Gene, 7 (1979) 355--362

355

© Elsevier/North-Holland Biomedical Press, P,m_~terdam -- Printed in The Netherlands

ASYMMETRIC LINgi~.R MOLECULES FOR RECOMBINANT DNA CONSTRUCTION8 (Chemical synthesis of DNA; dodecanucleotide; octanucleotide; £coRI) KJELD g. NORRIS, DIRK ISERENTANT*, ROLAND CONTRERAS* and WALTBR FIERS*

The Danish Institute of Protein Chemistry, 4 Venlighedwej, DK-2970 H~rsholm (Denmark) and *Laboratory of Molecular Biology, State University of Ghent (Belgium)

(Received May 2O+nd,1979) (Accepted July lVth, 19V9) SUMMARY

Asymmetric EcoRI DNA linkers consisting of an AATTC(A)7 dodecamer and a complementary G(T)~ octamer were synthesized. Ligation of such linkers to DNA fragments obviates the need for E¢oRI digestion prior to cloning in EcoRI~leaved vectors. Chemically synthesized oligonucleotides and DNA have played a crucial role in the elucidation of the genetic code (Khorana, 1968), in the study of transcription of an operon (Belegaje et al., 1978), and, more recently, as a tool for construction of recombinant DNA molecules (Bald et al., 1976; Scheller et al., 1977). In the latter studies, use was made of decameric selfcomplementary DNAs which contained in their center the recognition sequence of an appropriate restriction enzyme. These blunt+nd DNA blocks were joined to blunt.end DNA fragments by T4 DNA ligase. The resulting structures were treated with the restriction endonuclease to generate sticky ends at the added decameric blocks and the fmal product was then recombined with a plasmid vector. A disadvantage of this procedure is the necessity of a restriction endonuclease treatment, which could result in cleavage of the cloned DNA fragment itself if its restriction map is unknown. Such a difficulty particularly arises when the DNA of interest cannot be characterized, as a cDNA copy complementary to a mixture of mRNAs. To obviate the need for this subsequent digestion with a restriction enzyme, we have synthesized an ~mmmetric linker molecule which contains one-half of the recognition sequence of the £coRI endonuclease at one side and a bluntend at the other side (Fig. lb). The blunt, e n d can be ligated to the DNA fragment+ of interest and the attached EcoRI sticky end can be annealed to an £coRl-cleaved vector.

866

Chemical eynthem of the aymmetrie linker, The linker was eomdxucted by chemical synthesis of the two partly complementmy oligodeoxyribonucleotidas d( A-A-T-T~-A-A-A-A-A-A-& ) and d(T-T-T-T-T-T-T~]I). The dodecanucleotide was synthesized by conventional phosphodiastm fragment condensation in solution (Khoxena, 1968); the procedure is summaxized in Fig. la. The lipophillc 5'-phosplmte protecting group 2Ap-tritylphenyl)~dfonylethanol (TPSE) (Agarwal et al., 19/6) was used fox the syntheses of the di- and txinucleotide blocks d(pT-T), d(panCbzA), and d(pbxA-bzA-bzA). The blocks were conveniently isolated by solvent exlxacfion, as were the dinucleotide d[(MeOTr)bzA-bzA] and the tetra-

d (MeOTr ) bzA

(~

~d ['pbzA( Ac )] d [(HeOTr) bzA-bzA ] ~ d ~pT-T(Ac)] d [(HeOTr ) bzA- bzA-T-T] ~d [pa nC-bzA (Ac)] d E(HeOTr)bzA-bzA-T-T-a nC-bzA] ~d [pbzk- bzk-bzA (Ac) ] d [(HeOTr ) bzA-bzA-T-T-a nC- bzA- bzA-bzA-bzA] ~d [pbzA-bzk-bzA(Ac )] d [(HeOTr)bzA-bzA-T-T-anC-bzA-bzA-bzA-bzA-bzA-bzA-bzA] NH3 2. H+ d (A-A-T-T-C-A-A-A-A-A-A-A) 1.

5!

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AATTCAAAAAAA GTTTTTTT

s' s'

Fill. 1. (s) Flow chart outlining the chemical synthesis of the dodeemueleotide d(A-A-TT4~-A-A-A-A-&-&-A)by phoaphodiaster condensation in solution (Khorana, 1968). (b) Aaymmetriclinker DNA e o m m l l the dodecnueleotide chemically synthesized as described in (s) and an oetanucleotide prepmed by solid-phase synthesis (Nm~ng et it., 1977).

357

nucleotide d[(MeOTr)bzA-bzA-T-T]. Condensation of the tetranucleotide with the dinucleotide block gave the hexanucleotide d[(MeOTr)bzA-bzA-TT-anC-bzA], which was purified by DEAE-cellulose chromatography. The protected hexanucleotide was condensed twice successively with the same trinucleotide, d[pbzA-bzA-bzA(Ac)], to give the nonanucleotide and the desired dodecanucleotide. In each case the products were isolated by DEAEcellulose chromatography. After deprotection, the dodecanucleotide was further purified by DEAE~ellulose chromatography in the presence of 7 M urea. The dodecanucleotide, d(A-A-T-T-C-A-A-A-A-A-A-A), was characterized by labeling of the 5' end with [7-32P] ATP and T4 polynucleotide kinase followed by a "wandering spot" analysis (Fig. 2a). The octanucleotide was prepared by a recently developed solid-phase methodology on a functionalized cross-linked polyacrylmorpholide support (Narang et al., 1977). The procedure involved stepwise additions of protected mononucleotides, starting with 5'-O-(p-carboxymethyloxytrityl)-thymidine bound by an amide linkage to cross-linked polyacrylmorpholide beads (Enzacryl Gel K2, Koch-Light) after exchange of some of the polymer's morpholine groups with ethylenediamine. In the published synthesis of the pentanucleotide d(T-T-T-T-T), excellent coupling yields were obtained (Narang et al., 1977). Extension of the polymer-bound pentanucleotide to the octanucleotide gave also satisfactory although somewhat lower coupling yields. After addition of d[pibG(Ac)], the isobutyryl protecting group was cleaved by ammonia treatment. The octanucleotide, d(T-T-T-T-T-T-T-G), was liberated from the polymer by acid hydrolysis and purified by ion exchange chromatography on Dowex I using a gradient of ammonillm chloride in 40% aqueous ethanol (Asteriadis et al., 1976). After desalting, the octanucleotide was characterized by enzymatic breakdown and measurement of the ratio dT:dpT:dpG (observed, 1.02:6.00:1.05; calcd., 1:6:1), and, further, by a wandering spot analysis (Fig. 2b) as described above for the dodecanucleotide. Full details of the chemical synthesis of the two oligodeoxyribonucleotides will be published elsewhere (Norris, Narang, Bhat and Brumfeldt, manuscript in preparation).

Construction of recombinant DNA The purified linker molecule was used for the construction of a recombinant plasmid containing in a specific orientation the genetic information corresponding to the 3' end region of bacteriophage M82 RNA. This plasmid was needed for other studies, such as the characterization of a particular class of RNA-phage mutants, as will be discussed in a later publication. The MS2 DNA fragment was derived from the recombinant plasmid pMS2-7, which comprises a nearly full-size DNA copy of MS2 RNA as well as the insertion sequence IS1 integrated adjacent to the PstI site of the ampicWinresistance gene of plasmid pBR322 (Devos et al., 1979a,b). pMS2-7 DNA was digested with the restriction enzyme MbolI and the resulting fragments were dephosphorylated with bacterial alkaline phosphatase. Approx. 2 pmol

QD

359

of the M b o l l fragment containing the 3' end of MS2 DNA (Fig. 3) was isolated from a 4% polyacrylamide gel by extraction with 1 M NaCI. The purified f ~ g m e n t was treated with 4 units $1 endonuclease (purified as described by Britten et al., 1974) for 45 min at 37°C in 0.2 M NaCI, 0.001 M Zn(Ac)2, 0.05 M NaAc, pH 4.5, 0.005 M EDTA in order to generate flush ends. The mixture was extracted with phenol and the DNA precipitated from 0.20 M KAc with 2 vol. of ethanol. The blunt end of the linker molecule was ligated to the Sl-treated M b o I I f ~ g m e n t in a reaction mixture t h a t contained 30 pmol of linker molecules phosphorylated at the 5' end of the octanucieotide, 2 pmol of the M b o I I fragment, 20 pl of I mM ATP, 20 mM Tris-HCI, pH 7.6, 10 mM MgCI2, 6 mM DTT, and 10 units of T4 ligaee (Biolabs). The reaction was allowed to proceed for 15 h at 10°C. The linker molecules were added t o the ligation mixture directly after the kination reaction (without removal of the excess radioactive ATP), such that estimates of neither the specific radioactivity of the linkers nor of their ligation efficiency could be made. The ligation mixture was subsequently extracted twice with phenol, precipitated, digested with PstI endonuclease (Fig. 3) and fractionated on a 6% polyacrylamide gel. The incorporation of radioactivity into the two Psfl-generated fragments was nearly equal; therefore both ends seem to accept the linker molecule with comparable efficiency. Hence we believe that at least a fraction of the fragments before redigestion carried linkers at both ends.

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Fig. 3. The Mboll restriction fragment derived ~om recombinant D~A plesmid pM82-7 (Devns et aL, 1979s,b) and used for cloning by means of the ehemiesily synthesized linker is a composite structure of DNA originating from three sources: M82 DNA eorrespondinf to the 3' end (residues 8221 to 3569) of M82 RNA, a poly(dA)-poly(dT)

linker segment of about 150 base pairs used in the construction of pM82-7, and 450 b u , pairs of the left end of 181. The position of the Mboll eleavage sites and of the PsH cleavage site are indicated by vertical arrows. The scale is calibrated in units of 100 nueleotides.

Fig. 2. Partial snake venom phesphodiesterase digestion of the dodacanudeotide (a) and the octanueleotide (b) described in Fig. 1. After "P-labeling of the 5' ends both fragments were pm~iaily digested as described previously (Contreres et ai., 1977) and fractionated on a two-dimensionai system of eleetrophoresis on cellulose acetate at pH 3.5 in the rust dimension and homochromatography on 20 em x 40 cm DEAE-cellulose thinlayer plates in the second dimension.

360

Fig. 4. RastrietJon endonuelease dJtestion of plasmid pBR$22 DNA end of the recombinant phumid pM3'E DNA. pBR322 (a) end pld3'E (b) were disested with £coRI a~d Pstl in 30-~! reaction mixtures eontaininft 0.04 M NaCI, 0.012 l~i Tris.HCI, pH 7.6, 0.01 M MgCi=, 0.01 M ~-merceptoethenol, 1 ~g of DNA, I unit of EeoRl end I unit of PstI. The reactions were run for I h at 37°C. The products were elactrophoresed on a 1% aprose gel in a buffer containing 0.05 M Tri~acetate, pH 7.8, 0.02 M NaAe- and 0.002 M EDTA. The double digestion verifies the presence of the EeoRI and Pstl restriction sites in the pM3'E DNA, The 752 base pair fragment removed from pBR822 by double digestion during preparation of the vector DNA is replaced in pld3'E by a frefpnent of approximately 680 base pairs. MboH digestion of pBR322 (e) end of pM3'E (d) were carried out in 30~I reaction mixtures conteining 0.01 M Tris.HCl, pH 7.6, 0.007 Id Mk~I=, 0.007 M #-mercsptoethanol, 5 ~g of DNA and 4 units of MboH. ~ reactions were incubated for 2 h at 37°C. The resultin8 ~ n t s were separated on a 4% polyacrylamide gel in an eleetrophoresis buffer containing 0.09 M Tris-borate, pH 8.4, 0.0025 M EDTA. The two smaller MboH fragments of pBR322, which are situated between the Pstl site end the EeoRI site, disappear in the MbolI ditast of pM3'E. These fragments are replaced by the insert, which contains no internal MboIl recognition sequences. The net result is an elongation of the largest pBR322~3eelfie fragment. The insert, however, still maintains the MboH recoL~ition sequence at the EeoRl end due to the distance between the MboH recoi|nition sequence and the cleavage site (Endow, 1977). It should be noted that because pBR322 (as well as pM3'E) was Erown in E. eoli strain IIB101, four MboII sites of pBR322 are protected alialnst cleavqe by adenine methylation; this can be shown by eomperison of the MbolI restriction patterns of pBR322 DNA from adenine methylatint and nonmethyleting cells (J. van Emmelo, personal communication).

361

The vector DNA was obtained by digestion of plasmid pBR322 with PstI and EcoRI (which removes part of the/]-lactamase gene) and fractionation of the products on a 1% agarose gel. The vector was extracted from the gel by freezing and centrifugation. To construct recombinant plasmid DNA molecules, 0.4 pmol of the linker-~sgment DNA and 0.2 pmol of vector DNA were mixed in 7 #1 of 20 mM Tris- HCI, pH 7.6, 10 mM MgCI2 and 10 mM #-mercaptoethanol. The mixture was heated for 5 rain at 60°C and slowly cooled to 10°C. After addition of DTT and ATP to final concentrations of 6 mM and 1 mM, respectively, 5 units of T4 ligase was added and the mixture was kept at 10°C overnight. Transformation of E. coli cells was carried out essentially as described by Cohen et al. (1972). Colonies harboring recombinant plasmids were selected on the basis of resistance to tetracycline. Of a total of 194 colonies, 52 were found to be sensitive to ampicillin. The intactness and chimeric nature of the newly constructed plasmids were verified by size estimations and examination with restriction endonucleases Sinai, MbolI, EeoRI and PstI. One particular clone was taken as the prototype of the new construction and its plasmid is referred to as pM3'E. Analysis of this DNA showed that the EcoRI site and the Pstl site were indeed regenerated during construction of this molecule (Fig. 4). The results demonstrate that a chemically synthesized DNA segment can be used for constructing recombinant DNA molecules. The linker we have synthesized and employed contains one blunt end and one staggered end, the latter corresponding to an £¢oRI sticky end (Fig. 1). Ligation of the blunt end to a DNA fragment equips the fragment with the proper sticky end to anneal to an £¢oRI.cleaved vector, without requiring any further digestion with a restriction enzyme. Thus, DNA segments whose nucleotide sequence or susceptibility to restriction cleavage is unknown can be recomhined by means of chemically synthesized linkers without having to undergo a potentially destructive endonuclease digestion to generate sticky ends. It should also be noted that this linker molecule may be ~ d to convert an EcoRI sticky end into a blunt end. In the course of this work, similar adapter molecules were described by Bahl et al. (1978). ACKNOWLEDGEMENT

R.C. holds a Bevoegdverklaard Navorser fellowship from the Belgian Nationaal Fonds voor Wetenschappelijk Onderzoek. Research carried out at the University of Ghent was supported by grants from the "FKFO" and from the "Geconcerteerde Acties." REFERENCES

Agarwal, K.L., Berlin, Y.A., Fritz, H.-J., Gait, M.J., Kleid, D.G., Lees, R.G., Norris, K.E., RamaMoorthy, B. and Khorana, H.G., Studies on polynucleotides, CXLHL A rapid and convenient method for the synthesis of deoxyribooligonucleotides carrying 5'-phmphate end groups using a new protecting group, J. Am. Chem. Soc., 98 (1976) 1065--1072.

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Gene, 7 (1979) 355--362 355 © Elsevier/North-Holland Biomedical Press, P,m_~terdam -- Printed in The Netherlands ASYMMETRIC LINgi~.R MOLECULES FOR...
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