Improved nucleotide selectivity and termination of 3'-OH unblocked reversible terminators by molecular tuning of 2-nitrobenzyl alkylated HOMedU triphosphates.
Improved nucleotide selectivity and termination of 3'-OH unblocked reversible terminators by molecular tuning of 2-nitrobenzyl alkylated HOMedU triphosphates.
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DOI：
10.1093/nar/gkq1293
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发表时间：
2011-03
影响因子：
14.9
通讯作者：
Metzker ML
中科院分区：
文献类型：
--
作者：
Litosh VA;Wu W;Stupi BP;Wang J;Morris SE;Hersh MN;Metzker ML
我们描述了一种新型 3′-OH 未封闭的可逆终止子，具有提高下一代测序 (NGS) 技术的准确性和读取长度的潜力。该终止子基于 5-羟甲基-2'-脱氧尿苷三磷酸 (HOMedUTP)，这是一种天然存在于众多噬菌体和低等真核生物基因组中的超修饰核苷酸。根据我们之前使用光化学可裂解终止子的工作，合成了一系列 5-(2-硝基苄氧基)甲基-dUTP 类似物 (dU.I–dU.V)。这些 2-硝基苄基烷基化 HOMedUTP 类似物的特征包括掺入、单碱基终止、核苷酸选择性和光化学裂解特性。人们发现，2-硝基苄基的 α-亚甲基碳被尺寸增大的烷基取代是一个关键的结构特征，它提供了酶性质的分子调节，例如单碱基终止和比天然核苷酸更高的核苷酸选择性。 5-[(S)-α-叔丁基-2-硝基苯甲氧基]甲基-dUTP (dU.V) 被确定为一种有效的可逆终止子，由此，在循环可逆终止 (CRT) 实验中使用十个互补模板碱基的均聚物重复序列证明了测序的可行性，在光化学裂解步骤中没有检测到紫外线损伤。这些结果验证了我们创建 3'-OH 未封闭可逆终止剂试剂的总体策略，该试剂在光化学裂解后转回自然状态。基于5-羟甲基-嘧啶和7-脱氮-7-羟甲基-嘌呤的修饰核苷酸为开发用于NGS技术的一整套四个可逆终止子奠定了基础。
We describe a novel 3′-OH unblocked reversible terminator with the potential to improve accuracy and read-lengths in next-generation sequencing (NGS) technologies. This terminator is based on 5-hydroxymethyl-2′-deoxyuridine triphosphate (HOMedUTP), a hypermodified nucleotide found naturally in the genomes of numerous bacteriophages and lower eukaryotes. A series of 5-(2-nitrobenzyloxy)methyl-dUTP analogs (dU.I–dU.V) were synthesized based on our previous work with photochemically cleavable terminators. These 2-nitrobenzyl alkylated HOMedUTP analogs were characterized with respect to incorporation, single-base termination, nucleotide selectivity and photochemical cleavage properties. Substitution at the α-methylene carbon of 2-nitrobenzyl with alkyl groups of increasing size was discovered as a key structural feature that provided for the molecular tuning of enzymatic properties such as single-base termination and improved nucleotide selectivity over that of natural nucleotides. 5-[(S)-α-tert-Butyl-2-nitrobenzyloxy]methyl-dUTP (dU.V) was identified as an efficient reversible terminator, whereby, sequencing feasibility was demonstrated in a cyclic reversible termination (CRT) experiment using a homopolymer repeat of ten complementary template bases without detectable UV damage during photochemical cleavage steps. These results validate our overall strategy of creating 3′-OH unblocked reversible terminator reagents that, upon photochemical cleavage, transform back into a natural state. Modified nucleotides based on 5-hydroxymethyl-pyrimidines and 7-deaza-7-hydroxymethyl-purines lay the foundation for development of a complete set of four reversible terminators for application in NGS technologies.