Computer simulation in template-directed oligonucleotide synthesis

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.