Effect of Molecular Crowding on DNA Polymerase Reactions along Unnatural DNA Templates

Shuntaro Takahashi, Piet Herdwijn, Naoki Sugimoto
2020 Molecules  
Unnatural nucleic acids are promising materials to expand genetic information beyond the natural bases. During replication, substrate nucleotide incorporation should be strictly controlled for optimal base pairing with template strand bases. Base-pairing interactions occur via hydrogen bonding and base stacking, which could be perturbed by the chemical environment. Although unnatural nucleobases and sugar moieties have undergone extensive structural improvement for intended polymerization, the
more » ... olymerization, the chemical environmental effect on the reaction is less understood. In this study, we investigated how molecular crowding could affect native DNA polymerization along various templates comprising unnatural nucleobases and sugars. Under non-crowding conditions, the preferred incorporation efficiency of pyrimidine deoxynucleotide triphosphates (dNTPs) by the Klenow fragment (KF) was generally high with low fidelity, whereas that of purine dNTPs was the opposite. However, under crowding conditions, the efficiency remained almost unchanged with varying preferences in each case. These results suggest that hydrogen bonding and base-stacking interactions could be perturbed by crowding conditions in the bulk solution and polymerase active center during transient base pairing before polymerization. This study highlights that unintended dNTP incorporation against unnatural nucleosides could be differentiated in cases of intracellular reactions.
doi:10.3390/molecules25184120 pmid:32927591 fatcat:vs2voj4yyvhmfmfn3r6jcivhv4