(self-replicating systemyenantiomeric cross-inhibitionyL-nucleotidesyalternating D-L-oligonucleotides)

The oligomerization of activated Dand Land racemic guanosine-5*-phosphoro-2-methylimidazole on short templates containing Dand L-deoxycytidylate has been studied. Results obtained with D-oligo(dC)s as templates are similar to those previously reported for experiments with a poly(C) template. When one L-dC or two consecutive L-dCs are introduced into a D-template, regiospecific synthesis of 3*-5* oligo(G)s proceeds to the end of the template, but three consecutive L-dCs block synthesis. Alternating D-,L-oligomers do not facilitate oligomerization of the D-, L-, and racemic 2-guanosine-5*-phosphoro-2-methylimidazole. We suggest that once a ‘‘predominately D-metabolism’’ existed, occasional L-residues in a template would not have led to the termination of self-replication. All living organisms use the D-enantiomers of riboand deoxynucleotides as building blocks for their nucleic acids. Any abiotic synthesis of a nucleotide would yield equal amounts of the Dand Lisomers. It is not clear why only D-nucleotides were chosen during evolution (1–4). In the present study, we explore the way in which L-nucleotides in a prebiotic soup would effect a self-replicating system of D-nucleotides early in the development of nucleic acid replication. As a model system, we used the highly efficient template-directed oligomerization of activated guanosine mononucleotides on oligo(dC) templates (5). We report here on the oligomerization of activated Dand L-guanosine mononucleotides on templates containing Dand L-deoxycytidylate residues (Fig. 1). MATERIALS AND METHODS Unless otherwise noted, all chemicals were reagent grade, were purchased from commercial sources, and were used without further purification. D-Guanosine 59-monophosphate was obtained from Sigma. L-Guanosine 59-monophosphate was synthesized from N3-isobutyryl-9-(29,39-di-O-benzoyl-b-Lribofuranosyl)guanosine (6) by phosphitylation with bis(2cyanoethyl)-N,N-diisopropyl phosphoramidite, followed by oxidation and aminolysis (85% yield). The enantiomers of guanosine 59-phosphoro-2-methylimidazolide (2-MeImpG) were synthesized by a published procedure (7) with yields of 95%. b-L-Deoxycytidine(N-bz)b-cyanoethyl N,N-diisopropyl phosphoramidite was purchased from Chem-Genes (Waltham, MA). Oligodeoxyribonucleotide templates were synthesized on a 391A DNA synthesizer (Applied Biosystems), were deprotected in concentrated ammonia at 55°C, were purified by 20% PAGE, and were desalted on a Nensorb column (DuPontyNEN). The (dG)3G primer is an oligodeoxynucleotide terminated by a single ribonucleotide residue at the 39 terminus. It was obtained as described above but by using a ribo controlled pore glass GAc column (Glen Research, Sterling, VA) to introduce the 39-ribonucleotide. Nuclease P1 was obtained from Pharmacia. The primer was labeled with [g32P]ATP and T4 polynucleotide kinase (New England Biolabs) as described in ref. 8 and was purified on a Nensorb column. To determine the position of Gpp(dG)3G in PAGE it was synthesized as described in ref. 9. All PAGE separations were run on a denaturing (8 M urea) 20% polyacrylamide gel. The elution buffer was 50 mM Triszborate (pH 8.3) containing 1 mM EDTA. Loading buffer was prepared by mixing 900 ml of deionized formamide, 25 ml of xylene cyanol (2%), 25 ml of bromophenol blue (2%), and 50 ml of 103 Triszborate, EDTA buffer. Reaction conditions for the polymerization of 2-MeImpG on various templates were chosen to permit comparison with earlier published work (5, 10, 11). The reactions were run for 7 days at 0°C in 0.2 M 2,6-lutidine buffer (pH 7.9 at 25°C) containing 1.2 M NaCl, 0.2 M MgCl2, 0.5 mM template, and 0.1 M 2-MeImpG. When a mixture of Dand Lisomers was used, the concentration of each isomer was 0.1 M. The volume of the reaction mixture was 3 ml. To prepare the reaction mixture, the appropriate amounts of stock solutions of NaCl, MgCl2, and a template were mixed in an Eppendorf tube and were evaporated to dryness. The residue was redissolved in 3 ml of fresh, prepared solution of 2-MeImpG in 0.2 M 2,6lutidine buffer to start the reaction. HPLC analyses of the reaction mixtures were performed on an RPC5 column as described (7). Reaction products were eluted with a linear gradient of NaClO4 (pH 12, 0–0.06 M in 60 min) and were monitored by UV absorption at 254 nm. Reaction conditions for p(dG)3G primer extension with 2-MeImpG on different templates were chosen again to permit comparison with earlier published work (12, 13). The reactions were run for 5 days at 0°C in 0.2 M 2,6-lutidine buffer (pH 7.9 at 25°C) containing 1.2 M NaCl, 0.2 M MgCl2, 20 mM template, 20 nM primer, and 50 mM 2-MeImpG. When a mixture of Dand L-isomers was used, concentration of each isomer was 25 mM. The volume of the reaction mixture was 6 ml. To prepare the reaction mixture, appropriate amounts of stock solutions of NaCl, MgCl2, primer, and a template were mixed in an Eppendorf tube and were evaporated to dryness. The residue was redissolved in 3 ml of 0.2 M 2,6-lutidine buffer. The solution was heated to 95°C and was cooled to 25°C for 1 hour, then kept at 0°C for 20 min. Then, fresh prepared 2-MeImpG solution in 0.2 M 2,6-lutidine buffer (3 ml) was added to start the reaction. Reaction mixtures were analyzed by 20% PAGE.