Eur. J. Biochem. H3. 613— 620 (1984)
`1: FEBS 1984
`
`Single-step elongation of oligodeoxynucleotides
`using terminal deoxynucleotidyl transferase
`
`Herbert SCHOTT and Herbert SCIIRADE
`
`Institut l't'tr Orgattische Chemie. Universitat Tt‘tbingcn
`
`(Received March 26. 1984]
`
`- FJB 840317
`
`Procedures for the stepwise addition of one or more deoxyribonucleotidc residues to the 3' end of an
`oligodcoxyribonttcleoside phosphate acceptor using commercially available terminal deoxynucleotidyl transferase is
`described. 2—80 nmol of acceptors with a chain length of four, five or nine monomer units Were elongated with a
`single 2’-deoxyribonucleoside S’-triphosphate in yields ont) — 30 "-,,. The monomers carried no protecting groups and
`were used both radioactively labelled and unlabelled.
`The elongated oligodeoxynucleoside phosphates were isolated by reverse-phase (Nucleosil C13} high—
`performanee liquid chromatography or paper chromatography. The isolated products were sequenced by the
`fingerprint method. Advantages and disadvantages of this new methodology for the enzymatic synthesis of defined
`oligodeoxynuclcotidcs are discussed.
`
`Besides the well—known chemical synthesis. two different
`approaches for
`the controlled addition of monomers or
`oligomers to the 3’ end of an oligodeoxyribonuclcotidc primer
`are known.
`In one approach, deoxyribonucleotide residues
`derived from 2’—deoxyribonucleoside S’-diphosphates may be
`added to o]igodeoxyribonneleotide primers with polyntt-
`cleotide phosphorylasc. purified from Escherichia t‘oiiB [I —3].
`This method allows the synthesis ofoligodeoxyribonucleotidcs
`of defined sequences by repeated addition of single residues.
`applying conventional biochemical procedures. The other
`approach demonstrates conditions which allow the efficient
`addition of 2’-dcoxyribonucleoside 3',5’—bisphosphates
`to
`oligodeoxyribonuelcotides and of single—stranded oligodeoxy-
`ribonuclcotides to one another using T4 RNA ligase [4].
`Both methods have been used to synthesize a considerable
`number of oligonuelcotidcs. However. general application of
`these techniques is limited because the preparation of the pure
`polynucleotidc phosphorylase requires many critical puriti-
`cation steps and the condensation time (several days for one
`reaction step} with T4 RNA ligase takes too much time in
`comparison to the rapid chemical synthesis which can be done
`by an automated synthesirrer.
`H0wever, an enzymatic synthesis should be a very attractive
`tecltniquc for the simple preparation ofoligonucleotides in high
`purity. if the required enzymes are easily available and if the
`condensation time is short enough. A well known and com-
`mercially available enzyme is terminal deoxynucleotidyl trans-
`l'erase which catalyses the polycondensation of deoxynu-
`
`cleosidc triphosphates onto a suitable acceptor 3'-hydroxy
`group. The acceptor mttst have at
`least
`three phosphate
`residues. This enzyme has found wide application in DNA and
`RNA sequence analysis and is widely used for the purpose of
`construction of recombinant DNA in vitro. Depending on tlte
`presence ofeither Mg2 ' or Co2 ’ ions. the enzyme may be used
`to add a number- preferentially 15 w 20 nucleotide residues. to
`the 3' cntl of either singc—stranded or double-stranded DNA
`fragments [5 — 9].
`Several series ofhomologous oligomers were examined for
`initiator utilization and product distribution in the hotnopoly—
`condensations and the copolycondensations. bttt the stepwise
`elongation of oligonucleotides by the repeated addition of
`monomers using terminal dcoxynuclcotidyl transferase has not
`yet been reported.
`Usually this enzyme condenses a varying number of
`monomers onto the 3’ end ofan acceptor in a polycondensation
`reaction yielding oligonucleotides of different chain length.
`Even if the elongation could be shifted to a monoaddition. a
`complex reaction mixture would be obtained.
`We have investigated the enzymatic reaction conditions and
`the chromatographic separation procedure which allows the
`stepwise synthesis of defined oiigodeoxyntteleotides using
`terminal deoxynuclcotidyl transferasc.
`
`MATERIALS AND METHODS
`
`A (rep tors
`
`tricthylammonium hydrogen car-
`Abbreviations. EISNIICOJ.
`bonate: HPLC, high—performance liquid chromatography.
`Hazy-van. Terminal deoxynucleotidyl transferase (EC 2.1131);
`polynuctcotidyl phosphorylase {EC 2.17.8); T4 RNA ligasc (EC
`6.5.1.3): alkaline phosphatase (liC 3.1.3.1): snake venom phos-
`phodiesterase (EC 3.1.4.1): spleen phosphodicsterase [EC 3.1.4.18}:
`T4 polynuclcotide kinase {EC 2.11.78].
`
`The acceptors (dTJ4. {dC)4 and d{A—A-A—C) were synthe—
`sized in our laboratory (ll. Schott, unpublished); d{A—A—A—G)
`[l2]. d{(i—A-A~A} [12] and dtC4,'l'5) [13} were isolated from
`partial hydrolysates of chemically degraded herring sperm
`DNA. dTTP. dATPand dC'l‘P were obtained from Boehringer,
`Mannheim;
`[at—”HdATP (specific activity = 2Cifmmol).
`
`CU REVAC EX2040
`CUREVAC EX2040
`Page 1
`Page 1
`
`

`

`614
`
`[y-“PIATP and [8—311]dGTP [specific activity = 9 Citmmol)
`were purchased from Amersham Buchlcr. Braunschweig.
`
`Enzymes-
`
`Alkaline phosphatasc (calf intestine), snake venom phos-
`phodiesterase and spleen phosphodiesterase were obtained
`from Boehringer, Mannheim; T4 polynucleotide-kinase was
`purchased from Amersham Buchlcr and terminal deoxynu—
`cleotidyl transferase from Pharmacia P—L Biochemicals. Frei—
`burg.
`
`Chromatography
`
`DEAE-cellullose thin-layer plates: Polygram, Cel 300
`DEAE HR—EHS, were obtained from Macherey & Nagel,
`Diiren. and cellulose paper no. 23l6 from Schleicher & Schi'tll.
`Dassel.
`
`Cmm’mttrttiort reactions
`
`The elongation of acceptors was carried out at 37 "’C in the
`foll0wing buffer: 5011i H20. 70 ul 2 M TristHCl pH 7.6, 70 til
`2 M sodium cacodylate. 10 pl 0.1 M CoClz.
`1 |.tl 0.1 M di-
`lhioerythritol. The conditions ofthe condensation reactions are
`summarised in Table l. l U (condensations III —VI, IX) or 5 U
`[condensations VII. VIII} of alkaline phosphatase were added
`at the end ofthc reaction time and the incubation continued for
`l h.
`
`Separations of reaction mixtures from condensation I and
`ll were performed on DEAE-cellulose thin-layer plates by
`homochromatography using homornix V] according to Jay et
`a]. [14]. Elongated products Were detected autoradiographi—
`cally. eluted with IjtsNHCOJ buffer and identified by the
`nearest—neighbour analysis [1 t. 15] (see Fig. 1).
`Separation ofthc reaction mixture from condensation VII
`was achieved by descending paper chromatography. First the
`mixture was chroinatographed in solvent A: ethanolt‘l M
`ammonium acetate {pH 15‘: (7:3, vtv). Unreacted monomer.
`which migrates closely to the solvent front, was detected by
`liquid-scintillation counting ofpaper segments. The segment of
`the paper chi'omalograin which contained the spot of the
`monontcr was cut oil'. The rest ofthe paper chromatogram was
`dried and then rechromatographed in solvent B: l-propanol
`liconcentrated ammonia,twater (55: 10:35. vt'vr'v). Elongated
`oligonucleotidcs (Fig.4) were detected by liquid scintillation
`counting of paper segments. eluted from the paper and finally
`lyophilised.
`Separations of reaction mixtures from condensations III —
`VI. VII] and IX were carried out by HPLC (l-‘ig. 2). Nuclcosil
`C13. particle sire 7.0mm (Machcrey 8:. Nagel, Dijren) was used
`as the stationary phase. The column (250 x4.6 mm internal
`diameter) was eluted under isocratic conditions with a mixture
`of 72 3’", A and 28 TI, B. except condensation mixture [X which
`was separated using 68 '21; A and 32 'i--;, B. A = 0.] M ammonium
`acetate (pH 7.5); B = methanoltwater (60:40. va).
`A high—pressure liquid Chromatograph (Waters 6000 A} was
`equipped with an ultraviolet detector (Du Ponl Instruments
`model 836} and a high-pressure sampling valve {Waters U6K).
`The monitoring wavelength was 254 rim,
`the [low rate
`lmlftnin. The flow detector for radioactivity consisted of a
`detector unit [BF 5026) with a measuring ecll (BF 5029} and a
`measuring unit {BF 2240: ralemeter
`31-“ 2305. Berthold.
`Wildbad). The time constant used was 25.
`
`RES U LTS
`
`At first, chemically synthesised tetranueleoside trisphos-
`phatcs of different sequences, as well as these isolated from
`partial DNA hydrolysates, were used as acceptors and clon-
`gated with purine or pyrimidine mononucleotides. Two re—
`actions were evaluated qualitatively and four quantitatively. in
`condensation VII. all components of the mixture of sequence-
`isomeric nonanucleoside octakisphosphatcs dtCM T5), isolated
`from the partial hydrolysatc of depurinated DNA. were
`elongated together with a (16 residue. This reaction was also
`investigated quantitatively.
`in condensation VIII the syn-
`thetically obtained tetranucleoside trisphosphate th—A-A-C)
`was elongated to th-A-A—C‘—A) which was then converted to
`d(A-A—A—(T—A[33P]p'f) (condensation IX).
`
`Elongation of homologous pyrimidine nttt’ieoside phosphates
`
`For qualitative investigations. the two chemically synthe-
`sised homologous pyrimidine tetranueleosidc trisphosphates,
`(dC)4 and (d'l'h. were condensed with [a—JZP]dATP to obtain
`radioactively labelled elongated products. The acceptor was
`applied in a 40-fold molar excess (with respect to the monomer)
`favouring the formation of the monoaddition products dt'I‘—T-
`T-T[32P]pA} and dtC-C-C-CPzPlpA), respectively. The con—
`densation was carried out according to the conditions given in
`Table! with I U terminal deoxynucleotidyl transferasetnmol
`monomer. After incubation at 37 "(I for 4 h. the crude reaction
`mixtures were separated on DEA E—cellulose precoated sheets
`by homochromatography [IO] aceording to the chain length.
`Because of the incorporation ofI32PlpdA units. all elongated
`oligonuclcotide phosphates were detected by the autoradiog-
`raphy of the chromatograms. In the autoradiograms of each
`reaction mixtures, only one spot occurred which corresponded
`to the monoaddition products d(T—'I'-T—T[32P]p/\) and dtC-C-
`C—(Tl‘nPlpAl respectively. This assumption was confirmed by
`the ‘nearcst—neighhour analysis‘ [11. 15].
`To determine the number of added adcnylic acid units. the
`spot of the corresponding radioactively labelled oligonu—
`cleoside phosphate was scraped from the DEAF.—cellulose
`plate, eluted with the volatile FIJNHCO3 buffer and sub-
`sequently lyophilised. An aliquot ofthe freeze—dried substances
`was totally hydrolysed with spleen phosphodiesterase so that
`nu cleoside 3’~monophosphates were obtained. The hydrolysate
`was separated by paper chromatography and the radioactively
`labelled mononucleotides were identified. Only dT[32Plp could
`be found in the totally hydrolysed d{T-T—T—T[32P]pA) and only
`dC‘Ii‘zPlp in the hydrolysate of dtC-C—C—C[“P]pA) (liig. l).
`After the total hydrolysis of both elongation products with
`snake venom phOsphodjestcrase which liberates nucleoside
`S’-monophosphatcs_. only [32PlpdA was found, as expected.
`These results demonstrate that in the enzymatic reaction both
`acceptors are elongated. each by only a single adcnylie acid
`unit.
`
`Elongation ot'pw‘nn’ nur-t'eostde phosphates
`
`In the following two quantitatively evaluated reactions. the
`sequence—isomeric purine tetranucleoside trisphOSphates dtG-
`A-A-A) and d(A-A-A-G) were used as acceptors which had
`been isolated from depytimidinatcd herring sperm DNA [I2].
`Both acceptors were condensed with tiC’l‘P and dT‘TP re-
`spectively. In contrast to the preceding examples, the excess of
`acceptor relative to the amount ot'monorner was now less than
`2021,: however, again about I U enzymefnmol monomer was
`
`CU REVAC EX2040
`CUREVAC EX2040
`Page 2
`Page 2
`
`

`

`615
`
`Tablet. Conditions [lit-H.119 (”Hammin- elongation of oit'godt'o.\'_l'rihrmrrrl'r'ost‘de phosphates using terminal dt’t).\'J-’Htt(’it°fl¢'ftft‘f
`manonnt'ieoside ti'ipltosphrttes
`The monomer was obtained as a lyophilisate. The enzyme concentration was 10 Uiul; the reaction was carried out at 37 "'C
`
`trans-{erase and
`
`Conden-
`Acceptor
`Amount
`Monomer
`Amount
`linzymc Buffer
`Reaction
`Reaction
`sati on
`volume
`time
`
`
`l
`ll
`[ll
`1V
`V
`VI
`V1]
`VII]
`1x
`
`d(T-T-T—TJ
`d(C-C-C~C)
`(1(A—A—A—G]
`d(A—A—A—G}
`d(Ci—A—A—A}
`d(Ci—A—A—A)
`d(C4. T5)
`d(A-A-A-(.‘)
`th—A—A-C-A)
`
`nmol
`(A260 units)
`
`8.6 (0.08}
`8.1 (0.06)
`5.2 (0.3}
`5.2 (0.3)
`2.6 (0.15}
`2.6 (0. l5}I
`250 (4.0)
`30 (4.3]
`5 (0.35)
`
`[ot‘uPltlA-l—P
`[OLEPMATP
`dTTP
`dC'l‘l’
`d't‘I'P
`dC‘l'IJ
`[8—3H]dG‘I'P
`dATP
`[a—“HdTTP
`
`nmol
`
`units
`
`ul
`——
`
`0.2
`0.2
`4.4
`4.4
`2.2
`2.2
`4f}
`(10
`3.3
`
`0.2
`0.2
`4.5
`4.5
`2,0
`2.0
`20
`20
`3.0
`
`5
`5
`5
`S
`5
`5
`16
`5
`5
`
`25
`25
`7
`7
`7
`7
`50
`8
`7
`
`h
`
`4
`4
`4
`4
`3
`3
`4
`4
`3.5
`
`[3:10 pdA pdG
`
`pdT
`
`
`
`MIGRATION [cm]
`
`. Results of the nrarest—neighbour dimly-iris of ”P—z‘ah’h'ed perim-
`Fig. l
`mtt'lem‘ide leti'w'risplmsphates. Paper electrophoresis (a) of the four
`mononucleotides. (b) of the total enzymatic hydrolysate using spleen
`phosphodicsterase of pentanueleotidc telrakisphosphate dtT—T—TJI'
`[33PIpAJI and (c) of diC—C-C—L‘inmpA).
`In (b) can be seen the
`migration ol‘dTlflPjp and in [c] of dC[3ZI‘]p
`
`used (Table1). After 3 -—4h the reactions were stopped by
`addition of alkaline phosphatase, which dephosphorylates the
`applied monomers to nucleOSides. The crude reaction mixtures
`were separated by reverse-phase HPL.C (Nucleosil C13) under
`isocratic clution conditions. The peaks were monitored by
`ultraviolet detection : the resulting elution profiles are shown in
`Fig. 2. To simplify the interpretation of the elution profiles.
`so-called ‘blanks' were chromatographcd under identical con-
`ditions. These ‘blanks' consisted ofal] the reaction components
`except the transl‘erase and were treated identically: only one of
`the ‘blank’ runs is shown (Fig. 2a}. By comparing the ‘blank'
`(Fig. 2a) and the reaction mixture (Fig. 2b). the new product
`
`(peak 3 in Fig. 2b) can be identified easily. The clution profiles
`in Fig.2b— c demonstrate that
`the enzymatic elongation
`yielded different reaction products which are well separated.
`After
`the reaction of d(A-A~A-G} + dTTP.
`the elution
`profile (Fig. 2b) shows that only one additional main peak
`appears. but
`in the other three elation profiles two or three
`new peaks were present. The fractions of the numbered peaks
`were lyophilised and their products identified by the fingerprint
`method [[0].
`Aliquots of the freeze-dried products were therefore first
`phosphorylated enzymatically at the 5’—hydroxyl groups. The
`oligonucleotides were labelled, using [ir-azP]ATP and polynu-
`cleotide kinase. and were then partially hydrolysed with snake
`venom phosphodicsterase. The resulting hydrolysate was chro-
`matographed two—dimensionally. From the autoradiograms
`(fingerprints). which were obtained after chromatography, the
`purity as well as the sequence of the components could be
`determined. The fingerprints obtained are shown in Fig. 3.
`From the interpretation ofthe fingerprints ofthe enzymatieally
`elongated tetranucleoside trisphosphates, the following results
`are obtained, which are summarised in Table 2. In all cases, the
`unreactcd monomers were degraded with alkaline phosphatase
`to the corresponding nucleosides and inorganic phosphate.
`They are eluted in peaks 1 and la —c.
`in the reations Ill and V dTTP was used. and each of the
`acceptors th—A—A—G) and d{G-A-A-A} were elongated with
`only one monomer unit to th-A-A-G-T) and d(G-A-A-A-T)
`respectively. Ide‘TP was used, however, besides the monoad—
`dition products th-A-A-G-C) or d(G-A-A-A-C}.
`di— and
`triaddition products were also obtained (reactions IV‘ VI). in
`reaction V]
`[dtG—A—A—A) + dC‘TP]. besides the monoaddi-
`tion product. the nucleotide d(G—A A—A—C—C‘} was obtained
`{peak 11. Fig.2c}, and in reaction IV 'ld(A—A~A—G) + dCTP],
`the nucleotides diA—A—A—G—C-C) (peak 5, Fig. 2c) and th—A—
`A-G-C-C-C) (peak 6. Fig. 2c} were found. By reaction of dfG-
`A—A—A) with either dTTP or dCTP (reaction V and Vi res—
`pectively]. not only elongated products were found but also
`th-A-A) (peak 9, Fig. 2d, e}. Apparently the acceptor d(G—A—
`A-A} lost its 5’—terminal dG residue prior to the elongation step
`because neither d(A-A—A—T] nor d(A—A—A—C)could be foundin
`the reaction mixture. although these should have been present
`if the already elongated acceptor had been degraded. Un—
`expectedly, after
`the elongation of the sequence-isomeric
`
`CU REVAC EX2040
`CUREVAC EX2040
`Page 3
`Page 3
`
`

`

`[112
`
`0.03
`
`0.0:.
`
`0
`
`0.12
`
`.
`
`E “'08
`
`C.
`
`4 K
`
`i4:
`
`a}
`
`2
`
`t
`
`616
`
`0.12
`
`Ec
`.4
`3 008
`
`0.0:.
`
`0
`
`00“
`
`0
`
`(.0
`
`20
`
`0
`
`1.0
`
`20
`
`0
`
`TIME [ min 1
`
`TIME [ min 1
`
`
`
`
`
`
`TIME
`[ min ]
`
`
`TIME [ min 1
`
`
`E
`[‘3‘
`q
`
`0.08
`
`0.0!.
`
`9
`
`D
`
`.-
`
`
`1.0
`
`
`LEI-Jul
`
`20
`
`
`0
`
`12
`n
`
`I;
`l
`.1
`l
`
`n
`
`1.2
`
`50-8
`E?
`
`0:.
`
`13
`
`III-ll
`t
`J. 03-}
`
`100
`
`80
`
`50 _L alo— ‘
`TIFEEmin}
`
`Fl
`
`lb
`
`I:
`I
`
`I
`1|:
`MU
`I
`
`20—
`
`0
`
`E
`i
`”:3
`:1
`
`008
`.
`
`0.0!.
`
`0
`
`0.12
`
`E 0.00
`E.
`Q“
`
`0.0:.
`
`.
`
`1.0
`
`20
`
`_
`
`0
`
`TIME
`
`[min]
`
`Fig. 2. S'eparmirm quh‘tm‘m‘ off a tif(J.\'_l"flll{‘[£’0.$l"(l“(’ and m‘igwmct'msidc phosphates on a Nm‘t'cm‘il' C‘.H {71mm} when!” (250 x 4.6mm infernal
`(fiumt’h’rJ by :‘wcrarfc elation m rrmm warmer-mum. Flow rate:
`lmly'min, The mixtures were obtained after the enzymatic elongation of
`tctranucleoaidc trisphosphates (at—F) 0r pentanuclemide tetrakisphosphatcs (g} with nudges'tde triphosphates using the terminal dc-
`oxynuclcotidyl transferasc {except a) and were subsequently treated with alkaline phOSphatasc. The column was eluted with 'J‘E‘T-g A (A _- 0.] M
`ammonium acetate) and 28‘.';, B (B —- methanolfwatcr- 60:40}. Reaction mixtures: (a) : d[A-A-A—G) + dTTP treated only with alkaline
`phosphataseztb} = th—A—A~G)
`+ dTTP:(c) = th-A-A—G) + dCTP: (d) .— d(G-A-A—AJ + dTTP:(c} .— d(G-A-A-A) + dCTP:{f‘) = d(A—A-
`A-C) + dATP: (g) 2 th—A—A—C—A} + [a~:’2P]dT['P, the column eluted with 68?; A and 32“;3 B. Peaks: l = dT: la -: dC; lb = dA,
`10 = [“P]P0i
`:2 = d(A—A—A—Gl;3 = d(A—A—A-G-T}:4 —- d(A-A—A—U-(.‘); 5 = d(A—A~A G—C—C):6 2 d(A—A—A-(i-C CC): 1 2 d{G—A-A—A};
`8 = le—A-A-A—'I'); 9 = d{A—A—A): 10 = d{G—A~A-A—C): ll : d(G—A—A—A—C‘ C): 12 r: dlA-A—A—C); I3 = d[A—A-A-C—A): I4 = d[A-A—A—C—
`A-A}: IS ; d(A—A-A-(‘-A-A—A): I6 = d[A—A—(.‘-A); I? : dlA-A-A-C‘—A[”P}pT): 18 -.~. dim-A-A-C—A[”P]p’l‘[”P}pT)
`
`CU REVAC EX2040
`CUREVAC EX2040
`Page 4
`Page 4
`
`

`

`Table 2. Results rgfirhe communi- corrugation ofofigodcoa‘yri'homrt'Icosr'n’e phosphates rising rcrmi'mu’ dean-vtut'icotioirt rrrtnsf'crasc
`HPLC ofthe reaction mixture was carried out on a T—um Nucleosit CI 3 column {250 >~ 4.6 mm internal diameter)eluted with 72 "g A and 28 3-", B or,
`for reaction 1X, 689:; A and 32 '71, B: A = [1.1 M ammonium acetate: B = 60"1, C‘HJOHHJU 9:, H10
`
`617
`
` Reaction Acceptor t Monomer IIPLC‘. of the reaction mixture
`
`
`
`
`
`
`
`Identified by fingerprint
`
`Ill
`
`[V
`
`V
`
`V]
`
`th-A-A-G}+dTTP
`
`d1A—A—A—G}+ dC'l'P
`
`dttj—A—A—A)—t—d'l'TP
`
`d((}-A-A-AJ+dCTP
`
`V! H
`
`d(A-A-A-C) + dATP
`
`IX
`
`th-A-A-C-AH—[a—“PJdTTP
`
`peak
`
`Fig. 2
`
`retention
`time
`
`yield
`
`Fig. 3
`
`sequence
`
`2
`3
`
`4
`5
`2
`6
`
`7
`8
`0
`
`10
`7"
`11
`9
`
`12
`16
`l3
`14
`15
`
`13
`17"
`18
`
`b
`b
`
`c
`c
`c
`c
`
`cl
`ti
`d
`
`e
`e
`c
`c
`
`1‘
`f
`1'
`t'
`f'
`
`g
`g
`g
`
`lrlin
`22.7
`31.8
`
`14.7Ir
`20.5
`22.7
`32.0
`
`21.8
`2‘). 8
`36.0
`
`19.0
`21.15
`24.8
`36.0
`
`26.8
`55.0
`62.1
`81.0
`95.0
`
`29.3
`40.3
`78 .5
`
`"1-;
`68.5
`21.?
`
`26.0
`20.?
`47".0
`4.0
`
`51.4
`31.]
`12.0
`
`25.8
`45.8
`13.8
`3.9
`
`58.1
`2.9
`21.4
`8.7"
`2.1
`
`58.3
`31.9
`9.4
`
`a
`b
`
`c
`d
`a
`e
`
`F
`g
`—
`
`h
`i‘
`i
`—
`
`j
`k
`|
`m
`n
`
`|
`o
`p
`
`d(A—A—A—G}
`th—A-A—G—T)
`
`th-A-A-G-C)
`th-A-A-G-C)
`th-A-A-G]
`d(A-A-A-G-C-C-C}
`
`dtG—A-A—A)
`d(G—A-A—A—T)
`th-A—A)
`
`d(G-A-A—A-C'J
`dtG-A-A-A]
`d(G-A-A-A-C-CJ
`th-A~A)
`
`th-A—A—C)
`th-A—C-A)
`d(A-A-A-C—Al
`th-A-A-C-A-A}
`th-A-A-C-A A-A)
`
`th—A—A—C—A}
`th-A-A-C—A[“2P]pT)
`d(A -.’\-/\-C—/\[3 2I’]p'l‘[3 JI’]pT)
`
`acceptor d(A-A-A-G), a corresponding degradation product
`could not be detected (Table 2}.
`The desired mono-addition product was obtained in yields
`between 2091, and 300;, in all four reactions. Moreover, the
`unchanged acceptor could be recovered in chromatographi—
`cally pure form and could be used again for another elongation
`reaction. Using this possibility,
`the yield of the enzymatic
`elongation could be substantially increased.
`
`Elongation of segueiit'tJ-iis'omert'c- p_i-'ri'mrdt'm' nonmmt'i'eost'dc
`or:Mkimprints-photos
`
`In reaction VII. the mixture of sequence-isomeric pyrim—
`idine nonanucleoside octakisphosphates. d(C4, T5}. isolated
`From partially hydrolysed dcpurinated herring sperm DNA
`[13]. was elongated enzymatically with [8—3 H]dGTP under the
`conditions given in Table I by a single dCi residue. The acceptor
`was used in about 20 '7}, molar excess with respect
`to the
`monomers.
`l U of terminal deoxynucleotide transferase was
`applied for 2 nmol monomer in contrast to the proceeding
`reactions. i.e. only half as much enzyme was used with respect
`to the monomer. Again the reaction was stopped after 4h by
`addition of alkaline phosphatase. which dephosphorylates
`[8—"‘H]dGTP to [8-3H]dG.
`The reaction mixture could not be separated by [IPLC
`however. because the sequence isomers are partially separated
`by chromatography on Nucleosil C13 and it
`is not generally
`possible to separate the elongated products from the original
`acceptors. Therefore the separation was performed by paper
`chromatography in two different systems. First the mixture was
`chromatographed in the solvent system A where the nucleoside
`
`[8-3ll]dG runs within the front, whereas the unconverted
`sequence isomers and their elongated products remain at the
`origin. To determine the yield of the reaction. a strip 01' the
`paper chromatogram, containing an aliquot of the reaction
`mixture, was first cut in the direction of the run. and then cut
`perpendicularly into strips of 2cm width. Then the radioac—
`tivity of the strips was determined. Usually about 10".; of the
`applied radioactivity was located in the region ofthe elongated
`sequence isomers whereas about 90 f5; was recovered as
`[3-3ll]dG. For preparative isolation ot‘the elongated sequence
`isomers, first
`the upper region of the paper chromatogram
`containing [8-"H]dG was cut off. The paper chromatogram was
`then chromatographed in solvent system B in which the
`unchanged sequence—isomeric nonanucleoside octakisphos-
`phates Form a single spot. This spot moves faster than the
`mixture 01‘ the nucleotides elongated by one dG residue which
`also migrate as one spot. The positions of the unchanged
`oligonucleoside phosphates could be detected by their ul—
`traviolet absorption. whereas all the elongated sequence iso-
`mers were detected by their radioactivity (Fig. 4}. Only one
`radioactive spot appeared in the paper chromatogram which
`contained the monoaddition products ofthe sequence-isomeric
`oligonucleosidc phosphates d(C4,T5—[8—5H]G). It can therefore
`be concluded that only one dG residue was condensed onto the
`sequence isomers.
`
`Stepwise (Itoitgarion oft: rcrrmiuct’tlosfde trisphosphatc
`to rt hm‘antrt’teosidc pctitakrsphosplmre
`
`1n the two quantitatively evaluated examples. reactions
`VIII and [X (Table 2}. the synthetic tetranucleoside trisphos—
`
`CU REVAC EX2040
`CUREVAC EX2040
`Page 5
`Page 5
`
`

`

`24‘D
`
`E
`"if
`22
`I—‘f‘
`
`0
`
`30
`21]
`10
`MBRATlON [ em l
`
`Fig. 4. Papar—t'iiromatographit' separation attire reaction mixture {H C4,
`T5} + {S—AdeGTP catalysed by terminal tiaoljt-‘mtcieotidy! traits-
`ferasc.
`Thc paper was
`chromatographcd first
`in
`system A
`—- ethanol}! M ammonium acetate pH ?.5 [7: 3, viv) and after drying
`in system B = l—propanoii'NIhOI-l conc.twatcr (55:10:35: vltvtv).
`(a) Migration of d(C4, T5) detected by ultraviolet absorption: (b)
`migration of d{C4‘ 'I'S—[8—3H]G) detected by the radioactivity
`
`impurities ot‘ the chemically synthesised acceptor. Peak 13
`contained the desired monoaddition product d(A-A-A-C-A].
`The yield (a: 21 ‘3’”) was about twice as high as the one obtained
`by the elongation of the sequence isomers although less enzyme
`was applied. The peaks 14 and 15 containcd thc diaddition
`product th—A—A—C—A—A) and the triaddition product d{A—A-
`A—C—A—A—A), respectively, in yields 01‘9'5;J and 2"1]. in peak 16
`the tetranucleosidc trisphosphate d(A—/-\—C-A) was eluted,
`which was formed from the cl on gated acceptor th—A-A-C-A)
`by loss of a 5’-terminal adenylic acid residue. The degradation
`must have taken place after the elongation because a degraded
`acceptor, th-A-C‘), could not be elongated. This finding is
`remarkable because prior to [he elongation the acceptor, d{G-
`A—A—A], was partially degraded to d(A-A-A), These results
`indicate different kinetics which are presumably influenced by
`the sequence of the acceptor.
`in the second step (IX) 5 nrnol ofthc isolated monoaddition
`product d(A-A-A-C—A) was used as acceptor which was
`converted with 3.3 nmol [a—“PJdTTP and 3 U terminal dc-
`oxynucleotidyl transferase during 3.5 h. After the reaction had
`been interrupted by addition of alkaline phosphatase, which
`dephosphorylated [a—“PldTTP. thc crude mixture was sepa-
`rated likewise by HPLC on Nuclcosil C”; (Fig. 2g). To detect
`the radioactivity,
`in addition to the ultraviolet detector, a
`radioactivity measuring detector was used and both the
`ultraviolet absorbance and the radioactivity of the eluatc were
`monitored. For practical rcasons,
`the corresponding peaks
`were shifted to a minor extent. The interpretation ofthe elution
`profile was simplified considerably. because now all elongated
`products were labelled radioactively whereas the unlabelled
`acceptor showed only ultraviolet absorbance.
`The degradation products of the monomer were eluted in
`pck as 1 and 1c, which were formed by the action ofthc alkaline
`
`CU REVAC EX2040
`CUREVAC EX2040
`Page 6
`Page 6
`
`618
`
`at clan-two] or
`
`I
`
`b] on awe—n 13:
`
`o
`O
`
`_ i
`
`s
`
`o
`
`'
`
`‘_ .
`
`I
`
`at
`
`dlA—A—ArG-C-Cl
`
`[5]
`
`El dln-A-A—GJZ-C—CI
`
`{El
`
`II
`
`le—A—A—Al
`
`l7l
`
`.'
`
`,.
`
`Q
`
`O
`
`o
`
`9
`le—A—A—A—TI 15!
`
`g!
`
`hi
`
`C
`.
`OI
`‘
`dtG—A—A—Aucl
`
`I
`di‘G—A—A—A—(‘rcl
`
`ii
`
`1111
`
`[10!
`
`a
`I
`
`.
`
`ii
`
`th—A—A—Cl
`
`[12l
`
`kl
`
`dlA—A—C—Al
`
`115:
`
`ll dM—A—A—C-Al
`
`I13]
`
`0
`
`O
`
`'.'
`
`"
`
`‘
`
`ml dlA—A—A-CvA AI
`
`11¢]
`
`nl CllA A—A—C—A—A-A}
`
`[15]
`
`o]
`
`dln—A—«l—C—A—TI
`
`th’l
`
` . - o
`
`)_
`s
`
`gE
`8
`é
`N
`
`'.
`
`ELECIRDPt-imEEAS
`
`o
`'
`o
`c
`I
`
`.
`C
`I
`
`{18}
`O
`
`pl
`
`dlA—A—A—C-AF‘I‘T}
`
`.
`
`C O
`
`D
`
`Fig. 3. Determination of the sequences of titigoimctcosidc phosphates
`isolated by reversed-phase HPLC { Pit-{.2} after the an:__rittatit' rim;—
`gation of tiifli'rent acceptors rising terminal dear}'mart'eotidi-‘I trans—
`fer-axe. The isolated products were first labelled with 32P and then
`partially hydrolysed with snake venom phosphodicstcrasc. The result-
`ing partial hydrolysales were two—dimensionally chromatographed.
`The first dimension consisted of an electrophoretic separation on
`ccllullose acetate strips at pll 3.5, the second a homochromatography
`on DEAE-ccllulosc thin-layer plates. (a — p) The resulting auloradio—
`grams. The numbers following the determined sequence of the
`fingerprints correspond to the peak numbers of Fig, 2
`
`phate d(A-A-A-C) was converted (according to the conditions
`in 'l'ablel) enzymatically into the hexanucleOSide pentakis-
`phOSphate th-A-A-C-APZPmT} in two steps on a preparative
`scale.
`in the first step {VIII}. 80 nmol of the acceptor (HA-A-
`A-C‘] were treated with 60 nmol dATP and ZDU terminal
`deoxynucleotidyl transferase. After 4 h. the reaction was stop-
`ped by addition of alkaline phosphatase and the reaction
`mixture was separated by reverse-phase HPl.C on Nucleosil
`Cm (Fig. 2f}. The fractions of the numbered peaks were
`lyophiliscd and identified by the fingerprint method. These
`fingerprints are shown in Fig. 3. The quantitative evaluation of
`the reaction based on the comparison of the different peak
`areas is summarised in Table 2. About 58 3;, of the starting
`acceptor th—A—A—C} remained unchanged and was eluted in
`peak 12 of I‘ig. 2f. Both side peaks of peak l2 are probably
`
`

`

`phosphatase. Peak 1 contains dT and peak tc inorganic
`phosphate. The unchanged acceptor th-A-A-C-A) could be
`recovered from peak 13 and amounted to about 58 “1, of the
`reaction mixture. Peak [7 contained the desired elongation
`product
`th—A—A—C—AI”P]pT) with a yield of 329;. The
`diaddition product th—A—A—(T—M”P]p"l'["2P]pT) was found
`under peak 18 which amounted to 9 1’1, ofthc reaction mixture.
`The sequences ofthe isolated oligonuclcotidc phosphates were
`confirmed by the fingerprint method (Fig.3). Peak 17 was
`isolated and lyophilized and a white powder was obtained. It is
`remarkable that. in contrast to the first step of the synthesis,
`after the second step no degraded oligonuclcosidc phosphates
`were found.
`
`DISCUSSION
`
`The results show that oligodeoxyribonucleotides can be
`elongated enzymatically using terminal deoxynuclcotidyl
`transferase. Acoeptors must have at least four monomer units
`and be obtainable by chemical synthesis or by isolation from
`partial hydrolysates of chemically degraded DNA. The purity
`of the commercially available transferasc is fully sufficient for
`this purpose. The addition of purine nucleotides to a pyri-
`midine acceptor and vice versa is of special
`interest. since
`acceptors with both purine and pyrimidine monomer units are
`not yet available from partial hydrolysates of chemically
`degraded DNA.
`The reaction mixtures, which contain 2~80 nmol of aceep-
`tors yield 20— 302; of monoaddition products. Remarkably,
`the reaction is not affected by the chain length ofthe aceeptors
`used. which have four, five or nine monomer units in the
`reported examples. The yield can be increased by recycling the
`unchanged acceptors. which can be recovered easily in chro-
`matographic-ally pure form.
`The reaction mixtures are separated by different chroma-
`tographic procedures. of which thin—layer homochromatog—
`raphy on DEAF. cellulose sheets is most suitable for analytic
`purposes. Very small quantities of addition products are
`detectable when radioactively labelled monomers are used in
`the elongation reaction,
`larger quantities of products are
`separated by paper chromatography or IIPLC.
`The examples described show HPLC separations using
`reverse—phase chromatography on Nucleosil C18. The elution
`profiles indicate that the capacity ofthe analytical column {250
`x 4.6mm internal diameter) used is not yet fully utilized and
`that even larger batches could be separated in one run.
`In
`comparison to anion-exchange chromatography. which can
`also be used for the separation ofthe reaction mixtures, reverse-
`phase
`chromatography
`has
`the
`following advantages.
`Separations can be carried out with a volatile buffer system:
`therefore,
`the products can be isolated without a dcsalting
`procedure. By-products having the same net charge as the
`desired elongation product can also be removed by reverse-
`phase but not by anion-exchange chromatography. On the
`other hand, the elution profile of the reaction mixture is not
`determined by the net charge of
`the oligonucleotides.
`Normally, the highest peak ofthe newly appearing peaks is the
`monoaddition product. The correct identification, however,
`has to be performed by the fingerprint method.
`The reaction mixtures [except
`th-A-A-G) + dTTP]
`contained not only the mono— but also di- and triaddition
`products although the acceptor concentration was higher than
`that of the monomer.
`In two reaction mixtures. dilTerent
`
`619
`
`degraded products were found. The sequence of the oligonu—
`cleotide acceptor is probably important in determining if and
`when a 5’—nucleotide is enzymatically removed. For example.
`th—A—A—A—C) looses its 5’ monomer unit after elongation at
`the 3’ terminus. but dtG-A-A-A) looses its 5’-nucleotide before
`enzymatic elongation. The different composition ofa reaction
`mixture shOWs also that the condensation catalysed by terminal
`deoxynuclcotidyl
`transferase is influenced by the acceptor
`sequence and the monomer unit to be added to it. Diaddition
`products, which are found in the examined reaction mixtures
`up to 21 '31,. can be very useful in cases where the oligonuclcotide
`desired contains homologous segments. Polyaddition can be
`favoured by varying the reaction conditions, so that in a single
`reaction cycle two or three monomer units may be added to a
`given acceptor.
`The elongation of acceptors with monomer units as de-
`scribed here may be regarded as a new way to the stepwise
`enzymatic synthesis of defined oligonucleotides. The time
`needed for one condensation cycle, including the isolation of
`the reaction products,
`is about 5h. A comparison with
`automated chemical synthesis of defined oligonucleotides
`shows that the machines can do a single addition step in a
`shorter time. However. the resulting reaction product is less
`well defined and still carries protecting groups which have to be
`removed. The limiting factor in both procedures ofsynthesis is
`probably the separation of reaction products, which will be
`more and more difficult with increasing chain length. An
`important advantage ofthe enzymatic synthesis is the isolation
`of a pure, well defined product after each reaction cycle. In
`contrast,
`the product of an automated chemical synthesis is
`identified, without internal feed-back. only at the end of the
`synthesis. The difficulties in an isolation of the desired oligo-
`nucleotide are therefore considerably greater when the chemi