Frontier Institute for Biomolecular Engineering Research (FIBER), Konan University, 7-1-20 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan.
Medicinal Chemistry, KU Leuven, Rega Institute for Medical Research, Herestraat 49-box 1041, 3000 Leuven, Belgium.
Molecules. 2020 Sep 10;25(18):4120. doi: 10.3390/molecules25184120.
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 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.
非天然核酸是一种很有前途的材料，可以将遗传信息扩展到天然碱基之外。在复制过程中，底物核苷酸的掺入应该严格控制，以与模板链碱基实现最佳碱基配对。碱基配对通过氢键和碱基堆积发生，这可能会受到化学环境的干扰。尽管非天然碱基和糖基已经经历了广泛的结构改进以实现预期的聚合，但对反应的化学环境影响了解较少。在这项研究中，我们研究了分子拥挤如何影响包括非天然碱基和糖基的各种模板上的天然 DNA 聚合。在非拥挤条件下，Klenow 片段（KF）对嘧啶脱氧核苷酸三磷酸（dNTP）的优先掺入效率通常很高，但保真度较低，而嘌呤 dNTP 则相反。然而，在拥挤条件下，每种情况下的效率几乎保持不变，但偏好程度不同。这些结果表明，氢键和碱基堆积相互作用可能会在聚合前短暂碱基配对过程中被体相溶液和聚合酶活性中心的拥挤条件干扰。这项研究强调，在细胞内反应中，非天然核苷的非预期 dNTP 掺入可以得到区分。
