Template-directed synthesis on the pentanucleotide CpCpGpCpC

The pentanucleotide CpCpGpCpC facilitates the synthesis of oligomers containing G and C from a mixture of the two activated mononucleotides (guanosine 5'-phosphor)-2-methylimidazolide and (cytidine 5'-phosphor)-2-methylimidazolide. The major pentameric product of the template-directed reaction is all 3' to 5'-linked and has the sequence pGpGpCpGpG, which is complementary to that of the template. It can be obtained in a yield of up to 17%, based on the input of the template. The 3' to 5' isomer of GpG is elongated on the template to give GpGpC, GpGpCpG and GpGpCpGpG, while the 2' to 5' isomer does not initiate the synthesis of detectable amounts of longer oligomers.     

Non-enzymatic template-directed synthesis of RNA copolymers

Cytosine-rich RNA copolymers facilitate the template-directed synthesis of complementary oligomers from mononucleotide 5'-phospho-2-methylimidazolides (Fig. 1). The efficiency of this reaction falls sharply as the ratio of cytosine to non-cytosine in the template is decreased. This is a severe limitation for self-replication because it means that any cytosine-rich polynucleotide that can serve as a good template will produce a cytosine-poor complementary strand that is unable to direct further rounds of synthesis. Studies with low-ratio random copolymer templates have shown that the efficiency can be increased by adjusting initial monomer concentrations and by providing additional activated monomers during later stages of the reaction. The oligomeric reaction products can be studied in detail using high performance liquid chromatography. It is possible to separate oligonucleotides on the basis of chain length and base composition. Thus a wealth of information is available to characterize the distribution of products over the course of the reaction and under a variety of reaction conditions.     

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.     

Montmorillonite,Oligonucleotides, RNA and Origin of Life

Na-montmorillonite prepared from Volclay by the titration method facilitates the self-condensation of ImpA, the 5'-phosphorimidazolide derivative of adenosine. As was shown by AE-HPLC analysis and selective enzymatic hydrolysis of products, oligo(A)s formed in this reaction are 10 monomer units long and contain 67% 3',5'-phosphodiester bonds (Ferris and Ertem, 1992a). Under the same reaction conditions, 5'-phosphorimidazolide derivatives of cytidine, uridine and guanosine also undergo self-condensation producing oligomers containing up to 12-14 monomer units for oligo(C)s to 6 monomer units for oligo(G)s. In oligo(C)s and oligo(U)s, 75-80% of the monomers are linked by 2',5'-phosphodiester bonds. Hexamer and higher oligomers isolated from synthetic oligo(C)s formed by montmorillonite catalysis, which contain both 3',5'- and 2',5'-linkages, serve as catalysts for the non-enzymatic template directed synthesis of oligo(G)s from activated monomer 2-MeImpG, guanosine 5'-phospho-2-methylimidazolide (Ertem and Ferris, 1996). Pentamer and higher oligomers containing exclusively 2',5'-linkages, which were isolated from the synthetic oligo(C)s, also serve as templates and produce oligo(G)s with both 2',5'- and 3',5'-phosphodiester bonds. Kinetic studies on montmorillonite catalyzed elongation rates of oligomers using the computer program SIMFIT demonstrated that the rate constants for the formation of oligo(A)s increased in the order of 2-mer < 3-mer < 4-mer ... < 7-mer (Kawamura and Ferris, 1994). A decameric primer, dA(pdA)8pA bound to montmorillonite was elongated to contain up to 50 monomer units by daily addition of activated monomer ImpA to the reaction mixture (Ferris, Hill and Orgel, 1996). Analysis of dimer fractions formed in the montmorillonite catalyzed reaction of binary and quaternary mixtures of ImpA, ImpC, 2-MeImpG and ImpU suggested that only a limited number of oligomers could have formed on the primitive Earth rather than equal amounts of all possible isomers (Ertem and Ferris, 2000). Formation of phosphodiester bonds between mononucleotides by montmorillonite catalysis is a fascinating discovery, and a significant step forward in efforts to find out how the first RNA-like oligomers might have formed in the course of chemical evolution. However, as has been pointed out in several publications, these systems should be regarded as models rather than a literal representation of prebiotic chemistry (Orgel, 1998; Joyce and Orgel, 1999; Schwartz, 1999).     

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.     

Simple and rapid enzymatic method for the synthesis of single-strand oligonucleotides containing trifluorothymidine

To investigate the mechanism of trifluorothymidine (TFT)-induced DNA damage, we developed an enzymatic method for the synthesis of single-strand oligonucleotides containing TFT-monophosphate residues. Sixteen-mer oligonucleotides and 14-mer 5'-phosphorylated oligonucleotides were annealed to the template of 25-mer, so as to empty one nucleotide site. TFT-triphosphate was incorporated into the site by DNA polymerase and then ligated to 5'-phosphorylated oligonucleotides by DNA ligase. The synthesized 31-mer oligonucleotides containing TFT residues were isolated from the 25-mer complementary template by denaturing polyacrylamide electrophoresis. Using these single-strand oligonucleotides containing TFT residues, the cleavage of TFT residues from DNA, using mismatch uracil-DNA glycosylase (MUG) of E.coli origin, was compared with that of 5-fluorouracil (5FU) and 5-bromodeoxyuridine (BrdU). The TFT/A pair was not cleaved by MUG, while the other pairs, namely, 5FU/A, 5FU/G, BrdU/A, BrdU/G, and TFT/G, were easily cleaved from each synthesized DNA. Thus, this method is useful for obtaining some site-specifically modified oligonucleotides.     

Simple and Rapid Enzymatic Method for the Synthesis of Single-Strand Oligonucleotides Containing Trifluorothymidine

To investigate the mechanism of trifluorothymidine (TFT)-induced DNA damage, we developed an enzymatic method for the synthesis of single-strand oligonucleotides containing TFT-monophosphate residues. Sixteen-mer oligonucleotides and 14-mer 5′-phosphorylated oligonucleotides were annealed to the template of 25-mer, so as to empty one nucleotide site. TFT-triphosphate was incorporated into the site by DNA polymerase and then ligated to 5′-phosphorylated oligonucleotides by DNA ligase. The synthesized 31-mer oligonucleotides containing TFT residues were isolated from the 25-mer complementary template by denaturing polyacrylamide electrophoresis. Using these single-strand oligonucleotides containing TFT residues, the cleavage of TFT residues from DNA, using mismatch uracil-DNA glycosylase (MUG) of E.coli origin, was compared with that of 5-fluorouracil (5FU) and 5-bromodeoxyuridine (BrdU). The TFT/A pair was not cleaved by MUG, while the other pairs, namely, 5FU/A, 5FU/G, BrdU/A, BrdU/G, and TFT/G, were easily cleaved from each synthesized DNA. Thus, this method is useful for obtaining some site-specifically modified oligonucleotides.     

Template-directed synthesis of oligonucleotides under eutectic conditions

Summary One of the most important sets of model prebiotic experiments consists of reactions that synthesize complementary oligonucleotides from preformed templates under nonenzymatic conditions. Most of these experiments are conducted at 4°C using 0.01–0.1 M concentrations of activated nucleotide monomer and template (monomer equivalent). In an attempt to extend the conditions under which this type of reaction can occur, we have concentrated the reactants by freezing at –18°C, which is close to the NaCl–H₂O eutectic at –21°C.The results from this set of experiments suggest that successful syntheses can occur with poly(C) concentrations as low at 5×10^–4 M and 2MeImpG concentrations at 10^–3 M. It was also anticipated that this mechanism might allow the previously unsuccessful poly(A)-directed synthesis of oligo(U)s to occur. However, no template effect was seen with the poly(A) and ImpU system. The failure of these conditions to allow template-directed synthesis of oligo(U)s supports the previously proposed idea that pyrimidines may not have been part of the earliest genetic material.Because of the low concentrations of monomer and template that would be expected from prebiotic syntheses, this lower temperature could be considered a more plausible geologic setting for template-directed synthesis than the standard reaction conditions.     

The template properties of some oligodeoxynucleotides containing cytidine and guanosine

Summary the co-condensation of guanosine- and cytidine-5-phospho-2-methylimidazolide on various oligodeoxynucleotides containing C and G has been studied. We find that GC₇ is an effective template for the incorporation of C into products of the form G_nC, whereas C₇G does not act as a template for C incorporation. The template C₃GC₃GC₃GC₃ directs the synthesis of complementary products, but the yield of long oligomers is very small. Templates in which G residues are contiguous or separated by a single C residue are ineffective, while templates containing the sequence GCCG are very inefficient. The significance of these findings in the context of prebiotic chemistry is discussed.     

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.     

Thermolytic release of covalently linked DNA oligonucleotides and their conjugates from controlled-pore glass at near neutral pH

The functionalization of long chain alkylamine controlled-pore glass (CPG) with a 3-hydroxypropyl-(2-cyanoethyl)thiophosphoryl linker and its conversion to the support 7 has led to the synthesis of DNA oligonucleotides and their 3'- or (3',5')-conjugates. Indeed, CPG support 7 has been successfully employed in the synthesis of both native and fully phosphorothioated DNA 20-mers. Unlike conventional succinylated CPG supports, this distinctively functionalized support allows oligonucleotide deprotection and removal of the deprotection side products to proceed without releasing the oligonucleotide into the aqueous milieu. When freed from deprotection side products, the DNA oligonucleotide is thermolytically released from the support within 2 h under nearly neutral conditions (pH 7.2, 90 degrees C). The quality of these oligonucleotides is comparable to that of identical oligonucleotides synthesized from succinylated CPG supports in terms of shorter than full length oligonucleotide contaminants and overall yields. The versatility of the thermolytic CPG support 7 is further demonstrated by the synthesis of a DNA oligonucleotide (20-mer) and its conjugation with an azido and alkynyl groups at both 5'-and 3'-termini, respectively. The functionality of the (3',5')-heteroconjugated oligonucleotide 18 is verified by its circularization to the DNA oligonucleotide 19 under "click" chemistry conditions.     

Non-enzymatic template-directed synthesis of RNA copolymers

Cytosine-rich RNA copolymers facilitate the template-directed synthesis of complementary oligomers from mononucleotide 5′-phospho-2-methylimidazolides (Fig. 1). The efficiency of this reaction falls sharply as the ratio of cytosine to non-cytosine in the template is decreased. This is a severe limitation for self-replication because it means that any cytosine-rich polynucleotide that can serve as a good template will produce a cytosine-poor complementary strand that is unable to direct further rounds of synthesis. Studies with low-ratio random copolymer templates have shown that the efficiency can be increased by adjusting initial monomer concentrations and by providing additional activated monomers during later stages of the reaction. The oligomeric reaction products can be studied in detail using high performance liquid chromatography. It is possible to separate oligonucleotides on the basis of chain length and base composition. Thus a wealth of information is available to characterize the distribution of products over the course of the reaction and under a variety of reaction conditions.     

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.     

Kinetic Analysis of Oligo(C) Formation from the 5′-Monophosphorimidazolide of Cytidine with Pb(II) Ion Catalyst at 10–75°C

The temperature dependence of the rate constants for the formation of oligocytidylate (oligo(C)) from the 5′-monophosphorimidazolide of cytidine (ImpC) in the presence of Pb(II) ion catalyst has been investigated at 10–75°C. The rate constants for the formation of oligo(C) increased in the order of the formation of 2-mer < 3-mer ≤ 4-mer; this trend resembles the trend in the cases of the template-directed and the clay-catalyzed formations of oligonucleotides. While the rate constants of the formation of oligo(C) increased with increasing temperature, the yield of oligo(C) decreased with increasing temperature. This is due to the fact that the relative magnitude of the rate constants of the formation of 2-mer, 3-mer, and 4-mer to that of the hydrolysis of ImpC decreased with increasing temperature. This is probably due to the fact that association between ImpC with the elongating oligo(C) decreases with increasing temperature. The apparent activation energy was 61.9 ± 8.5 kJ mol⁻¹ for the formation of 2-mer, 49.3 ± 2.9 kJ mol⁻¹ for 3-mer, 51.8 kJ mol⁻¹ for 4-mer, and 66.8 ± 4.5 kJ mol⁻¹ for the hydrolysis of ImpC. The significance of the temperature dependence of the formation rate constants of the model prebiotic formation of RNA is discussed.     

Telomerase is processive.

Telomerase synthesizes tandem repeats of the sequence d(TTGGGG) onto input d(TTGGGG)n primer oligonucleotides (C. W. Greider and E. H. Blackburn, Cell 43:405-413). An intrinsic RNA component of the enzyme provides the template for d(TTGGGG)n repeat synthesis [C. W. Greider and E. H. Blackburn, Nature (London) 337:331-337, 1989; G.-L. Lu, J. D. Bradley, L. D. Attardi, and E. H. Blackburn, Nature (London) 344:126-132, 1990]. In a typical reaction, products greater than 2,000 nucleotides were synthesized in 60 min. Dilution and primer challenge experiments showed that these long products were synthesized processively. The apparent processivity was not due to a higher affinity of the enzyme for long d(TTGGGG) products over the shorter competitors. The degree of processivity was quantitated; telomerase synthesized approximately 520 nucleotides before half of the enzyme had dissociated. After dissociating, telomerase reinitiated d(TTGGGG)n synthesis on new primer oligonucleotides. The products from a telomerase reaction have a characteristic 6-nucleotide banding pattern (C. W. Greider and E. H. Blackburn, Cell 51:887-898, 1987). A strong pause in the reaction occurs after the addition of the first G in the sequence d(TTGGGG). Both the processivity and the banding pattern analysis imply that in the elongation mechanism there must be a translocation step after the 9 nucleotides of internal template RNA have been copied to the extreme 5' end.     

Synthesis and properties of oligodeoxyribonucleotids with mono- and diphosphoryldisulfide internucleotide groupings

The synthesis of oligodeoxyribonucleotides bearing mono- and diphosphoryldisulfide internucleotide links was optimized. Oligonucleotide 3'-thiophosphorothioates were modified using the thiophosphoryl-disulfide exchange with preactivated 5'-deoxy-5'-mercaptooligonucleotides or 5'-phosphorothioate derivatives both with and without a complementary template. The lack of template was shown to differently affect the product ratio (homo- and heterodimers) in the reactions of mono- and diphosphoryldisulfide-containing oligonucleotides. A replacement of one natural phosphodiester bond in 15-16-mer duplexes by a mono- or diphosphoryldisulfide group causes a slight thermal destabilization of the corresponding duplex. The disulfide recombination of the resulting compounds was studied.     

2'-modified nucleosides for site-specific labeling of oligonucleotides.

We report the synthesis of 2'-modified nucleosides designed specifically for incorporating labels into oligonucleotides. Conversion of these nucleosides to phosphoramidite and solid support-bound derivatives proceeds in good yield. Large-scale synthesis of 11-mer oligonucleotides possessing the 2'-modified nucleosides is achieved using these derivatives. Thermal denaturation studies indicate that the presence of 2'-modified nucleosides in 11-mer duplexes has minimal destabilizing effects on the duplex structure when the nucleosides are placed at the duplex termini. The powerful combination of phosphoramidite and support-bound derivatives of 2'-modified nucleosides affords the large-scale preparation of an entirely new class of oligonucleotides. The ability to synthesize oligonucleotides containing label attachment sites at 3', intervening, and 5' locations of a duplex is a significant advance in the development of oligonucleotide conjugates.     

Site-specific binding constants for actinomycin D on DNA determined from footprinting studies.

We report site-specific binding constants for the intercalating anticancer drug actinomycin D (Act-D), binding to a 139-base-pair restriction fragment from pBR 322 DNA. The binding constants are derived from analysis of footprinting experiments, in which the radiolabeled 139-mer is cleaved using DNase I, the cleavage products undergo gel electrophoresis, and, from the gel autoradiogram, spot intensities, proportional to amounts of cleaved fragments, are measured. A bound drug prevents DNase I from cleaving at approximately 7 bonds, leading to decreased amounts of corresponding fragments. With the radiolabel on the 3' end of the noncoding strand (A-label), we measured relative amounts of 54 cleavage products at 25 Act-D concentrations. For cleavage of the 139-mer with the label on the 3' end of the coding strand (G-label), relative amounts of 43 cleavage products at 11 Act-D concentrations were measured. These measurements give information about approximately 120 base pairs of the restriction fragment (approximately 12 turns of the DNA helix); in this region, 14 strong and weak Act-D binding sites were identified. The model used to interpret the footprinting plots is derived in detail. Binding constants for 14 sites on the fragment are obtained simultaneously. It is important to take into account the effect of drug binding at its various sites on the local concentration of probe elsewhere. It is also necessary to include in the model weak as well as strong Act-D sites on the carrier DNA which is present, since the carrier DNA controls the free-drug concentration. As expected, the strongest sites are those with the sequence (all sequences are 5'----3') GC, with TGCT having the highest binding constant, 6.4 x 10(6) M-1. Sites having the sequence GC preceded by G are weak binding sites, having binding constants approximately 1 order of magnitude lower than those of the strong sites. Also, the non-GC-containing sequences CCG and CCC bind Act-D with a binding constant comparable to those of the weak GGC sites. The analysis may reveal drug-induced structural changes on the DNA, which are discussed in terms of the mechanism of Act-D binding.     

Kinetic analysis of the template effect in ribooligoguanylate elongation

We have undertaken a complete kinetic analysis of the template-directed oligoguanylate synthesis originated in Orgel's laboratory (Inoue and Orgel, 1982). The reaction of guanosine 5′-phospho-2-methylimidazolide, 2-MelmpG, with ribooligoguanylates all 3′–5′ linked, designatedn ³ withn=7−12, was studied in the presence/absence of the complementary template polycytidylic acid, poly(C). Conditions were chosen where poly(C) and 2-MelmpG are in large excess over the oligoguanylate. In the absence of the template at 37 °C the reaction leads to three isomeric oligomers that are elongated by one monomer unit. They are the 3′–5′ linked, (n+1)³, the 2′–5′ linked, (n+1)², and the pyrophosphate product, (n+1) ^p , formed in an approximate ratio 1:2:5. In the presence of the template the reaction is 20-fold faster and yields productsn+1,n+2,n+3 etc. as long as 2-MelmpG is available. Most importantly the formation of the natural, 3′–5′ linked isomer, is enhanced selectively by 140-fold at 37 °C. Qualitative observations allow the conclusion that this enhancement is temperature dependent and increases with decreasing temperature. For example, at 1 °C only the 3′–5′ linked isomers were detected. Initial rates for the disappearance of then ³ oligoguanylate were determined at 1, 23, and 37 °C. It was found that the pseudo-first order rate constant for oligoguanylate elongation was linearly proportional to the 2-MelmpG concentration. This implies that the reaction complex poly(C)·n ³·2-MelmpG does not accumulate under the reaction conditions, a conclusion which is also supported by infrared data (Miles and Frazier, 1982). The implication of the above results with respect to chemical evolution is that lower temperatures, i.e., close to freezing, enhance the regioselectivity of these template-directed reactions and that one way to improve replication models may be sought in finding conditions that favor stable reaction complexes. NASA — National Research Council Research Fellow.