction takes place. To our knowledge, this is the first example
TIBTECH- NOVEMBER 1989 [Vol. 7]
of a direct transfer of chemical i n f o r m a t i o n b e t w e e n the two grooves of DNA 34 (Fig. 5). The w a y s ahead Highly sequence-specific artificial nucleases have b e e n designed and, for single-stranded nucleic acids, these can be targeted to any sequence. For d u p l e x DNA, h o w e v e r , the specificity of the reaction is p r e s e n t l y limited. T h e r e is a n e e d to d e v e l o p ligands that recognize either longer sequences from the m i n o r groove or more diverse sequences t h a n h o m o p u r i n e - h o m o p y r i m i d i n e sequences from the major groove. T h e challenge to chemists is to use the basic k n o w l e d g e that is a c c u m u l a t i n g on local DNA structures to a c c o m p l i s h this. Cleavage reactions also n e e d i m p r o v e m e n t . The available cleaving reagents have m a n y drawbacks: metal chelates effect oxidative attack of the sugar moiety, but reaction with targets other t h a n nucleic acids c a n n o t be avoided; p h o t o c l e a v i n g reagents are inactivated during irradiation; in addition, several bonds are cleaved in all described cases despite the selectivity of base seq u e n c e recognition. F u r t h e r m o r e , the termini w h i c h are generated b y artificial n u c l e a s e cleavage are not suitable as substrates for some enzymes (e.g. ligases) u s e d in genetic engineering. One solution to these difficulties w o u l d be to follow the c h e m i s t r y of natural nucleases (i.e., to design reagents w h i c h h y d r o l y s e p h o s p h o d i e s t e r b o n d s 35. Attaching such h y d r o l y t i c reagents to oligon u c l e o t i d e s c o u l d p r o d u c e 'oligozymes'; these c o u l d be used in v i v o if the o l i g o n u c l e o t i d e was nuclease resistant. Sequence-specific modifications and cleavage of nucleic acids have m a n y potential applications. Artificial e n d o n n c l e a s e s c o u l d be used as tools in m o l e c u l a r and cellular biology. Natural ribonucleases have v e r y limited base sequence specificity; sequence-specific cleavage by artificial nucleases might, therefore, be used to d e t e r m i n e w h e t h e r a particular nucleic acid s e q u e n c e was accessible (e.g., in n u c l e o p r o t e i n complexes) or to selectively destroy a selected mRNA or viral RNA. Restric-
tion e n d o n u c l e a s e recognition sites are limited to 8 base pairs; oligonucleotides e q u i p p e d w i t h molecular scissors could be used to recognize m u c h longer target sequences. A p p l i c a t i o n s for gene mapping on long DNA fragments or for site-directed mutagenesis can be c o n t e m p l a t e d . In addition, sequencespecific modifications and/or cleavage of selected sequences on mRNA, viral RNAs or d u p l e x DNA opens the possibility of controlling gene expression at different levels. This might also p r o v i d e a rational basis for the design of drugs that c o u l d selectively inhibit DNA replication and the t r a n s c r i p t i o n or translation of selected genes. A n e w era o f ' g e n o m i c p h a r m a c o l o g y ' might emerge. References 1 Toulm~, J. J. and H~l~ne, C. (1988) Gene 72, 51-58 2 Cazenave, C., Loreau, N., Thuong, N. T., Toulm~, J. J. and H~l~ne, C. (1987) Nucleic Acids Res. 15, 4717-4736 3 Boutorin, A. S., Vlassov, V. V., Kazakov, S. A., Kutiavin, I. V. and Podyminogin, M. A. (1984) FEBS Lett. 172, 43-46 4 Chu, B. C. F. and Orgel, L. E. (1985) Proc. Natl Acad. Sci. USA 82, 963-967 5 Dreyer, G. B. and Dervan, P. E. (1985) Proc. Natl Acad. Sci. USA 82, 968-972 6 Boidot-Forget, M., Chassignol, M., Takasugi, M., Thuong, N. T. and H61~ne, C. (1988) Gene 72, 361-371 7 Chen, C. H. B. and Sigman, D. S. (1986) Proc. Natl Acad. Sci. USA 83, 7147-7151 8 Chen, C. H. B. and Sigman, D. S. (1988) J. A m . Chem. Soc. 110, 6570-6572 9 Francois, J-C., Saison-Behmoaras, T., Chassignol, M. et a]. (1988) Biochemistry 27, 2272-2276 10 Fran§ois, J-C., Saison-Behmoaras, T., Chassignol, M., Thuong, N. T. and H61~ne, C. (1989)J. Biol. Chem. 264, 5891-5898 11 Le Doan, T., Perrouault, L., Chassignol, M., Thuong, N. T. and H61~ne, C. (1986) Biochemistry 25, 6736-6739 12 Le Doan, T., Perrouault, L., Praseuth, D. et al. (1987) Nucleic Acids Res. 15, 8643-8659 13 Asseline, U., Delarue, M., Lancelot, G. et al. (1984)Proc. Natl Acad. Sci. USA 81, 3297-3301
14 Le Doan, T., Perrouault, L., Praseuth, D. et al. (1987)NucleicAcids Res. 15, 7749-7760 15 Praseuth, D., Perrouault, L., Le Doan, T. (1988) Proc. Nat] Acad. Sci. USA 85, 1349-1353 16 Praseuth, D., Le Doan, T., Chassignol, M. et al. (1988) Biochemistry 27, 3031-3038 17 H~l~ne, C., Le Doan, T. and Thuong, N. T. (1989) in Photochemical Probes in Biochemistry (Nielsen, P. E., ed.), pp. 219-229, Kluwer Academic Press 18 Corey, D. and Schultz, P. (1987) Science 238, 1401-1403 19 Zuckermann, R. N. and Schultz, P. G. (1989) Proc. Natl Acad. Sci. USA 86, 1766-1770 20 Cech, T. R. (1987) Science 237, 1532-1539 21 Haseloff, J. and Gerlach, W. L. (1988) Nature 334, 585-591 22 Morvan, F., Rayner, B., Imbach, J. L. et al. (1987) Nucleic A c i d s Res. 15, 3421-3437 23 Thuong, N. T., Asseline, U., Roig, V., Takasugi, M. and H61~ne, C. (1987) Proc. Nail A~ad. Sci. USA 84, 5129-5133 24 H~l~ne, C. and Thuong, N. T. (1988) in Nucleic Acids and Molecular Biology, Vol. 2) (Eckstein, F. and
25 26 27 28 29 30
31 32
33 34 35
Lilley, D. M. J., eds), pp. 105-123, Springer-Verlag Sun, J. S., Fran§ois, J-C., Lavery, R. et al. (1988) Biochemistry 27, 6039-6045 Chert, C. H. B. and Sigman, D. S. (1987) Science 237, 1197-1201 Sluka, J. P., Horvath, S. J., Bruist, M. F., Simon, M. I. and Dervan, P. B. (1987) Science 238, 1129-1132 Mack, D. P., Iverson, B. L. and Dervan, P. B. (1988) J. Am. Chem. Soc. 110, 7572-7574 Dervan, P. B. (1986) Science 232, 464-471 Buchardt, O., Karup, G, Egholm, M. eta]. (1989) in Photochemical Probes in Biochemistry (Nielsen, P. E., ed.), pp. 209-218, Kluwer Academic Press Moser, H. E. and Dervan, P. B. (1987) Science 238, 645-650 Fran§ois, J-C., Saison-Behmoaras, T., Chassignol, M., Thuong, N. T. and H~l~ne, C. (1988) C. R. Acad. Sci. Paris (1988) 307 (s6rie III), 849-854 Strobel, S. A., Moser, H. E. and Dervan, P. B. (1988)J. A m . Chem. Soc. 110, 7927-7929 Francois C., Saison-Behmoaras, T., Barbier, C. et al. Proc. Nail Acad. Sci. USA (in press) Basile, L. A., Raphael, A. L. and Barton, J. K. (1987) J. Am. Chem. Soc. 109, 7550-7551

Cleaving Pdf Free Download For Windows 7

  1. An apparatus for cleaving a section of a bar of brittle material is provided. The apparatus comprises a support adapted to hold the section of the bar in a position to be cleaved, a blade, an actuator coupled to the blade for driving the blade at least partially through the bar to create a cleaved portion of the bar, and a follower for engaging the end of the bar during cleaving.
  2. Cleaving A Diamond Free PDF eBooks. Posted on January 17, 2015. Cleaving the Halqeh-ye-nur diamonds: a dynamic fracture analysis. Read/Download File Report Abuse.
  3. Artificial sequence-specific nucleic acid cleaving reagents provide new opportunities in nucleic acid chemistry and biol. Email: email protected. 657KB Sizes 2 Downloads 32 Views. Recommend Documents. Sugar non-specific endonucleases.

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