Total synthesis of a tyrosine suppressor transfer RNA gene. XVII. Transcription, in vitro, of the synthetic gene and processing of the primary transcript to transfer RNA.

Primer- and promoter-dependent transcription of the synthesis gene had been studied. Primer-dependent transcription gave, as a major product, an end-to-end transcript which was strand-specific. The transcript was characterized rigorously by two-dimensional separation and analysis of the oligonucleotides formed on digestion with T1-RNase and pancreatic RNase and by nearest neighbor analyses of the oligonucleotides obtained when different alpha-32P-labeled ribonucleoside triphosphates were used as substrates. Minor products accompanying the major transcript were characterized similarly. The major transcript, when treated with an Escherichia coli S-100 extract, was processed to the tRNATyr with correct 5'- and 3'-ends. The nucleolytic cleavages occurring at the 3'-end were characterized. In promoter-dependent transcription, transcription of a restriction fragment containing phi80psu+III gene and the synthetic gene with and without the promoter were compared. Transcription of the synthetic gene was promoter-dependent and strand-specific, the initiation of transcription occurring at the same point as previously found in vivo. Although the synthetic gene contains only 16 base pairs corresponding to the natural sequence following the C-C-A end, processing of the transcript at the 3'-end occurred normally, the endonucleolytic cleavage being followed by exonucleolytic cleavages. The products of promoter-dependent transcription were completely characterized. An examination of the base modifications of the primary transcript during treatment of the latter with E. coli S-100 extract showed couplete modification of uridine to pseudouridine and partial methylation of uridine to ribosylthymine in TpsiCG sequence and partial formation of pseudouridine in the anticodon loop. However, hardly any formation of 2'-O-methylguanosine or of 2-methylthio-6-isopentenyl adenosine could be detected.


Chemical syntheses of the two dodecanticleotides d(T-C-A-A-C-G-T-A-A-C-A-C)andd(A-C-G-T-T-G-A-G-A-A-A-G), the two undecanucleotides d(T-T-T-A-C-A-G-C-G-G-C) and d(T-G-T-A-A-
A noteworthy feature of the present syntheses was the use of reverse phase high pressure liquid chromatography for the rapid and efficient separation of synthetic reaction mixtures. In the preceding paper (l), a plan was described for synthetic work in the promoter region of the Escherichia tyrosine suppressor tRNA gene. The DNA duplex (Fig. 1) to be synthesized included the first 51 nucleotides in the promoter region and the EcoRI restriction enzyme sequence at the appropriate 5'-end. As shown in Fig. 1, a total of 10 deoxyribo-oligonucleotide segments were to be synthesized and of these, syntheses of Segments P-l to P-5 were documented in the preceding paper (1). The present paper describes the synthesis of the Segments P-6 to P-10 corresponding to the nucleotide sequence -30 to -56, thus completing the chemical work necessary for the total synthesis of the promoter region.
The present paper also describes a modification of the previously synthesized 23-unit-long DNA corresponding to the sequence adjoining the C-C-A end of the tyrosine tRNA gene. The previously synthesized DNA and the segments which were used for its synthesis (2) are shown in Fig. 2A. The modified duplex which now forms the terminus distal to the promoter region is shown in Fig. 2B. The decision to add the latter duplex to the C-C-A end of the structural gene previously synthesized (3) was arrived at from the following considerations.
Previous studies with the E. coli tyrosine suppressor tRNA gene, which has formed the topic of present investigations, have been carried out mainly after its integration into the transducing bacteriophage $30. By transduction, the "doublet" strain (diagram, Fig. 3A) which contains the suppressor tRNA,"'" (su+) and the suppressor-negative tRNA1""' (sum) genes in tandem has been isolated. Unequal recombination between the two tRNA,"'" genes in this strain gives rise to a "singlet" strain $3Ops~& (diagram, Fig. 3B), in which only one of the two genes is present (4). Determination of the nucleotide sequences in the promoter region and at the ends adjoining the C-C-A sequence of the tRNA"'" genes using the doublet and the singlet strains gave the results which are shown in Fig. 3 (5). Work on the in vivo and in vitro transcription of the above genes shows that termination of transcription does not occur either at or near Sequence 3 or the region between the tandem tyrosine tRNA genes.' Therefore, the primary transcript of the tyrosine tRNA genes must undergo processing and, clearly, the tRNA precursor discovered by Altman and Smith (6) must have undergone processing at the 3'-end prior to isolation. In the present goal of total synthesis, it was a prerequisite to determine the length of the DNA region adjoining the C-C-A end which would contain the required processing signal. Therefore, transcription experi- ments were carried out, in uitro, with the synthetic structural gene (3) to which the 23-nucleotide-long DNA ( Fig. 2A) had been joined (7). This work showed that the processing signal for nucleolytic cleavages downstream from the C-C-A sequence is indeed contained within the relatively short synthetic DNA shown in Fig. 2. Further sequence work at the 3'end was therefore unnecessary from the standpoint of the synthesis of a biologically functional gene.
With the previously synthesized Segments 25 to 29 ( Fig.  24 in hand, the main concern was to add the appropriate EcoRI restriction enzyme sequence at the terminus with minimal additional synthetic work. Clearly, the simplest possibility was to replace Segment 29 with d (A-A-T-T-C-T-T-T-C), which contains the required recognition sequence at the 5'-end and which had already been synthesized as the terminal segment (Segment P-10) for the promoter duplex (Fig. 1). Thus, in the modified plan shown in Fig. 28 the only additional synthetic work involved modification of the original Segment 28 ( Fig. 2A) to Segment 28'. Since the protected pentanucleotide, d[ (MeOTr)bzA -mbG -T -anC -anC], was available from previous work, completion of the work involved only the two steps shown in Chart 6.
During the progress of the present work, the use of reverse phase high pressure liquid chromatography was being developed concurrently (8) and a distinctive and practically important feature of the syntheses herein described has been the use of this technique for the rapid and efficient separation of synthetic oligonucleotide reaction mixtures.   compounds. Therefore, a portion of the crude hexanucleotide was further purified by preparative hplc (Fig. 4B) (see "Materials and Methods" for description of the preparative hplc system).
The pure protected hexanucleotide was then condensed successively with the dinucleotide d[pibG-anC(Ac)] and the trinucleotide d[pibG-ibG-anC(Ac)] to give the octanucleotide and the undecanucleotide, respectively. The product after each condensation was isolated by preparative hplc, the separation patterns being shown in Figs. 5 and 6. The deprotected undecanucleotide without purification was phosphorylated by polynucleotide kinase using [Y-""PIATP. Purity and sequence of the resulting preparation was confirmed by the two-dimensional fingerprint (Fig. 7) of a partial snake venom phosphodiesterase digest of the labeled undecanucleotide (9).
It should be noted that the preparative purification of the  has been described previously (10). For this work, it was further purified by preparative hplc (Fig. 8). Synthesis of Segment  Synthesis of Tyrosine Suppressor tRNA Gene phase column (Fig. lo), but it could not be completely freed from the unreacted pentanucleotide.
Since the next step required the addition of the same dinucleotide, d[pibG-bzA(Ac)], to the heptanucleotide, addition of the dinucleotide to the contaminating pentanucleotide would give the same heptanucleotide and, therefore, no new unwanted product would be produced. Hence, the contaminated heptanucleotide was used directly in the next condensation with the protected dinucleotide d[pibG-bzA(Ac)]. The resulting nonanucleotide, was purified by anion exchange chromatography and the fractions which contained only the nonanucleotide as shown by hplc were pooled.
The nonanucleotide was finally condensed with the trinucleotide d[pbzA-bzA-ibG(Ac)] to give the protected dodecanucleotide.
The product along with the unreacted nonanucleotide was separated from the excess of trinucleotide and its pyrophosphate by preparative hplc (Fig. 11). After complete Steps in the synthesis Fig. 1). as described under "Experimental Section." A final purification of this trinucleotide was achieved by hplc (Fig. 14).

Synthesis of Segment P-7, d(T-G-T-A-A-A-G-T-G-T-T)-
The steps used in this synthesis are shown in Chart 3.
and the latter was next condensed with d[pT(Ac)].
The protected di-and trinuclectides were isolated rapidly by solvent extraction. The The reaction products were fractionated with gradients of acetonitrile in 0.  finally condensed with the tetranucleotide d[pT-ibG-T-T(Ac)] to give the protected undecanucleotide, which was isolated by DEAE-cellulose anion exchange chromatography (Fig. 17). The tetranucleotide used was prepared by conden- with d[pT-T(Ac)]. The undecanucleotide, after removal of the protecting groups, was finally chromatographed on an anion exchanger column in the presence of 7 M urea. The pattern obtained is shown in Fig. 18. The material pooled as shown was further characterized by the two-dimensional fingerprinting method following phosphorylation of the Y-OH group by polynucleotide kinase using [y-,"P]ATP (Fig. 19).

SynthesisofSegmentP-8,d(T-C-A-A-C-G-T-A-A-C-A-C)-
The plan for the synthesis of this dodecanucleotide is shown in The product after each of these condensations phy (Figs. 20 and 21). The octanucleotide was finally condensed with the tetranucleotide d[pbzA-anC-bzA-anC(Ac)] to give the desired protected dodecanucleotide.
Because of the lipophilic 5'-0-monomethoxytrityl group on the 5'-termini of the octa-and the dodecanucleotides, these oligonucleotides were conveniently separated from the tetranucleotide block and its pyrophosphate by preparative hplc. However, it wtiis not possible to separate the octa-and dodecanucleotides on this column. This mixture was fully deprotected and the required dodecanucleotide, d(T-C-A-A-C-G-T-A-A-C-A-C), was isolated by DE-52 column chromatography in the presence of 7 M urea (Fig. 22) and characterized by the twodimensional fingerprinting method (Fig. 23). The tetranucleotide d[pbzA-anC-bzA-anC(Ac)] which was purified by anion exchange chromatography (Fig. 24). Condensation of the pentanucleotide with d[pT-T(Ac)] gave the required heptanucleotide. This product was purified by hplc using the preparative reverse phase column (Fig. 25). Because of the small lipophilic contribution of the thymine residues relative to the standard hydrophilic contribution of the two-phosphate dissociations of the added dinucleotide d[pT-T(Ac)] there was a relatively large difference in retention time between the product and unreacted starting material. This permitted isolatfod of the heptanucleotide completely free of the pentanucleotide. The heptanucleotide was finally condensed with d[pT-anC(Ac)], and the nonanucleotide was isolated by anion exchange chromatography (Fig. 26). It was further purified after complete deprotection by chromatography in the presence of 7 M urea (Fig. 27). Analysis by the fingerprinting

28) after ['2P]phosphorylation
of the 5'-OH group and by hplc using an analytical column showed the product to be pure.

Synthesis of Segment 28', d(A-G-T-C-C-G-A-A-A-G)-Starting with the pentanucleotide, d[ (MeOTr)bzA-mbG-T-anC-anC]
(2), the decanucleotide was synthesized as shown in Chart 6. Thus, condensation with the dinucleotide d[pibG-bzA(Ac)] gave the corresponding heptanucleotide, which was isolated by anion exchange chromatography on DEAE-cellulose (Fig. 29). The heptanucleotide was then condensed with the trinucleotide d[pbzA-bzA-ibG(Ac)], which was also available from the synthesis of Segment P-9, described above, to give the desired protected decanucleotide.
The product was purified by anion exchange chromatography using DEAEcellulose (Fig. 30). In both of the above condensations, appropriate fractions were pooled after checking them by hplc on the analytical column.
Characterization of Synthetic Deoxyribo-oligonucleotides-As described, the nucleotide sequences in the final synthetic compounds were confirmed by two-dimensional fingerprinting of the partial digests obtained upon degradation with snake venom phosphodiesterase.
The other methods used for characterization of several protected and unprotected deoxyribo-oligonucleotides are described in earlier papers (lo-13). In the present work they were analyzed by reverse phase high pressure liquid chromatography on the analytical hplc  column. This method could be used in all phases of oligonucleotide synthesis and was especially suitable for detecting any loss of the amino protecting groups. The details of these procedures have been described elsewhere (8). Concluding Remarks on Synthetic Procedures-Chemical synthesis of deoxyribonucleotides corresponding to the entire two strands constitutes the first and the most demanding phase of total synthesis of a DNA. The separation, purification, and characterization of the synthetic intermediates and of the final products are the most time-consuming aspects. The introduction of hydrophobic protecting groups permitted the use of solvent extraction procedures and this general technique is now used routinely for the isolation of protected short oligonucleotides (1,10,14). However, a main feature of the present work has been the use of reverse phase high pressure liquid chromatography in rapid and highly efficient separations of the synthetic intermediates.
While the development of the method as an analytical tool in synthetic work has been described elsewhere (8) components were separated in pure form in less than 3 h (Fig.  6A). A similar separation on an anion exchanger column would have taken a number of days. However, separations of the longest condensation products, e.g. undeca-and dodecanucleotides, from the starting methoxytritylated oligonucleotides were not always so successful by preparative hplc using the current protecting groups. The introduction of the strongly hydrophobic t-butyldiphenylsilyl group (14) as the protecting group for 3'-OH group in place of the classical 3'-0-acetyl group has provided the solution to the above problem. The resulting elongated chain has much more lipophilic character than the starting oligonucleotide with the result that these two classes of compounds are well resolved by preparative hplc.
In addition to reducing the time required for separation of oligonucleotides, the preparative hplc method has higher resolving power than anion exchange chromatography in separating the trityl-containing components of reaction mixtures. As was noted for Segment P-6 and has been observed for the other protected oligonucleotide end products purified by the reverse phase system,4 the deprotected analog is often totally free of impurities and does not require any additional purifi-' Ref. 14 and R. Belagaje, unpublished work cation. In the past the synthetic products that were isolated by anion exchange chromatography routinely were further purified, after deprotecting, on a DEAE-cellulose column in the presence of 7 M urea. Even then, the oligonucleotide products often contained impurities, and in order to be used in the polynucleotide ligase-catalyzed joining reactions they had to be further purified by hplc.

EXPERIMENTAL SECTION:'
All materials and methods used for the synthesis and isolation of oligonucleotides by anion exchange chromatography have been described in earlier papers (10)(11)(12)(13). Reverse phase high pressure liquid chromatography (hplc) was performed on a system consisting of the following components available from Waters Associates: two M6000A solvent delivery systems, a 660 solvent programmer, a U6K injector, a 440 UV detector operating at wavelengths of 254 and 280 nm, a PBondapak Clx column (0.4 x 30 cm), and a Houston Instruments Omniscribe TM chart recorder. For preparative separations, the column and detector of the above system were replaced with a PBondapak C&Porasil B column (0.7 x 183 cm) and an Altex model 151 UV detector equipped with a preparative flow cell operating at a wavelength of 254 or 280 nm. Pumps and solvent programmer were operated in such a way that one pump delivered aqueous buffer and the other acetonitrile.
The resulting eluent is a mixture (volume/volume) of the two components which, for convenience, is expressed as a percentage of acetonitrile in 0.1 M ammonium acetate or 0.1 M TEAA. Other materials and methods used for hplc have been described earlier (8).
Whenever hplc was used for the separation of a condensation reaction mixture, the following general procedure was adopted. After quenching the condensation reaction mixture with water and DIEA, the mixture was evaporated to a gum and taken up in 0.2 M TEAB as usual. Non-nucleotidic components were partially removed by four manual extractions, two using ethyl acetate and two using ethyl acetate with 5 to 10% of 1-butanol. The contents of the aqueous phase were isolated by evaporation with pyridine and precipitation with dry ether. At this stage, two samples (100 to 300 pg each) were taken from the precipitate, one was subjected to deacetylation with 1 N sodium hydroxide in the usual manner. The two samples were compared by hplc on the PBondapak C,, column using conditions that give good resolution of the tritylated oligonucleotides (30 to 35% acetonitrile in 0.1 M ammonium acetate) (8). In most cases, the product peak was easily identified by its shift to shorter retention time after alkaline treatment.
The main sample, precipitated as above, was dissolved in TEAA/ethanol (l:l, v/v) and centrifuged in an IEC clinical centrifuge for about 10 min to spin down all insoluble material. This solution was kept in an ice bath and applied to the preparative column as soon as possible.
For preparative separations, the PBondapak CJPorasil B column" was used with a flow rate of 9.9 ml/min. A column (0.2 x 61 cm) packed with the same support was employed to define the following conditions needed for the preparative separation: (a) a concentration (A) of acetonitrile in 0.1 M TEAA that elutes polar impurities and pyridine reasonably fast but strongly retains the acetylated di-, tri-, or tetranucleotide on the column; (b) a concentration (B) of acetonitrile that elutes the oligonucleotide but keeps the tritylated material on the column; (c) finally, a concentration (C) that elutes the mixture of tritylated compounds. In general, two linear gradients (A to B and B to C) were used to fractionate the precipitated reaction mixture on the preparative column. After that, a step gradient to 20% more acetonitrile was applied and the column washed for 10 min.
When the fractions from preparative hplc were concentrated, special attention was paid to ensure that a sufficient amount of pyridine was present at all times during evaporation of solvents.

Protected Dinucleotides
Carrying 5'-Phosphate Groups All of the protected dinucleotides carrying 5'-phosphate groups used in this work were prepared by using the TPSEprotecting group, as described earlier (10). In a few instances, the compounds were further purified by hplc.
The Trinucleotide, d@ibG-ibG-anC) The synthesis of this trinucleotide has been described (10). For the present work, the acetylated trinucleotide was further purified by preparative hplc (Fig. 8 , was extracted into dichloromethane/l-butanol (9:1, v/v, two times). These extracts were combined and concentrated in uacuo, evaporated several times with pyridine and finally dissolved in pyridine/ethanol/water (4:3:3, v/v, 100 ml). This solution was cooled to 0°C and 100 ml of precooled 2 N sodium hydroxide was added. After 5 min at 0°C pyridinium Dowex 50-X8 was added. The resulting mixture was filtered through a column of fresh Dowex. The eluant was concentrated in uacuo, extracted with diethyl ether (three times) to remove the elimination product of the TPSE protecting group, concentrated, and then precipitated into ether. The crude trinucleotide, d(pbzA-bzA-ibG), (2.07 g, 1.32 mmol) was obtained in 46% yield. The precipitated material was dissolved in anhydrous pyridine (40 ml) and acetylated by the standard procedure. The crude product was isolated as a dry powder (2.44 g) and subjected to preparative hplc (six injections, approximately 410 mg for each injection). The tracing from one injection is shown in Fig. 14A. The effluent corresponding to Peak I contained the trinucleotide.
After concentration and precipitation, pure d[pbzA-bzA-ibG(Ac)] (0.5 mmol) was isolated in 18% overall yield. The purity of this product was checked by hplc analysis (Fig. 14B). (2.2 g, 5.0 mmol) were allowed to stand at room temperature in the presence of TPS (2.88 g, 9.5 mmol) in dry pyridine (20 ml) for 5% h. After the usual work-up and alkaline hydrolysis, the solution was concentrated to a gum and 200 ml of 0.2 M TEAB was added and extracted continuously with diisopropyl ether at 4°C overnight. The aqueous phase was extracted with l-butanol/dichloromethane (1:9, 5 x 250 ml). The organic phases were combined and evaporated to an anhydrous pyridine solution (30 ml). The desired product was then precipitated by dropwise addition of this solution to an excess of anhydrous ether (1600 ml). Yield of the dinucleotide was 91% (2.06 mmol). The product was homogeneous on tic in Solvents K and Q. The UV data of the protected and unprotected compounds are given in Table I (2.85 g, 5.7 mmol) were allowed to stand at room temperature in the presence of TPS (4.09 g, 13.5 mmol) in pyridine (15 ml) for 5% h. After the usual work-up and alkaline hydrolysis, the solution was concentrated to 40 ml of pyridine solution and then diluted to 250 ml with 0.2 M TEAB. The solution was extracted continuously at 4°C overnight with ethyl acetate. The aqueous layer was evaporated to a pyridine solution (30 ml) and diluted to 850 ml with 0.02 M Tris-HCl (pH 7.5), containing 5% ethanol. This was applied to a DEAE-cellulose column (column pattern not shown). The fractions which contained the desired tetranucleotide were pooled and desalted by membrane filtration using an Amicon 2000 apparatus fitted with a UM05 membrane. The retentate (approximately 200 ml) was passed slowly through a column of pyridinium Dowex 50-X8 (50 ml) in the presence of 20% pyridine. The eluate was evaporated to an anhydrous pyridine solution (30 ml) and product precipitated by dropwise addition to anhydrous ether (800 ml). The yield of tetranucleotide was 52% (0.72 mmol). The product showed only traces of impurity on tic in Solvent Q. UV data of the protected and unprotected compounds are given in Table I (1.92 g, 1.6 mmol) were allowed to stand at room temperature in the presence of TPS (1.35 g, 4.47 mmol) in pyridine (7 ml) for 5% h. After the usual work-up and alkaline hydrolysis, the solution was extracted with ethyl acetate (250 ml). The organic layer was backwashed with 20% aqueous pyridine (30 ml). The aqueous layer and backwashings were combined and evaporated to a pyridine solution (20 ml). This was diluted with 250 ml of 0.02 M Tris-HCl (pH 7.5) containing 5% ethanol and applied to a DEAE-cellulose column. The details of chromatography and the elution profile are shown in Fig. 4A. Peak III which contained the desired hexanucleotide was pooled as shown by the dotted lines and desalted by membrane filtration.

Synthesis of d(T-T-T-A-C-
The retentate was made 20% with re-spect to pyridine and passed slowly down a pyridinium Dowex 50-X8 column (100 ml), in the presence of 20% pyridine. The eluant was evaporated to an anhydrous pyridine solution (20 ml) and the product precipitated by dropwise addition to anhydrous ether (1 liter). The yield of hexanucleotide was 46% (0.17 mmol). Analysis of this compound by hplc indicated a significant amount of an impurity.
A portion of the above hexanucleotide was further purified by preparative hplc (Fig. 4B), as described under "Discussion of Methods." Twenty micromoles (1500 A& of pure hexanucleotide was isolated which was homogeneous on hplc as well as on tic in Solvent Q. The UV data of the protected and unprotected compounds are given in Table I (20 pmol, 1500 A& and pyridinium d[pibG-anC(Ac)] (0.2 mmol) was allowed to stand with TPS (0.7 mmol) for 6 h at room temperature.
The reaction was terminated by the standard method using 1.4 ml of 1 M solution of DIEA in pyridine solution followed by addition of water (1.4 ml). After concentrating to a thick gum, 100 ml of 0.2 M TEAB was added and the solution was extracted with a mixture of 1-butanol/ethyl acetate (1:9) (4 x 50 ml). The aqueous phase was evaporated to an anhydrous pyridine solution (10 ml) and precipitated into anhydrous ether by dropwise addition. The mixture, as a dry powder, was dissolved in 4 ml of ethanol 0.1 M TEAA (1:l) and separated by hplc using the preparative reverse phase column (in 2 portions). The details of this chromatography and the elution profile are shown in Fig. 5A. Peak V which contained the desired octanucleotide was collected. The yield of this compound after precipitation was 30% (6 pmol, 620 A&. The product was homogeneous on hplc (Fig. 5B) and on tic in Solvent Q. Its spectral properties are given in Table I. The Undecanucleotide, d[(MeOTr) T-T-T-bzA-anC-bzA-ibG-anC-ibG-ibG-anC]-An anhydrous pyridine solution (1 ml) of the octanucleotide (5 pmol, 530 Azxo and pyridi;iium d[pibG-ibG-anC(Ac)] (114 pmol) was treated with TPS (0.5 mmol) for 5% h at room temperature.
After the usual work-up and termination by the standard method, the reaction mixture was evaporated to a gum and 0.2 M TEAB (100 ml) was added. This solution was extracted with a mixture of lbutanol/ethyl acetate (1:9) (3 x 50 ml). The aqueous layer was evaporated to an anhydrous pyridine solution (10 ml) and the crude product precipitated into ether from its anhydrous pyridine solution. The reaction mixture as dry powder was dissolved in 11.6 ml of ethanol, 0.1 M TEAA (1:l) and separated by hplc using the preparative column as described under "Discussion of Methods." The details of chromatography and the elution profile are shown in Fig. 6A. Peak V which contained the desired undecanucleotide was collected and the product was precipitated into anhydrous ether (500 ml) by dropwise addition of anhydrous pyridine solution (5 ml). The yield was 40% (2 pmol, 300 A,,). The product was homogeneous on hplc, as shown in A portion of the above undecanucleotide was phosphorylated enzymatically and then digested with snake venom phosphodiesterase and analyzed by two-dimensional homochromatography.
The fingerprint (Fig. 7) was consistent with the nucleotide sequence, as expected.