Additive‐Free Enzymatic Phosphorylation and Ligation of Artificial Oligonucleotides with C‐Nucleosides at the Reaction Points
Additive‐Free Enzymatic Phosphorylation and Ligation of Artificial Oligonucleotides with C‐Nucleosides at the Reaction Points
复制标题
无添加剂酶促磷酸化以及人工寡核苷酸与 C-核苷在反应点的连接
DOI：
10.1002/cbic.201900217
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发表时间：
2019
期刊：
影响因子：
3.2
通讯作者：
Inouye Masahiko
中科院分区：
文献类型：
--
作者：
Oda Yutaro;Chiba Junya;Kurosaki Fumihiro;Yamade Yusuke;Inouye Masahiko
我们报道了通过T4多核苷酸激酶和T4 DNA连接酶的作用，酶磷酸化和无添加剂连接含有非天然C核苷酸残基的DNA。每个人工单元都由炔基脱氧核糖组分和一种非天然核碱基D*、T*、G*和C*组成，从氢键模式的观点来看，它们分别对应于天然的a、T、G和C。嵌合dna中所有人工单位的5′端磷酸化都有定量进展。在D*和G*核苷酸残基的情况下，连接也在5 '端顺利进行，但在T*和C*核苷酸残基的情况下，连接几乎可以忽略不计。最后两个单元的化学重新设计成功地提高了连接效率，因此在切口处的3 ' -自然⋅5 ' -人工、3 ' -人工⋅5 ' -自然和3 ' -人工⋅5 ' -人工末端组合中，酶的连接对所有人工单元都很有效。
We report enzymatic phosphorylation and additive‐free ligation of DNAs containing unnatural C‐nucleotide residues through the action of T4 polynucleotide kinase and T4 DNA ligase. The artificial units are each made up of an alkynyl deoxyribose component and one of the unnatural nucleobases D*, T*, G*, and C*, corresponding—from a viewpoint of hydrogen‐bonding patterns—to natural A, T, G, and C, respectively. Phosphorylation progressed quantitatively at the 5′‐end in the cases of all of the artificial units in the chimeric DNAs. Ligation also smoothly progressed at the 5′‐end in the cases of the D* and G* nucleotide residues, but only negligibly in those of their T* and C* counterparts. Chemical redesign of the last two units successfully improved the ligation efficiency, so that enzymatic ligation worked well for all of the artificial units in every 3′‐natural⋅5′‐artificial, 3′‐artificial⋅5′‐natural, and 3′‐artificial⋅5′‐artificial terminal combination at the nicks.