Centre for Chemical Biology, Department of Chemistry, Sheffield Institute for Nucleic Acids, University of Sheffield, Sheffield S3 7HF, UK.
QuantuMDx Group, Lugano Building, 57 Melbourne Street, Newcastle upon Tyne NE1 2JQ, UK.
Molecules. 2021 Apr 13;26(8):2250. doi: 10.3390/molecules26082250.
Modified 2'-deoxyribonucleotide triphosphates (dNTPs) have widespread applications in both existing and emerging biomolecular technologies. For such applications it is an essential requirement that the modified dNTPs be substrates for DNA polymerases. To date very few examples of C5-modified dNTPs bearing negatively charged functionality have been described, despite the fact that such nucleotides might potentially be valuable in diagnostic applications using Si-nanowire-based detection systems. Herein we have synthesised C5-modified dUTP and dCTP nucleotides each of which are labelled with an dianionic reporter group. The reporter group is tethered to the nucleobase via a polyethylene glycol (PEG)-based linkers of varying length. The substrate properties of these modified dNTPs with a variety of DNA polymerases have been investigated to study the effects of varying the length and mode of attachment of the PEG linker to the nucleobase. In general, nucleotides containing the PEG linker tethered to the nucleobase via an amide rather than an ether linkage proved to be the best substrates, whilst nucleotides containing PEG linkers from PEG to PEG could all be incorporated by one or more DNA polymerase. The polymerases most able to incorporate these modified nucleotides included Klentaq, Vent(-) and therminator, with incorporation by Klenow() generally being very poor.
修饰的 2'-脱氧核苷酸三磷酸 (dNTPs) 在现有的和新兴的生物分子技术中有着广泛的应用。对于这些应用，一个基本的要求是修饰的 dNTPs 必须是 DNA 聚合酶的底物。迄今为止，尽管具有负电荷功能的 C5 修饰的 dNTP 可能在使用硅纳米线检测系统的诊断应用中具有潜在价值，但很少有描述带有负电荷功能的 C5 修饰的 dNTP 例子。在此，我们合成了 C5 修饰的 dUTP 和 dCTP 核苷酸，它们每个都标记有一个二阴离子报告基团。报告基团通过聚乙二醇（PEG）连接子连接到碱基上，连接子的长度不同。研究了这些修饰的 dNTPs 与各种 DNA 聚合酶的底物性质，以研究改变 PEG 连接子与碱基的连接长度和方式的影响。一般来说，含有通过酰胺而不是醚键连接到碱基上的 PEG 连接子的核苷酸被证明是最好的底物，而含有从 PEG 到 PEG 的 PEG 连接子的核苷酸都可以被一种或多种 DNA 聚合酶掺入。最能掺入这些修饰核苷酸的聚合酶包括 Klentaq、Vent(-)和 therminator，而 Klenow()的掺入通常很差。
