Fluorescent Tricyclic Cytidine Analogues as Substrates for Retroviral Reverse Transcriptases
Fluorescent Tricyclic Cytidine Analogues as Substrates for Retroviral Reverse Transcriptases
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荧光三环胞苷类似物作为逆转录病毒逆转录酶的底物
DOI：
10.1002/cplu.202000140
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发表时间：
2020
期刊：
影响因子：
3.4
通讯作者：
Purse, Byron W.
中科院分区：
文献类型：
--
作者：
Turner, M. Benjamin;Purse, Byron W.
我们报告了来自禽成髓细胞瘤病毒（AMV）、莫洛尼鼠白血病病毒（M-MLV）和人类免疫缺陷病毒1（HIV-1）的逆转录酶（RT）使用荧光三环胞苷类似物d（tC）TP和d（DEAtC）TP作为底物生成标记DNA的能力。这些类似物插入的Michaelis-Menten动力学显示Vmax/Km为天然dCTP的0.09-5倍，这取决于聚合酶以及模板是RNA还是DNA。这些类似物在RNA和DNA模板中都容易从腺苷上错误插入。类似物插入后的延伸对于RNA模板是有效的，但是类似物在插入DNA模板后引起停滞。使用HIV-1-RT的模型逆转录试验，包括RNA依赖性DNA合成，RT的RNase H活性降解RNA模板，以及合成第二条DNA链以形成荧光标记的dsDNA，表明d（tC）TP和d（DEAtC）TP与体外完整的逆转录循环相容。
We report on the ability of the reverse transcriptases (RTs) from avian myeloblastosis virus (AMV), Moloney murine leukemia virus (M‐MLV), and human immunodeficiency virus 1 (HIV‐1) to generate labeled DNA using the fluorescent tricyclic cytidine analogues d(tC)TP and d(DEAtC)TP as substrates. Michaelis‐Menten kinetics for the insertion of these analogues showVmax/KMfrom 0.09–5 times that of natural dCTP across from G, depending on the polymerase and whether the template is RNA or DNA. The analogues are prone to misinsertion across from adenosine with both RNA and DNA templates. Elongation after analogue insertion is efficient with RNA templates, but the analogues cause stalling after insertion with DNA templates. A model reverse transcription assay using HIV‐1‐RT, including RNA‐dependent DNA synthesis, degradation of the RNA template by the RT's RNase H activity, and synthesis of a second DNA strand to form fluorescently labeled dsDNA, shows that d(tC)TP and d(DEAtC)TP are compatible with a complete reverse transcription cyclein vitro.