NONENZYMATIC SYNTHESIS OF OLIGOADENYLATES ON A POLYURIDYLIC ACID TEMPLATE* BY J. SULSTON, R. LOHRMANN, L. E. ORGEL, AND H. TODD M\1ILES

SALK INSTITUTE FOR BIOLOGICAL STUDIES, SAN DIEGO, CALIFORNIA, AND NATIONAL INSTITUTES OF HEALTH, BETHESDA, MARYLAND Communicated by Renato Dulbecco, November 24, 1967 A number of nucleosides and mononucleotides have been shown to interact with complementary polynucleotides to form ordered helices of well- defined stoichiometry.1-3 These latter are believed to have structures closely resembling those formed by corresponding pairs of polynucleotides. The condensation of preformed hexathymidylate residues to the dodecadeoxynu- cleotide in the presence of polyadenylic acid has already been reported.4 We shall show that this reaction can be extended to the monomers adenosine and adenylic acid when they are incorporated in an ordered helix with poly U. Experimental and Results.-(a) Physical methods: Paper chromatography was carried out on Whatman 3 MM paper by the descending technique. The solvent systems were: I, isopropanol-concentrated - (7:1:2, v/v); II, n-propanol-concentrated ammonia-water (55:10: 35, v/v); III, saturated aque- ous sulphate-0.1 M sodium acetate-isopropanol (79:19:2, v/v); VIII, n-butanol-5 M (2: 1, v/v). Thin-layer chromatography (system IV) was performed on Brinkmann MN-Polygram Cel 300 PEI, by the ascend- ing technique.5 The sheets (20 X 20 cm) were prewashed with water and air- dried before application of substrate; they were developed for one hour in 0.5 M NaCl, then for two to three hours in 1.0 M NaCl. A paper wick applied to the upper edge allowed the solvent front to overrun. Paper electrophoresis was carried out on Whatman 3 MM paper, using varsol or CCl4 as coolant. The buf- fers were: V, potassium phosphate, 0.03 M, pH 7.1; VI, -ammonium formate, 0.05 M, pH 2.6; VII, sodium borate, 0.05 M, pH 9.0. - Chromatographic and electrophoretic data are collected in Tables 1 and 2. Chromatograms and electrophoretograms of radioactive samples were cut into strips and passed through a Baird-Atomic RSC-363 scanner, to give a graphic record. Relevant areas were cut out and their radioactivities measured more precisely in a liquid scintillation system (Beckman LS-200). The papers were placed directly in Liquifluor diluted with toluene. (b) Materials: Poly U was obtained from Schwarz BioResearch. Before use, it was dialyzed against 0.5 M NaCl, then against distilled water, and finally lyophilized in order to yield the sodium salt, free of small . The prod- uct was checked for oligomers in system II and found to contain none. Nucleo- sides and nucleotides, including those isotopically labeled, were supplied by Schwarz. 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (CDI) was obtained from the Ott Chemical Co. The carbodiimide content of a sample was estimated immediately before use by reaction with a known excess of oxalic acid, followed by back titration with standard potassium permanganate.6 Purity 726 Downloaded by guest on September 25, 2021 VOL. 59, 1968 CHEMISTRY: SULSTON ET AL. 727

TABLE 1. Chromatographic data. Rf rel. Rf rel. .- Rf -- to PA to A Rf I II III IV VIII A 0.60 0.67 1.00 0.39 A31pA 0.35 0.56 0.13 0.82 A2/pA 0.30 0.51 0.32 1.06 A5'pA 0.25 0.47 A3/pA3'pA 0.15 0.40 A31p 0.19 0.45 0.59 A21p 0.19 0.45 0.82 pA 0.15 0.41 1.00 0.07 pA3'pA 0.33 0.28 pA2tpA 0.33 0.35 A51ppA 0.44 0.01 ApA 0.30 0.51 0.32 1.06 (Products from ApApA 0.13 0.39 1.04, 0.90 template synthesis) pApA 0.33 0.35 A5'ppApA 0.33 A5'ppAp 0.25

TABLE 2. Relative electrophoretic mobilities.

V VI VII A 0.00 -0.83 pA +1.00 +0.06 +1.00 Ap +1.04 +0.89 2'-deoxyadenosine 0.00 U 0.00 Up +1.00 A3'pA 4-0.34 -0.39 +0.59 A2'pA +0.39 +0.63 Ar'pA +0.40 +0.83 As'pA3'pA +0.56 -0.18 pA3'pA +1.00 +0.13 pA2IpA +1.07 +0.20 pAp +1.56 +0.89 A5'ppA +0.78 - ApA +0.38 +0.63 ApApA +0.62 -0.14 (Products from template synthesis) pApA +1.07 +0.20 A5'ppApA +0.92 A5'ppAp +1.37 Plus indicates migration towards anode. Minus indicates migration towards cathode.

varied from 80 to 90 per cent; allowance was made for this in calculating the equivalent weight. Samples of A2' pA, pA2' pA, and pA3' pA were generously given by A. M. Michelson. Other adenylate oligoribonucleotides were obtained from Miles Laboratories, and each batch was characterized by chromatography and electro-

phoresis before use. (The conventional use of "...p.." to express a (3' -* 5'), phosphodiester linkage is modified as follows. pA2' pA: 5'-phosphoadenylyl- (2'-- 5')-adenosine; pA: adenosine-5'-phosphate; A3' p: adenosine-3'-phos- phate; A5' p pA: P1, P2-diadenosine-5'-pyrophosphate; etc.) Enzymes were supplied by Worthington Biochemical Corporation except for ribonuclease T2 which was generously provided by G. W. Rushizky. Downloaded by guest on September 25, 2021 728 CHEMISTRY: SULSTON ET AL. T.PROc. N. A. S.

(c) Degradation of products: Spleen phosphodiesterase is an exonuclease specific for (3' -- 5') phosphodiester linkages, and inactive towards (2' - 5') linkages.7 8 It is without effect upon chains bearing a 5' terminal phosphate. Attack on a chain always proceeds from the 5'-OH end, and yields nucleoside-3'-phosphates. A stock solution of the enzyme was prepared by dissolving 16.5 units in 1 ml water. Each digestion was carried out in a mixture containing 1 optical density unit (odu) of substrate, 0.3 M ammonium acetate pH 6.0 (10 ,ul) and enzyme stock solution (5 ,4). After incubation at 370 for 30 minutes, the products were separated in system II or V. Where little digestion occurred, an internal control was used to eliminate the possibility of enzyme inhibition. Thus, it was shown, for example, that although A2' pA was not hydrolyzed, complete digestion of A3' pA3' pA3' pA occurred in its presence. This enzyme preparation contained a contaminant which deaminated adenosine. Venon phosphodiesterase is an exonuclease which digests a nucleic acid chain from the 3'-OH end, yielding nucleoside-5'-phosphates. Both (3'-- 5') and (2' -* 5') phosphodiester linkages are attacked.8 The stock solution contained 5 mg of enzyme in 1 ml water. Digestion was carried out in a mixture containing sub- strate (1 odu), 0.1 M MgCl2 (10 1l), 0.1 M tris-acetate pH 8.8 (10 1l), and enzyme solution (2 ,l). After incubation at 370 for 30 minutes, the products were sepa- rated in system II or V. Alkaline phosphatase (Worthington BAPF) is a highly purified phosphomono- esterase derived from E. coli. The enzyme was supplied as a suspension (1 mg/ ml) in 65 per cent saturated ammonium sulphate. Digestion was carried out in a mixture containing substrate (5 odu, free of inorganic phosphate), 0.1 M tris- HC1 pH 8.2 (40 Ml), and enzyme suspension (20 Ml). After incubation at 370 for 30 minutes, the products were separated in system II. Ribonuclease T2 is an endonuclease specific for (3'-* 5') phosphodiester linkages, and inactive towards (2' -> 5') linkages.9 The stock solution contained 0.4 unit of enzyme in 1 ml of 0.1 M ammonium acetate pH 4.5.10 Digestion was carried out in a mixture containing substrate (1-2 odu) and enzyme-solution- (20 MIl). After incubation at 370 for ca. 15 hours, the products were separated in system V. KOH : Oligoadenylates are exceptionally resistant to alkaline hy- drolysis." Trials showed that A3' pA (1 odu) in 1 M KOH (15 ,M) was completely hydrolyzed in less than 48 hours at 21°C. Under the same conditions, A" pA was unaffected. (d) Condensation of adenosine with adenosine-5'-phosphate: The following re- agents were dissolved in water to give a final volume of 8 ml with the concentra- tions shown: adenosine-8-C14 (0.0125 M, specific activity ca. 0.1 mc/mM), adenosine-5'-phosphate (0.0125 M), poly U (ca. 0.05 M), MgCl2 (0.15 N), NaCl (0.2 N). The mixture was titrated to pH 6.0 with 1 M , and cooled to 0°C; CDI (210 mg, 10 equiv rel. to pA) was added. One drop of toluene was placed in the flask to retard bacterial action, and the reaction mixture was kept at ca. 0WC. After 14 days a further portion of CDI (210 mg) was added. After a total of 31 days, the mixture was fractionated as shown in the process flow sheet (Fig. 1). At every step the radioactivity of each region was measured, Downloaded by guest on September 25, 2021 VOL. 59, 1968 CHEMISTRY: SULSTON ET AL. 729

TABLE 3. Specific activities. Calculated Specific specific activitya Hyper- activity Experiment Material (cpm/odu) chromicityb (cpm/odu) *A + pA condensation *A (starting material) 7450 0 7450 *ApA 4120 18 4400 *ApApA 3010 24c 3080 p*A self-condensation p*A (starting 4250 0 4250 material) p*Ap*A 4960 18 5010 a Optical density measured at pH 7, 259 mu. b Michelson, A. M., The Chemistry of Nudeosides and Nucleotides (New York: Academic Press, 1963), p. 446. c Value inferred from that for triadenylic acid having mixed (2' -) 5') and (3' -. 5') linkages. Asterisk indicates radioactive label. before elution with water. (Specific activities of the principal products are col- lected in Table 3.) Only radioactive products were examined. The formation of A2' pA (yield 10%) as the major component of the product was not expected, and its identity was therefore established with some care. The product was identical with authentic A2' pA in four chromatographic systems,'2 including two which separated it from A3' pA. System III demonstrated ca. 0.3 per cent of A"' pA, in agreement with the results of spleen phosphodiesterase degradation. Ultraviolet spectra at pH 2, pH 7, and pH 12 were indistinguishable from those obtained from A3' pA, and selected degradation products also gave appropriate spectra. The chain length of ApApA (yield 1.4%) was demonstrated by the degradation of samples with venom phosphodiesterase (adenosine, 0.37 odu, radioactive; pA, 0.82 odu, not radioactive) and with KOH (adenosine, 0.36 odu, not radioactive; Ap, 0.70 odu, radioactive). Degradation with ribonuclease T2 and with spleen phosphodiesterase showed that about 18 per cent of the phosphodiester linkages were (3' 5'). This is in agreement with the results of chromatography in sys- tem IV; in other systems the isomers of ApApA did not separate. A"' pA was formed in ca. 5 per cent over-all yield. Its identity was established by degradation with venom phosphodiesterase (adenosine, 51% of radioactivity; pA, 49% of radioactivity) and by its failure to hydrolyze in 1 M KOH. TABLE 4. Per cent of radioactitvity in ApA region. 3 days 7 days - Poly U Very low 1.0 + Poly U 5.2 9.5 Effect of poiy U: In a preliminary experiment two reactions, similar to that described above but on a smaller scale, were set up. Poly U was omitted from one of them. The products were analyzed by chromatography in system I (Table 4). The product from the reaction containing poly U was 81 per cent de- graded by KOH and 95 per cent degraded by venom phosphodiesterase, but was largely resistant to spleen phosphodiesterase. The control reaction after seven days contained no more than 0.3 per cent of (A2' pA + A3' pA) and no more than 0.6 per cent of A" pA. Downloaded by guest on September 25, 2021 730 CHEMISTRY: SULSTON ET AL. PROC. N. A. S.

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(e) Self-condensation of adenosine-6'-phosphate: The following reagents were dissolved in water to give a final volume of 1 ml with the concentrations shown: adenosine-5'-phosphate-8-C14 (0.025 M, specific activity ca. 0.05 mc/mM), poly U (ca. 0.05 M), MgCl2 (0.15 N), NaCl (0.2 N). The mixture was cooled to 00C, received 24 mg of CDI (3.5 equiv. rel. to pA), and was titrated with 1 N NaOH to pH 6.5. One drop of toluene was added, and the reaction mixture was kept at ca. 0C. After three days a further portion of CDI (30 mg, 4.5 equiv.) was added. A control experiment was performed in an identical manner, except for the omission of poly U. A sample was taken from the mixture containing poly U, and its optical density at 260 ms was plotted as a function of temperature from 0 to 30'C. A satisfactory sigmoid melting curve was obtained, with Tm 12'C.3 After 17 days 0.5 ml of the reaction mixture containing poly U was analyzed as shown in Figure 2. pA2' pA (yield 14%) was distinguished from the (3'-- 5') iso- mer (yield 2.5%) in system IV; A"' p pA was present in about 25 per cent yield and A5' ppA pA in 6 per cent yield. UV spectra of these materials and some deg- radation products agreed closely with those of authentic materials. It seems likely that fraction "(pA)2" contained one or more trimers but insufficient mate- rial was available to establish their identity. The control reaction contained about 25 per cent of A5' p pA and less than 1 per cent of (pA pA + A5' p pA pA). Discussion.-Both A and pA are known to form triple-helices with poly U under the conditions employed in our experiments.2 3 Presumably the same is true for an equimolar mixture of A and pA. We have therefore established that when pA is held in a triple-helix, it may be activated by carbodiimide to form in- ternucleotide and pyrophosphate bonds. Under similar conditions in the absence of poly U internucleotide bond formation is much slower but the yield of pyro- phosphate is comparable. Preliminary experiments show that neither uridine nor cytidine competes with A in poly U-directed condensation with pA. The identification of A2' pA and pA2' pA as major products proves that initiation of oligonucleotide synthesis is possible. The presence of trinucleotides in the reaction mixture shows that chain propagation also occurs. Thus, in-principle, high-molecular-weight oligomers could be constructed by our method. The most surprising result is the finding that the initial linkage in the dinucleo- tide is largely (2' -> 5') and (5'-. 5'). Infrared observations have shown that (2'-> 5') oligo A forms three-stranded helices with poly U which are almost as stable as those formed by the corresponding (3'-> 5') oligomers.9' 13 Clearly in the transition state the adduct formed by carbodiimide assumes an orientation in the helix favoring the condensation with a 2' rather than a 3' hydroxyl group. The appearance of (5' -- 5') dinucleoside monophosphate and (5' -- 5') pyro- phosphate suggests that even when adenine is -bonded in the triple helix, considerable freedom remains in the orientation about the glycosidic bond. It remains to be seen whether this is true for other condensing agents. ApApA could be formed either (a) by the condensation of pA with ApA or (b) by the condensation of A with pApA. If we suppose that (3'-- 5') and (2'-- 5') linked dimers react equally readily, and that processes (a) and (b) proceed at equal rates, we deduce from the results presented in Figure 1 that the proportion of (3'-- 5') linkage formed is about 45 per cent in process (a) and about 21 per cent Downloaded by guest on September 25, 2021 VOL. 59, 1968 CHEMISTR Y: SULSTON ET AL. 733 in process (b). However, since these experiments were carried out on small amounts of trinucleotide obtained directly from monomers, we prefer to wait until the addition of pA to the isomeric ApA's has been studied before drawing far-reaching conclusions. The findings reported in this paper have obvious relevance to the laboratory synthesis of polynucleotides and to prebiotic polynucleotide synthesis. Summary.-Under conditions which lead to the formation of triple-helices 2 poly U:adenosine and 2 poly U:adenosine-5'-phosphate, poly U facilitates the formation of (2' -- 5') and (5' -- 5') internucleotide bonds between adenosine resi- dues. Under the same conditions poly U has no effect on the condensation of adenosine-5'-phosphate with C or U. These results establish that nonenzymatic chain initiation and extension can be directed by a polynucleotide template. * This work was supported in part by grant 13435 from the National Institutes of Health. 'Howard, F. B., J. Frazier, M. N. Lipsett, and H. T. Miles, Biochem. Biophys. Res. Commun., 17, 93 (1964). 2Howard, F. B., J. Frazier, M. F. Singer, and H. T. Miles, J. Mol. Biol., 16, 415 (1966). 3 Miles, H. T., F. B. Howard, and J. Frazier, Federation Proc., 25, 1853 (1966) (abstract). 4Naylor, R., and P. T. Gilham, Biochemistry, 5, 2722 (1966). Randerath, K., in Thin-Layer Chromatography (Berlin: Springer Verlag, 1963), p. 195. 6 Zetzsche, F., and G. Baum, Ber., 75B, 100 (1942); Zetzsche, F., and A. Friedrich, Ber., 72B, 363 (1939). 7Hilmore, R. J., J. Biol. Chem., 235, 2117 (1960). 8 Razzell, W. E., in Methods in Enzymology, ed. S. P. Colowick and N. 0. Kaplan (New York: Academic Press, 1963), vol. 6, p. 236. 9 Michelson, A. M., in press. 10 Rushizky, G. W., and H. A. Sober, J. Biot. Chem., 238, 371 (1963). "Lane, B. G., and G. C. Butler, Biochem. Biophys. Acta, 33, 381 (1959). M2After purification in systems VII and II (Fig. 1), the products were isolated as borate complexes. This was shown to have no effect on the enzymic degradations. For chromato- graphic analysis small samples were freed from borate by electrophoresis in system VI. 13 Miles, H. T., unpublished results. Downloaded by guest on September 25, 2021