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Non-enzymic synthesis of deoxyadenylate oligonucleotides on a polyuridylate template
J. Mol. Biol. (1968) 37, 151-155
Non-enzymic Synthesis of Deoxyadenylate Oligonucleotides on a Polyuridylate Template H. SCHNEIDER.BERNLOEHR, R. LOHRMANN, J. SULSTON B. J. WEEKANN, L. E. ORGEL The Salk Imtitute for Bidogical Studies San Diego, California 92112, U.S.A. AND
H. TODD &ES Laboratory of Molecular Biology National Institute of Arthritis and Metabolic Disease8 National Institutes of Health Bethesda, Maryland, U.S.A. (Received 31 May 1968) Deoxyadenosine-5’.phosphate condenses with deoxyadenosine on a polym-idylatc template to give a product containing mainly 6’ + 6’ linkages. Similarly, the self-condensation of deoxyadcnosine-5’.phosphate on a polymidylate template gives mainly the pyrophosphate.
1. Introduction pAt condenses with A on a poly U template in the presence of 1.ethyl-3.(3dimethylaminopropyl).carbodiimide hydrochloride. 65% of the ApA formed is Aa’pA, 33% is A5’pA and only 2% is the natural 3’ + 5’ isomer. In the self-condensation of pA, 60% of the dimer is A5’ppA, 34% is pAsspA and only 6% is pA3’pA (Sulston, Lohrmann, Orgel & Miles, 196%). We have now extended these reactions to the deoxy compounds to see whether the proportion of the 3’ -+ 5’ isomer is greater in the absence of the 2’ hydroxy group.
2. Experimental
Procedure and Results
(a) Materials and methods Paper chromatography was carried out on Whatman 3MM paper by the descending technique .The solvent systems were: I, isopropanol-concn ammonia-water (7 : 1 : 2, by vol.); II, saturated aqueous ammonium sulfate-O-1 M-sodium acetat&sopropanol (79 : 19 : 2, by vol.). Paper electrophoresis was carried out on Whatman 3MM paper using Varsol or Ccl, as coolant. The buffers were: III, formic acid-ammonium formate O-05 M, pH 2.7; IV, O-03 M-triethylammonium bicarbonate, pH 7-5 approx. Other met,hods and sources have been described (Sulston et al., 1968o,b). t The conventional use of. . . p . . ., to express a (3’ +- 5’) phosphodiester linkage is modified as follows: pA*‘pA: 5’.phosphoadenylyl-(2’ + 5’)adenosine; pA: adenosine-5’.phosphate; A3’p: adenosine-3’.phosphate; A6’ppA: PI, P,.diadenosine-5’.pymphosphate; etc. 151
152
H.
ET AL.
SCHNEIDER-BERNLOEHR
(b) Condenmtion of deoxyadenoaine with deoxyadenoaine-5’-phosphate Two reaction mixtures (05 ml.) were prepared. Each contained: O-0125 M-[S-~*C] deoxyadenosine (specific activity approx. O-2 me/m-mole); O-0125 M-dpA; O-075 MMgCl,; O-2 M-NaCI. In addition, reaction 1 contained 0.05 M-pO]y U. The solutions were adjusted to pH 5.7, cooled to O”C, and CDIt added to give a concentration Of 0.2 M. After eight days at 0°C the pH of reaction 1 had increased to 7-5 and 38% of the CD1 remained. The reaction mixture was chromatographed in system I. The chromatogram revealed several ultraviolet-absorbing bands, three of which contained radioactive material (Fig. 1). These were the starting material dA (81%, specific activity 26,700 disintegrations/min/optical density unit), dApA (18%) and a small amount of dApApA (1.4%). Degradation of a sample of the dApA with venom phosphodiesterase yielded dpA (1.4 O.D. units, 14,000 disinteg./min) and dA (l-4 O.D. units, 23,600 disinteg./min). The distribution of radioactivity between dpA and dA indicated that about 25% of
81%
22 dA*
Mobility + rel. to dpA
1.0
1 %A
2 dpA*
+ dA*;z,&,a
Fro. 1. Analysis of produots from the condensation of [8-“Cldeoxyadenosine with dpA in the presence of poly U. Only radioactive products were studied in detail. Asterisk denotes radioeotive label. yc denotes over-all yield bssed on total radioactivity. Roman numerals refer to ohromatographic or eleotrophoretio systems, + indicates migration towards anode, - towards cathode. VPH: venom phosphodiesterase; SPH: spleen phosphodiesterase; a: dA*pApA and dApA*pA may be present. t Abbrevietion
used: CDI,
I-ethyl-3-(3-dimethyleminopropyl)-oarbodiimide
hydrochloride.
NON-ENZYMIC
SYNTHESIS
OF
OLIGONUCLEOTIDES
153
the material was dAa’pA and the rest dA6’pA. This was conilrmed by treatment of a further sample with spleen phosphodiesterase, which degraded 21 y0 of the material. The specific activity of the dApA was found to be 17,100 disinteg./min/o.n. unit. The theroretical value is 15,600 disinteg./min/o.n. unit, when allowance is made for a 17% hyperchromic effect. This latter was determined by comparing the optical density before and after degradation with venom phosphodiesterase. The ultraviolet spectra of the dApA were determined at pH values of 2,7 and 12. The positions of the maxima agreed closely with those of A. The dApApA was purified by electrophoresis 6rst in system IV and then in system III. It was degraded by venom phosphodiesterase to give dpA (O-48 O.D. unit) and dA (O-24 O.D. unit). After eight days at O’Y!, the pH of reaction 2 had increased to 6.7 and no CD1 remained. Analysis showed a 2% yield of dApA, 95% of which was resistant to spleen phosphodiesterase. The degradation with venom phosphodiesterase yielded dA (50% of radioactivity) and dpA (50% of radioactivity). (0) Self-condensation of deoxyfz4$eno8ine-5’-ph~h&e Two reaction mixtures (0.5 ml.) were prepared. Each contained: O-025 M-[~-‘*C] deoxyadenosine-5’-phosphate (specific activity approx. O-1 me/m-mole), O-075 MMgCl,; O-2 M-NaCl. In addition, reaction 1 contained 0.05 M-poly U. The pH was adjusted to 5.7, the mixture was cooled to 0°C and CD1 was added to give a concentration of 0.2 M. After eight days the pH of reaction 1 had increased to 7.5 and 26% of the CD1 was left. A preliminary separation of the products was obtained by chromatography in system I for 72 hours (Fig. 2). The slower running radioactive bands were eluted and rechromatographed for five days in the same system. The main product was shown to be dA6’ppA (71%) by comparison of its behavior in systems I to IV with that of an authentic sample which we synthesized by the method of Smith, Drummond & Khorana (1960). Its identity was confirmed by degradation with venom phosphodiesterase, which gave dpA as sole product. A second band contained a compound (yield 5-5%, X,,, 258 rnp) which is believed to be dA5’ppApA, since it was not degraded by alkaline phosphatase but gave dpA with venom diesterase. dpApA was formed in a yield of 1.9%. It was dephosphorylated by alkaline phosphatase and gave dApA as the sole product. The fastest moving product (Fig. 2) was shown, by electrophoresis in system III and chromatography in system I, not to be the 3’,5’-cyclic phosphate. Its structure was not investigated further since it appeared in the control in comparable yield. After eight days the pH of reaction 2 had increased to 7.3 and 8% of the CD1 remained. Chromatography in system I showed 90% dA5’ppA, but virtually no dAS’ppApA or dpApA. The unknown product described above also appeared with a yield of about 3 %. 3. Discussion Since the template-directed condensation of pA with A yields principally A2’pA (Sulston et al., 1968u), it has been suggested (Anon., 1968) thatDNAmay have preceded RNA in biochemical evolution. This point of view is not supported by the finding that the condensation of dpA with dA gives predominantly dA5’pA, while the self-
154
H.
SCHNEIDER-BERNLOEHR /REACTION
ET
AL.
1
I, 72 hr
dpA - 1
R, rel. to 1 2.5
I I.5
1.0
I 0.82
IO.45
0.21
4.7%
I, 5 days
I, 5 days
dApA VPH
I \
VPH -dpA complete
+ dA
FIG 2. Analysis of products from the self-condensation of [S-“C]deoxyadenoeine-5’-phosphate in the presence of poly U. Roman numerals refer to chromatographic systems. “/:, denotes over-all yield based on total radioactivity. VPH: venom phosphodiesterase; APH: alkaline phosphatase.
condensation of dpA gives a high yield of dA6’ppA. Since chain-reversing 5’ -+ 5’ linkages cannot be used extensively in a polynucleotide chain, the condensation of clpA to useful polynucleotides would be ineficient. It remains to be seen whether this conclusion applies to reactions with other condensing agents. Furthermore, condensations with dAp might lead to useful oligomers although it is hard to see how this isomer could have arisen. Comparison of the yields in experiments in the deoxy and ribo series reveals a number of quantitative differences : (1) In the absence of poly U, the proportions of isomers making up the ApA formed from pA and A are: AShA, 66%; AshA, 12%; Aa’pA, 32%. In the deoxy series, less than 5% of the dApA formed is the 3’ + 5’ isomer. Thus, in free solution, the proportion of the 3’ -+ 5’ isomer is even less for the deoxy series than for the ribo series. (2) The ratio of the yields of dA3’pA and dAb’pA formed in the presence of a poly U template is 1 : 3, much greater than in the ribo series (about 1 : 15). (3) In the deoxy series clpApA is a very minor product of template synthesis (1.9% of starting material) compared with dA5’ppA (71% of starting material). This should be compared with yields of 2~5% and 25% for pA3’pA and A5’ppA in the ribo series. These experiments, together with those described in previous papers, make it clear that the yields of different isomers obtained from a pair of nucleoticle derivatives
NON-ENZYMIC
SYNTHESIS
OF
OLIGONUCLEOTIDES
155
are very sensitive to the nature both of the sugar and of the base. We believe this to reflect slight differences in relative orientation of the monomers in the complexes, but we do not have a detailed theory. This work of Health.
was supported
in part
by grant
number
13435 from
the National
Institutes
REFERENCES Anon. (1968). Nature, 218, 623. S&ton, J., Lohrmann, R., Orgel, L. E. & Miles, H. T. (1968a). Proc. Nat. Acud. Sci., Wash. 59, 726. Sci., Sulston, J., Lohrmann, R., Orgel, L. E. & Miles, H. T. (19683). Proc. Nat. Acd Wash. 69, 409. Smith, M., D rummond, G. I. & Khorana, H. G. (1960). J. Amer. Chem. Sot. 83, 698.
Non-enzymic Synthesis of Deoxyadenylate Oligonucleotides on a Polyuridylate Template H. SCHNEIDER.BERNLOEHR, R. LOHRMANN, J. SULSTON B. J. WEEKANN, L. E. ORGEL The Salk Imtitute for Bidogical Studies San Diego, California 92112, U.S.A. AND
H. TODD &ES Laboratory of Molecular Biology National Institute of Arthritis and Metabolic Disease8 National Institutes of Health Bethesda, Maryland, U.S.A. (Received 31 May 1968) Deoxyadenosine-5’.phosphate condenses with deoxyadenosine on a polym-idylatc template to give a product containing mainly 6’ + 6’ linkages. Similarly, the self-condensation of deoxyadcnosine-5’.phosphate on a polymidylate template gives mainly the pyrophosphate.
1. Introduction pAt condenses with A on a poly U template in the presence of 1.ethyl-3.(3dimethylaminopropyl).carbodiimide hydrochloride. 65% of the ApA formed is Aa’pA, 33% is A5’pA and only 2% is the natural 3’ + 5’ isomer. In the self-condensation of pA, 60% of the dimer is A5’ppA, 34% is pAsspA and only 6% is pA3’pA (Sulston, Lohrmann, Orgel & Miles, 196%). We have now extended these reactions to the deoxy compounds to see whether the proportion of the 3’ -+ 5’ isomer is greater in the absence of the 2’ hydroxy group.
2. Experimental
Procedure and Results
(a) Materials and methods Paper chromatography was carried out on Whatman 3MM paper by the descending technique .The solvent systems were: I, isopropanol-concn ammonia-water (7 : 1 : 2, by vol.); II, saturated aqueous ammonium sulfate-O-1 M-sodium acetat&sopropanol (79 : 19 : 2, by vol.). Paper electrophoresis was carried out on Whatman 3MM paper using Varsol or Ccl, as coolant. The buffers were: III, formic acid-ammonium formate O-05 M, pH 2.7; IV, O-03 M-triethylammonium bicarbonate, pH 7-5 approx. Other met,hods and sources have been described (Sulston et al., 1968o,b). t The conventional use of. . . p . . ., to express a (3’ +- 5’) phosphodiester linkage is modified as follows: pA*‘pA: 5’.phosphoadenylyl-(2’ + 5’)adenosine; pA: adenosine-5’.phosphate; A3’p: adenosine-3’.phosphate; A6’ppA: PI, P,.diadenosine-5’.pymphosphate; etc. 151
152
H.
ET AL.
SCHNEIDER-BERNLOEHR
(b) Condenmtion of deoxyadenoaine with deoxyadenoaine-5’-phosphate Two reaction mixtures (05 ml.) were prepared. Each contained: O-0125 M-[S-~*C] deoxyadenosine (specific activity approx. O-2 me/m-mole); O-0125 M-dpA; O-075 MMgCl,; O-2 M-NaCI. In addition, reaction 1 contained 0.05 M-pO]y U. The solutions were adjusted to pH 5.7, cooled to O”C, and CDIt added to give a concentration Of 0.2 M. After eight days at 0°C the pH of reaction 1 had increased to 7-5 and 38% of the CD1 remained. The reaction mixture was chromatographed in system I. The chromatogram revealed several ultraviolet-absorbing bands, three of which contained radioactive material (Fig. 1). These were the starting material dA (81%, specific activity 26,700 disintegrations/min/optical density unit), dApA (18%) and a small amount of dApApA (1.4%). Degradation of a sample of the dApA with venom phosphodiesterase yielded dpA (1.4 O.D. units, 14,000 disinteg./min) and dA (l-4 O.D. units, 23,600 disinteg./min). The distribution of radioactivity between dpA and dA indicated that about 25% of
81%
22 dA*
Mobility + rel. to dpA
1.0
1 %A
2 dpA*
+ dA*;z,&,a
Fro. 1. Analysis of produots from the condensation of [8-“Cldeoxyadenosine with dpA in the presence of poly U. Only radioactive products were studied in detail. Asterisk denotes radioeotive label. yc denotes over-all yield bssed on total radioactivity. Roman numerals refer to ohromatographic or eleotrophoretio systems, + indicates migration towards anode, - towards cathode. VPH: venom phosphodiesterase; SPH: spleen phosphodiesterase; a: dA*pApA and dApA*pA may be present. t Abbrevietion
used: CDI,
I-ethyl-3-(3-dimethyleminopropyl)-oarbodiimide
hydrochloride.
NON-ENZYMIC
SYNTHESIS
OF
OLIGONUCLEOTIDES
153
the material was dAa’pA and the rest dA6’pA. This was conilrmed by treatment of a further sample with spleen phosphodiesterase, which degraded 21 y0 of the material. The specific activity of the dApA was found to be 17,100 disinteg./min/o.n. unit. The theroretical value is 15,600 disinteg./min/o.n. unit, when allowance is made for a 17% hyperchromic effect. This latter was determined by comparing the optical density before and after degradation with venom phosphodiesterase. The ultraviolet spectra of the dApA were determined at pH values of 2,7 and 12. The positions of the maxima agreed closely with those of A. The dApApA was purified by electrophoresis 6rst in system IV and then in system III. It was degraded by venom phosphodiesterase to give dpA (O-48 O.D. unit) and dA (O-24 O.D. unit). After eight days at O’Y!, the pH of reaction 2 had increased to 6.7 and no CD1 remained. Analysis showed a 2% yield of dApA, 95% of which was resistant to spleen phosphodiesterase. The degradation with venom phosphodiesterase yielded dA (50% of radioactivity) and dpA (50% of radioactivity). (0) Self-condensation of deoxyfz4$eno8ine-5’-ph~h&e Two reaction mixtures (0.5 ml.) were prepared. Each contained: O-025 M-[~-‘*C] deoxyadenosine-5’-phosphate (specific activity approx. O-1 me/m-mole), O-075 MMgCl,; O-2 M-NaCl. In addition, reaction 1 contained 0.05 M-poly U. The pH was adjusted to 5.7, the mixture was cooled to 0°C and CD1 was added to give a concentration of 0.2 M. After eight days the pH of reaction 1 had increased to 7.5 and 26% of the CD1 was left. A preliminary separation of the products was obtained by chromatography in system I for 72 hours (Fig. 2). The slower running radioactive bands were eluted and rechromatographed for five days in the same system. The main product was shown to be dA6’ppA (71%) by comparison of its behavior in systems I to IV with that of an authentic sample which we synthesized by the method of Smith, Drummond & Khorana (1960). Its identity was confirmed by degradation with venom phosphodiesterase, which gave dpA as sole product. A second band contained a compound (yield 5-5%, X,,, 258 rnp) which is believed to be dA5’ppApA, since it was not degraded by alkaline phosphatase but gave dpA with venom diesterase. dpApA was formed in a yield of 1.9%. It was dephosphorylated by alkaline phosphatase and gave dApA as the sole product. The fastest moving product (Fig. 2) was shown, by electrophoresis in system III and chromatography in system I, not to be the 3’,5’-cyclic phosphate. Its structure was not investigated further since it appeared in the control in comparable yield. After eight days the pH of reaction 2 had increased to 7.3 and 8% of the CD1 remained. Chromatography in system I showed 90% dA5’ppA, but virtually no dAS’ppApA or dpApA. The unknown product described above also appeared with a yield of about 3 %. 3. Discussion Since the template-directed condensation of pA with A yields principally A2’pA (Sulston et al., 1968u), it has been suggested (Anon., 1968) thatDNAmay have preceded RNA in biochemical evolution. This point of view is not supported by the finding that the condensation of dpA with dA gives predominantly dA5’pA, while the self-
154
H.
SCHNEIDER-BERNLOEHR /REACTION
ET
AL.
1
I, 72 hr
dpA - 1
R, rel. to 1 2.5
I I.5
1.0
I 0.82
IO.45
0.21
4.7%
I, 5 days
I, 5 days
dApA VPH
I \
VPH -dpA complete
+ dA
FIG 2. Analysis of products from the self-condensation of [S-“C]deoxyadenoeine-5’-phosphate in the presence of poly U. Roman numerals refer to chromatographic systems. “/:, denotes over-all yield based on total radioactivity. VPH: venom phosphodiesterase; APH: alkaline phosphatase.
condensation of dpA gives a high yield of dA6’ppA. Since chain-reversing 5’ -+ 5’ linkages cannot be used extensively in a polynucleotide chain, the condensation of clpA to useful polynucleotides would be ineficient. It remains to be seen whether this conclusion applies to reactions with other condensing agents. Furthermore, condensations with dAp might lead to useful oligomers although it is hard to see how this isomer could have arisen. Comparison of the yields in experiments in the deoxy and ribo series reveals a number of quantitative differences : (1) In the absence of poly U, the proportions of isomers making up the ApA formed from pA and A are: AShA, 66%; AshA, 12%; Aa’pA, 32%. In the deoxy series, less than 5% of the dApA formed is the 3’ + 5’ isomer. Thus, in free solution, the proportion of the 3’ -+ 5’ isomer is even less for the deoxy series than for the ribo series. (2) The ratio of the yields of dA3’pA and dAb’pA formed in the presence of a poly U template is 1 : 3, much greater than in the ribo series (about 1 : 15). (3) In the deoxy series clpApA is a very minor product of template synthesis (1.9% of starting material) compared with dA5’ppA (71% of starting material). This should be compared with yields of 2~5% and 25% for pA3’pA and A5’ppA in the ribo series. These experiments, together with those described in previous papers, make it clear that the yields of different isomers obtained from a pair of nucleoticle derivatives
NON-ENZYMIC
SYNTHESIS
OF
OLIGONUCLEOTIDES
155
are very sensitive to the nature both of the sugar and of the base. We believe this to reflect slight differences in relative orientation of the monomers in the complexes, but we do not have a detailed theory. This work of Health.
was supported
in part
by grant
number
13435 from
the National
Institutes
REFERENCES Anon. (1968). Nature, 218, 623. S&ton, J., Lohrmann, R., Orgel, L. E. & Miles, H. T. (1968a). Proc. Nat. Acud. Sci., Wash. 59, 726. Sci., Sulston, J., Lohrmann, R., Orgel, L. E. & Miles, H. T. (19683). Proc. Nat. Acd Wash. 69, 409. Smith, M., D rummond, G. I. & Khorana, H. G. (1960). J. Amer. Chem. Sot. 83, 698.