Non-enzymatic, template-directed ligation of 2'-5' oligoribonucleotides. Joining of a template and a ligator strand.

Decauridylate containing exclusively a 2'-5' phospho-diester bond ([2'-5']U10) served as a template for the synthesis of oligoadenylates [oligo(A)s] from the 5'-phosphorimidazolide of 2'-5' diadenylate (ImpA-2'p5'A). Joining of [2'-5']U10and ImpA2'p5'A also took place in substantial amounts to yield long-chain oligoribonucleotides in the template-directed reaction. An unusual CD spectrum ascribed to helix formation between [2'-5']U10and [2'-5'](pA)2was observed under the same conditions as that of the template-directed reaction. The 3'-5' linked decauridylate ([3'-5']U10) also promoted the template-directed synthesis of oligo(A)s from ImpA2'p5'A, but more slowly compared with [2'-5']U10. The results indicate that short-chain RNA oligomers with a 2'-5' phosphodiester bond could lead to longer oligoribonucleotides by template-directed chain elongation.     

Non-enzymatic template-directed synthesis on RNA random copolymers. Poly(C, U) templates

Poly(C, U) random copolymer templates direct the oligomerization of 2-MeImpG and 2-MeImpA, resulting in the production of a variety of oligo/(G,A)s. The efficiency of monomer incorporation into newly synthesized oligomers is greater for 2-MeImpG than for 2-MeImpA, and decreases for both monomers as the uracil content of the template increases. The relatively poor incorporation of adenine is partly due to an intrinsically less efficient incorporation reaction, and partly due to the masking of uracil sites by G X U non-complementary pairing. The efficiency of adenine incorporation can be improved by decreasing the concentration of 2-MeImpG and increasing the concentration of 2-MeImpA in the reaction mixture. The oligomeric product distribution can be characterized in detail using high-pressure liquid chromatography on an RPC-5 column. Oligomers are separated on the basis of chain length, base composition, and phospho-diester-linkage isomerism. The 3'----5' regiospecificity of monomer addition to template-bound oligomers is lower for 2-MeImpA than for 2-MeImpG. The presence of an adenine residue at the 2'(3') terminus of the acceptor strand lowers the regiospecificity of 2-MeImpA addition even further.     

Non-enzymic template-directed synthesis on RNA random copolymers. Poly(C, G) templates

Poly(C, G) random copolymer templates direct the oligomerization of 2-Me-ImpG and 2-MeImpC, resulting in the production of a variety of oligo(G, C)s. The efficiency of monomer incorporation into newly synthesized oligomers is greater for 2-MeImpG than for 2-MeImpC, and decreases for both monomers as the guanine content of the template increases. The relatively low efficiency of oligomerization on guanine-rich templates is largely a consequence of intra- and intermolecular template self-structure. The problem of template self-structure is clearly a major obstacle to the development of a system of self-replicating polynucleotides. The distribution of oligomeric products can be characterized in detail using high-pressure liquid chromatography on an RPC-5 column. Oligomers are separated on the basis of chain length, base composition and phosphodiester-linkage isomerism. Oligomers up to about the 12-mer, with base composition Gn, Gn-1C and Gn-2C2, have been identified. The 3' to 5' regiospecificity of the products is high, particularly for oligomers with base composition Gn.     

Non-enzymatic template-directed synthesis on RNA random copolymers. Poly(C,A) templates

Poly(C,A) random copolymer templates direct the oligomerization of 2-MeImpG (2-MeImpX is the 5'-phospho-2-methylimidazolide of the nucleoside X) and 2-MeImpU, resulting in the production of a variety of oligo (G,U)s. This reaction is less efficient than comparable reactions involving poly(C,U) or poly(C,G) templates. The efficiency of monomer incorporation into newly synthesized oligomers is lower for 2-MeImpU than 2-MeImpG, and cannot be improved by increasing the concentration of 2-MeImpU relative to 2-MeImpG. This suggests that RNA templates containing runs of consecutive adenine residues would not be suitable for use in a chemical self-replicating system. The distribution of oligomeric products can be characterized in detail using high-pressure liquid chromatography on an RPC-5 column. Oligomers are separated on the basis of chain length, base composition, and phosphodiester-linkage isomerism. Oligomers up to about the 13-mer, with base composition Gn, Gn-1, U, and Gn-2, U2, have been identified.     

Infrared study of G · C complex formation in template-dependent oligo(G) synthesis

G · C complex formation was studied by infrared spectroscopy for a system that has been shown by Inoue & Orgel (1982) to give efficient, template-dependent synthesis of oligo(G). Guanosine-5′-phosphor-2-methylimiazolide (2-MeImpG) exhibits rapid formation with poly(C) of a G · C double helix at pD ~ 8 and of a C · G · CH⁺ triple helix at pD ~ 6.5 in the presence of Na⁺. Significant oligo(G) synthesis does not occur under these conditions. In the presence of synthetically effective concentrations of Mg²⁺ G · C complex formation is much slower but eventually goes to completion. The rate of complex formation parallels that of chemical synthesis. Infrared spectra and melting curves confirm that oligo(G) of high molecular weight is formed in high yield. The bulk of the G · C complex at any given time during the reaction is composed of G residues that have already been polymerized and not of the monomer 2-MeImpG. Evidence indicates that synthesis proceeds primarily at growing points at the ends of the G · C helical regions and not randomly on a fully occupied template.     

Computer simulation in template-directed oligonucleotide synthesis

Summary A computer simulation (KINSIM) modeling up to 33 competing reactions was used in order to investigate the product distribution in a template-directed oligonucleotide synthesis as a function of time and concentration of the reactants. The study is focused on the poly(C)-directed elongation reaction of an oligoguanylate (a 7-mer is chosen) with guanosine 5-monophosphate-2-methylimidazolide (2-MeImpG), the activated monomer. It is known that theelongation of oligoguanylates to form oligomeric products such as 8-mer, 9-mer, 10-mer, etc., is in competition with (1) thedimerization and further oligomerization reaction of 2-MeImpG that leads to the formation of dimers and short oligomers, and (2) thehydrolysis of 2-MeImpG that forms inactive guanosine 5-monophosphate, 5-GMP. Experimentally determined rate constants for the above three processes at 37°C and pH 7.95 were used in the simulation; the initial concentrations of 2-MeImpG, [M]_o, and of the oligoguanylate primer, [7-mer]_o, were varied, and KINSIM calculated the distribution of products as a function of time until equilibration was reached, i.e., when all the activated monomer has been consumed. In order to sort out how strongly the elongation reaction may be affected by the competing hydrolysis and dimerization, we also simulated the idealized situation in which these competing reactions do not occur. Simulation of the idealized system suggests that (1) the fraction of [7-mer]_o that has reacted as well as the product distribution after equilibration do not depend on the absolute concentrations of the reactants, but only on their ratio, [M]_o/[7-mer]_o; (2) the rate of elongation is proportional to [7-mer]_o and not to [M]_o; and (3) as the [M]_o/[7-mer]_o ratio increases longer oligomers are formed. The results of the computer simulation with the experimental system, i.e., elongation in the presence of both hydrolysis and dimerization, are similar to the ones obtained with the idealized system as long as dimerization and hydrolysis are not responsible for consuming a substantial fraction of 2-MeImpG.     

Template-directed polymerization of oligoadenylates using cyanogen bromide

Cyanogen bromide (BrCN) condensed oligoadenylates [oligo(A)] on a poly(uridylic acid) [poly(U)] template in an aqueous solution. Imidazole and divalent metal ions such as Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Mg2+, and Fe2+ were required for the condensation. Chain length of oligo(A) and reaction temperature affected the coupling yield. Hexaadenylate [(pA)6] was converted to (pA)12, (pA)18, (pA)24, (pA)30, (pA)36, (pA)42, and (pA)48 in a 68% overall yield for 20 h at 25 degrees C. The coupling yield increased with increase in the poly(U) concentration. Five- to sevenfold molar excess of uridylyl residues of poly(U) to adenylyl residues of oligo(A) gave the best yield (68%). Metal ions affected the formation of linkage isomers of the phosphate bonds: The 2',5'- and 3',5'-phosphodiester bonds were predominant in the presence of Co2+, Zn2+, and Ni2+ and the 5',5'-pyrophosphate bond was predominant in the presence of Mn2+. In particular, Ni2+ gave the highest ratio of the 3',5'-phosphodiester bond (30%). N-Cyanoimidazole (1), N,N'-iminodiimidazole (2), and N-carboxamidoimidazole (3) were formed in a reaction of imidazole with BrCN in an aqueous solution. 1 and 2 had much the same condensing activity for the polymerization of adenylates as BrCN. A reaction pathway was proposed in which 1 and 2 are not only intermediates for the production of 3 but also the true condensing agent in the coupling reaction of oligo(A). Phosphorimidazolide derivative was detected in a reaction of 5'-AMP with either 1 or 2. The condensation would proceed by way of N-cyanoimidazole-phosphate adduct, the phosphorimidazolide derivative, or both.     

Oligomerization of (guanosine 5′-phosphor)-2-methylimidazolide on poly(C): An RNA polymerase model

(Guanosine 5′-phosphor)-2-methylimidazolide (2-MeImpG), unlike guanosine 5′-phosphorimidazolide (ImpG), undergoes an efficient, buffer-independent, template-directed oligomerization in the presence of poly(C) at pH values above 7.6. The reaction occurs in a Watson-Crick double helix and yields predominantly 3′-5′-linked oligomers up to the 50-mer in above 80% yield. Synthesis proceeds in the 5′ → 3′ direction and has high fidelity in the sense that nucleotides other than G are not incorporated significantly into oligomers. Under some conditions, oligomers corresponding to approximately one and two turns of the helix are obtained in higher yield than somewhat longer or somewhat shorter oligomers.In the protonated triple-helical structure formed below pH 7, the efficiency of the oligomerization is much lower. Oligomers up to about the 10-mer are obtained. The most abundant products are “capped” at the 5′ terminus with a GppG pyrophosphate group.     

Natural 2',5'-phosphodiester bonds found at the ligation sites of peach latent mosaic viroid

Peach latent mosaic viroid (PLMVd) is a circular RNA pathogen that replicates in a DNA-independent fashion via a rolling circle mechanism. PLMVd has been shown to self-ligate in vitro primarily via the formation of 2',5'-phosphodiester bonds; however, in vivo the occurrence and necessity of this nonenzymatic mechanism are not evident. Here, we unequivocally report the presence of 2', 5'-phosphodiester bonds at the ligation site of circular PLMVd strands isolated from infected peach leaves. These bonds serve to close the linear conformers (i.e., intermediates), yielding circular ones. Furthermore, these bonds are shown to stabilize the replicational circular templates, resulting in a significant advantage in terms of viroid viability. Although the mechanism responsible for the formation of these 2',5'-phosphodiester bonds remains to be elucidated, a hypothesis describing in vivo nonenzymatic self-ligation is proposed. Most significantly, our results clearly show that 2',5'-phosphodiester bonds are still present in nature and that they are of biological importance.     

Oligoribonucleotides containing 2',5'-phosphodiester linkages exhibit binding selectivity for 3',5'-RNA over 3',5'-ssDNA.

Oligoribonucleotides containing 2',5'-phosphodiester linkages have been synthesized on a solid support by the 'silyl-phosphoramidite' method. The stability of complexes formed between these oligonucleotides and complementary 3',5'-RNA strands have been studied using oligoadenylates and a variety of oligonucleotides of mixed base sequences including phosphorothioate backbones. In many cases, particularly for 2',5'-linked adenylates, the UV melting profiles are quite sharp and exhibit large hyperchromic changes. Substituting a few 3',5'-linkages with the 2',5'-linkage within an oligomer lowers the Tm of the complex and the degree of destabilization depends on the neighboring residues and neighboring linkages. The 2',5'-linked oligoribonucleotides prepared in this study exhibited remarkable selectivity for complementary single stranded RNA over DNA. For example, in 0.01 M phosphate buffer--0.10 M NaCl (pH 7.0), no association was observed between 2',5'-r(CCC UCU CCC UUC U) and its Watson-Crick DNA complement 3',5'-d(AGAAGGGAGAGGG). However, 2',5'-r(CCC UCU CCC UUC U) with its RNA complement 3',5'-r(AGAAGGGAGAGGG) forms a duplex which melts at 40 degrees C. The decamer 2',5'-r(Ap)9A forms a complex with both poly dT and poly rU but the complex [2',5'-r(Ap)9A]:[poly dT] is unstable (Tm, -1 degree C) and is seen only at high salt concentrations. In view of their unnatural character and remarkable selectivity for single stranded RNA, 2',5'-oligo-RNAs and their derivatives may find use as selective inhibitors of viral mRNA translation, and as affinity ligands for the purification of cellular RNA.     

Chemical reactions in double-stranded nucleic acids. The nature of the bond formed during chemical ligation using a cis-diol group

Chemical and enzymatic ligation between the 5'-terminal phosphate of one oligonucleotide and the 3'-terminal 2',3'-cis-diol group of the other oligonucleotide on a complementary template was studied. Carbodiimide, imidazolide and N-hydroxybenzotriazole ester methods were used for chemical activation of the phosphate group, and T4 DNA ligase for enzymatic ligation. All the chemical activation methods produced 3',5'- and 2',5'-phosphodiester bonds (40-45 and 55-60%, resp.), whereas enzymatic ligation gave the product only with 3',5'-phosphodiester bond.     

Template-directed synthesis on short oligoribocytidylates

Summary The oligonucleotides C(pC)_n, n=4, 5, 6, 7, are efficient templates for the oligomerization of guanosine-5-phospho-2-methylimidazole (2-MeImpG). They yield oligomeric products that are substantially less regiospecific than those obtained on polycytidylate [poly(C)]. The overall distributions of products obtained on oligo(C)s are generally similar to those of products obtained on oligodeoxycytidylates [oligo(dC)s], but there are substantial differences in the ratios of isomers.The 3–5-linked dinucleoside monophosphate GpG efficiently initiates oligomer formation with 2-MeImpG on oligo(C) templates. The pattern of products obtained by chain extension parallels closely that of products obtained directly from 2-MeImpG, except that the former products lack the 5-terminal phosphate group.     

Template-directed synthesis on the oligonucleotide d(C7-G-C7)

When the deoxynucleotide template d(C7-G-C7) is incubated with the activated nucleotides 2-MeImpG and 2-MeImpC, a series of oligomers of G up to the sevenmer and a series of copolymers of composition GnC with n = 3 to 13 are formed. Oligomers GnC with n greater than 7 are completely degraded by pancreatic ribonuclease, establishing that they contain a 3' to 5' internucleotide bond between 5'-C and 3'-G within a sequence of the form (pG)ipC(pG)j. As expected, (pG)7-Cp and (pG)6-Cp are major hydrolysis products. Detailed analysis of the product distribution shows that a substantial fraction of the oligomeric products are of the type (pG)ipC(pG)j with i less than 7. This shows that product synthesis does not necessarily begin at the 3' terminus of the template. The significance of this finding in terms of the origin of molecular replication is discussed.     

Effect of Inhibitors on the Montmorillonite Clay-Catalyzed Formation of RNA: Studies on the Reaction Pathway

The Langmuir adsorption isotherms of the phosphoroimidazolides of adenosine (ImpA) and uridine (ImpU), dA^5'ppdA and N⁶, N⁶-dimethyladenine binding on montmorillonite are consistent with their forming a monolayer on the clay surface. This suggests the condensation of ImpA and ImpU to oligomers proceeds on the surface of the clay and not in groups of monomers stacked on the clay surfaces. The binding and reactions of ImpU and ImpA on montmorillonite are blocked by N⁶, N⁶-dimethyladenine and dA^5'ppdA. dA^5'ppdA is a better inhibitor of oligomer formation than N⁶, N⁶-dimethyladenine because both adenine rings of dA^5'ppdA bind to the clay surface and block adjacent catalytic sites. An upper limit of 5–10 × 10¹⁵catalytic sites on 50 mg of clay was estimated from the binding of ImpU and the inhibition of oligomer formation by dA^5'ppdA.     

A new affinity reagent for the site-specific, covalent attachment of DNA to active-site nucleophiles: application to the EcoRI and RsrI restriction and modification enzymes.

A modified oligodeoxyribonucleotide duplex containing an unnatural internucleotide trisubstituted 3' to 5' pyrophosphate bond in one strand [5'(oligo1)3'-P(OCH3)P-5'(oligo2) 3'] reacts with nucleophiles in aqueous media by acting as a phosphorylating affinity reagent. When interacted with a protein, a portion of the oligonucleotide [--P-5'(oligo2)3'] becomes attached to an amino acid nucleophilic group through a phosphate of the O-methyl-modified pyrophosphate linkage. We demonstrate the affinity labeling of nucleophilic groups at the active sites of the EcoRI and RsrI restriction and modification enzymes with an oligodeoxyribonucleotide duplex containing a modified scissile bond in the EcoRI recognition site. With the EcoRI and RsrI endonucleases in molar excess approximately 1% of the oligonucleotide becomes attached to the protein, and with the companion methyltransferases the yield approaches 40% for the EcoRI enzyme and 30% for the RsrI methyltransferase. Crosslinking proceeds only upon formation of a sequence-specific enzyme-DNA complex, and generates a covalent bond between the 3'-phosphate of the modified pyrophosphate in the substrate and a nucleophilic group at the active site of the enzyme. The reaction results in the elimination of an oligodeoxyribonucleotide remnant that contains the 3'-O-methylphosphate [5'(oligo1)3'-P(OCH3)] derived from the modified phosphate of the pyrophosphate linkage. Hydrolysis properties of the covalent protein-DNA adducts indicate that phosphoamide (P-N) bonds are formed with the EcoRI endonuclease and methyltransferase.     

Clay Catalysis of Oligonucleotide Formation: Kinetics of the Reaction of the 5'-Phosphorimidazolides of Nucleotides with the Non-Basic Heterocycles Uracil and Hypoxanthine

The montmorillonite clay catalyzed condensation of activated monocleotides to oligomers of RNA is a possible first step in the formation of the proposed RNA world. The rate constants for the condensation of the phosphorimidazolide of adenosine were measured previously and these studies have been extended to the phosphorimidazolides of inosine and uridine in the present work to determine if substitution of neutral heterocycles for the basic adenine ring changes the reaction rate or regioselectivity. The oligomerization reactions of the 5'-phosphoromidazolides of uridine (ImpU) and inosine (ImpI) on montmorillonite yield oligo(U)s and oligo(I)s as long as heptamers. The rate constants for oligonucleotide formation were determined by measuring the rates of formation of the oligomers by HPLC. Both the apparent rate constants in the reaction mixture and the rate constants on the clay surface were calculated using the partition coefficients of the oligomers between the aqueous and clay phases. The rate constants for trimer formation are much greater than those dimer synthesis but there was little difference in the rate constants for the formation of trimers and higher oligomers. The overall rates of oligomerization of the phosphorimidazolides of purine and pyrimidine nucleosides in the presence of montmorillonite clay are the same suggesting that RNA formed on the primitive Earth could have contained a variety of heterocyclic bases. The rate constants for oligomerization of pyrimidine nucleotides on the clay surface are significantly higher than those of purine nucleotides since the pyrimidine nucleotides bind less strongly to the clay than do the purine nucleotides. The differences in the binding is probably due to Van der Waals interactions between the purine bases and the clay surface. Differences in the basicity of the heterocyclic ring in the nucleotide have little effect on the oligomerization process.     

Studies of oligoadenylate formation on a poly(U) template

We have studied a variety of condensation reactions involving poly (U) as template and isomeric adenosine dinucleotides as substrates. We find that [3'-5']-linked dinucleotides such as A3pA and pA3pA are better acceptors than the corresponding [2'-5']-linked compounds, while ImpA2pA is a better donor than ImpA3pA. The reaction between A2pA and ImpA3pA, for example, yields only 4% of product while the reaction of A3pA with ImpA2pA yields 86% of product. The more efficient condensation reactions of dimers are about as efficient as the self-condensation of ImpA. They yield a few percent of material in which five or more substrate molecules are linked together. The percentage of the natural [3'-5']-linkage in the product varies greatly, from as little as 1% to as much as 45%.     

Oligo(methionyl) proteins. Enzymatic hydrolysis of the model isopeptides N epsilon-oligo(L-methionyl)-L-lysine

A number of model isopeptides containing oligo(methionine) chains varying in length (2-5 residues) covalently linked to the epsilon-amino group of lysine were synthesized by solid-phase procedures. Hydrolysis of these peptides by pepsin, chymotrypsin, cathepsin C (dipeptidyl peptidase IV) and intestinal aminopeptidase N was investigated using high-performance liquid chromatography to identify and quantify the hydrolysis products. Methionine oligomers grafted onto lysine were cleaved to tripeptides by pepsin. Chymotrypsin preferentially hydrolyzed the methionyl-methionine bond preceding the isopeptide bond. Cathepsin C released dimethionyl units from the covalently attached polymers. Intestinal aminopeptidase caused efficient hydrolysis of both peptides and isopeptide bonds although free methionine decreased the cleavage of the latter bond. Hydrophobic characteristics of oligo(methionine) chains promoted enzyme-catalyzed transpeptidations resulting probably from acyl-transfer-type reactions. Complementary hydrolysis of the isopeptides by these digestive enzymes suggests that covalent attachment of oligo(amino acid)s to food proteins may improve their nutritional value.     

Montmorillonite-catalysed formation of RNA oligomers: the possible role of catalysis in the origins of life

Large deposits of montmorillonite are present on the Earth today and it is believed to have been present at the time of the origin of life and has recently been detected on Mars. It is formed by aqueous weathering of volcanic ash. It catalyses the formation of oligomers of RNA that contain monomer units from 2 to 30-50. Oligomers of this length are formed because this catalyst controls the structure of the oligomers formed and does not generate all possible isomers. Evidence of sequence-, regio- and homochiral selectivity in these oligomers has been obtained. Postulates on the role of selective versus specific catalysts on the origins of life are discussed. An introduction to the origin of life is given with an emphasis on reaction conditions based on the recent data obtained from zircons 4.0-4.5Ga.     

The Prebiotic Leslie Orgel: Some of His Contributions to Our Understanding of the Origins of Life

In the previous recollection Jack Dunitz presented a delightful account of Leslie's pre-prebiotic life as a theoretical inorganic chemist. In this account I will present my recollections of his contributions, as a chemist-molecular biologist, to the field of origins of life.Leslie officially started his prebiotic research upon arrival at the Salk Institute in September 1964. Anna Beck and I, who arrived there at that time and Rolf Lohrmann and Robert Sanchez who joined the group within first year, were the vanguard of the large group of postdoctorals and graduate students that have spent time in Leslie's laboratory. John Sulston and Carl Woese were among the notables that spent time in Leslie's lab during his initial years at Salk.Leslie's emphasis on the new and different, which was described by Jack Dunitz, continued at Salk. He would greet me each day with well Jim, what's new! Or he would come upon an unusual reaction from the organic chemistry literature and ask me to explain it. Occasionally something new would occur in the lab and we would discuss its ramifications at great length throughout the day. He would often come up with a key suggestion on how this new finding should be extended to provide insight into an entirely different area. On other occasions he would suggest an empirical approach, such as setting up a series of reaction mixtures putting one of each of the metal salts in the lab inventory into each reaction, to determine if it was catalyzed by metal ions. This latter approach always seemed to me to be inconsistent with his background as a theoretician since I have yet to find a theoretician who would follow such a blatantly empirical approach to the discovery of new chemical reactions.Leslie claims to have no competence in the laboratory but he certainly understands what is required to obtain meaningful experimental data. First, he can often see through a forest of data and suggest a pivotal simple experiment to prove or disprove a hypothesis. Second, he has demanding standards for quality experimental design and execution. I have heard him on many occasions patiently (occasionally impatiently if it was a repeat offender) chastising a researcher in his laboratory ... for not performing the proper controls... to establish the validity of their experimental findings.Leslie started working on the prebiotic synthesis of nucleic acids and their replication when he came to Salk and was extremely successful in both studies. He improved the Oro synthesis of adenine (Oro, 1960) by extending it to dilute solutions of HCN (Ferris and Orgel, 1966) and to a general prebiotic synthesis of purines (Sanchez et al., 1966a). He also recognized that cyanoacetylene contained the carbon backbone of the pyrimidine ring and used it as a starting material for the prebiotic synthesis of pyrimidines (Sanchez et al., 1996b). Approaches to the prebiotic syntheses of nucleosides and nucleotides flowed logically from these studies (Beck et al., 1967; Lohrmann and Orgel, 1968, 1971; Fuller et al., 1972).Leslie conducted research on the nonenzymatic template-directed synthesis of RNA concurrently with studies on prebiotic synthesis. This research was initiated by the important discovery that the carbodiimide-driven condensation of A with pA gives higher yields of A²pA in the presence of poly(U) than in its absence (Sulston et al., 1968). Some minor reaction products included A³pA, A⁵pA and trimers. This discovery was followed by the substitution of an imidazole activated pA (ImpA) for the carbodiimide and pA in the reaction with A on a poly(U) template which gave much better yields of dimers and trimers (Weimann et al., 1968). In the following year this reaction was extended to the synthesis of oligo(G)s on a poly(C) template (Sulston et al., 1969), the system that was eventually shown to be optimal for probing the scope of nonenzymatic template-directed syntheses. The development of the RPC-5 column for the analysis of the oligonucleotides formed by template-directed synthesis was pivotal in characterizing the length and the regioselectivity of the phosphodiester bonds formed in these reactions (Lohrmann et al., 1980). A further advance was the development of the 2-methylimidazole activating group which resulted in the formation of oligo(G)s by template-directed synthesis that contain predominantly 3', 5'-linked phosphodiester bonds. This reaction proceeds in the solution phase (Inoue and Orgel, 1981) in contrast to the Zn²⁺- and Pb²⁺-catalyzed reactions which, while regioselective for the formation of the 3', 5'- and 2', 5'-linked phosphodiester bonds respectively, proceed via an insoluble metal complex (Lohrmann et al., 1980). Thus the 2-methylimidazole activating group made it possible to explore the efficiency of template-directed synthesis using templates containing different bases.The first self-replicating system, that was based on template-directed synthesis, was prepared by Günter von Kiedrowski in Leslie's laboratory in 1986 (von Kiedrowski, 1986). Leslie devised a replicater, which exhibited autocatalytic kinetics, that was also based on a templating reaction (Zielinski and Orgel, 1987).While prebiotic peptide synthesis was not a dominant theme in his laboratory Leslie did make important contributions to this field of work. He investigated the mechanism of peptide synthesis in aqueous solution using carbonyl diimidazole as the condensing agent (Ehler and Orgel, 1976) and he was able to make polypeptides containing 55 mers on mineral surfaces (Ferris et al., 1996). He proposed that the condensation of amino acids on the primitive Earth could have resulted in short polypeptides with alternating structures that would have formed small -sheets (Brack and Orgel, 1975). Subsequent studies have shown that some -sheets catalyze the hydrolysis of RNA (Barbier and Brack, 1992).Leslie, collaboratively with Francis Crick, has also published some theoretical papers dealing with the origin of life. The most perceptive was their anticipation of the RNA world before the name was coined by Gilbert (1986). In companion papers (Crick, 1968; Orgel, 1968), they suggested that RNA could have catalytic activity in addition to its ability to store genetic information, so that it could have been the basis for the first life. They noted that life based on protein was less likely because of its inability to preserve its sequence information by replication. In another very provocative paper they proposed that life on Earth may have been seeded here by intelligent aliens in a process they call Directed Panspermia (Crick and Orgel, 1973). They reckoned that the seeds of life might not survive the trip through open space but these initial microorganisms could have survived a trip through space if properly shielded in a spacecraft. In this scenario the Earth is a giant Petri dish and these aliens have performed an experiment using it. I have always felt that this paper had a major tongue-in-cheek component but it is often seriously mentioned as one possible explanation for the presence of life on Earth.In addition to his search for the novel and unusual Leslie also finds it challenging to search for the practical (again, not the usual practice of a theoretician!). He has the remarkable ability to appreciate the practical applications of his research and I know of at least two ideas that resulted in useful patents. (Some of the income from these patents is used to finance group parties at first rate restaurants.) One patent stemmed from the research on the prebiotic synthesis of pyrimidine nucleosides which Bob Sanchez developed into a an efficient preparation of the antileukemia agent cytosine arabinoside from cyanoacetylene, cyanamide and arabinose (Sanchez and Orgel, 1970). This successful patent is an excellent example of how the so called esoteric work in prebiotic chemistry led to a simple and new route to the synthesis of drugs or other useful materials. Another valuable patent came from the work of Barbara Chu which has been developed into a test for the genes from pathogens. She found that it was possible to prepare the 5'-imidazolides of high molecular weight QB RNA without affecting its autocatalytic replicating ability. This made it possible to attach cleavable reporter groups such as biotin or DNA probes to QB via the imidazolide intermediate, the selected DNA-QB probe could then be amplified by QB replicase, and easily detected after amplification (Chu et al., 1986).The search for useful therapeutic agents continues. In 1997 Leslie identified yet another possible application of Barbara's work. She made use of results from prebiotic chemistry showing that oligomers of glutamic acid bind irreversibly to hydroxylapatite, the predominant mineral in bone. She then attached potential therapeutic agents to the amino acid oligomers and demonstrated that they bound to the hydroxylapatite. This approach may be used to bind potential therapeutic agents to hydroxylapatite with the possibility that this might be a way to bring these agents to particular target site in bones (Chu and Orgel, 1997).The honors and awards that Leslie has received underline the high esteem in which he is held by his fellow scientists. He was recognized as a brilliant scientist by the British colleagues when he was awarded the Harrison prize of the Royal Society of Chemistry at the tender age of 30. Only five years later was he elected to the Royal Society. Among the awards he has received in the US are his election to the American Academy of Arts and Sciences in 1985, the National Academy of Sciences in 1990 and the Harold C. Urey medal of the International Society for the Study of the Origins of life in 1993. We in the field of the origins of life have not only benefitted from his major contributions to this field but the reputation of the field has also benefitted from having such a distinguished scientist as one of our own.Happy 70th birthday, Leslie! I thank you for baptizing me with HCN and sending me forth into the prebiotic world. It has been great fun as well as rewarding for me and I look forward to continuing our discussions of the origins of life for many years to come.