A high-throughput screening method for evolving a demethylase enzyme with improved and new functionalities.
A high-throughput screening method for evolving a demethylase enzyme with improved and new functionalities.
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DOI：
10.1093/nar/gkaa1213
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发表时间：
2021-03-18
影响因子：
14.9
通讯作者：
Pan T
中科院分区：
文献类型：
--
作者：
Wang Y;Katanski CD;Watkins C;Pan JN;Dai Q;Jiang Z;Pan T
AlkB是一种DNA/RNA修复酶，可从DNA和RNA中去除碱基烷基化，如n1 -甲基腺苷（m1A）或n3 -甲基胞嘧啶（m3C）。AlkB酶已被用作促进tRNA测序和鉴定mRNA修饰的关键工具。作为一种工具，人们迫切需要具有更好反应性和新功能的AlkB突变体；然而，以前对这类AlkB突变体的鉴定是基于经典的靶向诱变方法。在这里，我们介绍了一种高通量筛选方法来评估AlkB变体库在RNA和DNA底物上的去甲基化活性。该方法基于具有内部修饰的RNA/DNA残基的荧光性RNA适体，该适体可以阻断逆转录或引入导致cDNA产物固有荧光丧失的突变。AlkB变体的去甲基化消除了阻塞或突变，从而恢复了荧光信号。我们将我们的筛选方法应用于大肠杆菌AlkB蛋白的D135和R210位点，并鉴定出一种比先前已知的RNA中n1 -甲基鸟苷（m1G）的高活性突变体具有更高活性的变体。我们还将该方法应用于o6 -甲基鸟苷（O6mG）修饰的DNA底物，并鉴定出具有去甲基化活性的候选AlkB变体。我们的研究为任何脱甲基酶的体外进化提供了一种高通量筛选方法。
AlkB is a DNA/RNA repair enzyme that removes base alkylations such as N1-methyladenosine (m1A) or N3-methylcytosine (m3C) from DNA and RNA. The AlkB enzyme has been used as a critical tool to facilitate tRNA sequencing and identification of mRNA modifications. As a tool, AlkB mutants with better reactivity and new functionalities are highly desired; however, previous identification of such AlkB mutants was based on the classical approach of targeted mutagenesis. Here, we introduce a high-throughput screening method to evaluate libraries of AlkB variants for demethylation activity on RNA and DNA substrates. This method is based on a fluorogenic RNA aptamer with an internal modified RNA/DNA residue which can block reverse transcription or introduce mutations leading to loss of fluorescence inherent in the cDNA product. Demethylation by an AlkB variant eliminates the blockage or mutation thereby restores the fluorescence signals. We applied our screening method to sites D135 and R210 in the Escherichia coli AlkB protein and identified a variant with improved activity beyond a previously known hyperactive mutant toward N1-methylguanosine (m1G) in RNA. We also applied our method to O6-methylguanosine (O6mG) modified DNA substrates and identified candidate AlkB variants with demethylating activity. Our study provides a high-throughput screening method for in vitro evolution of any demethylase enzyme.
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影响因子：
62.1
作者：
Zheng, Guanqun;Fu, Ye;He, Chuan
通讯作者：
He, Chuan
影响因子：
8.6
作者：
Svensen N;Jaffrey SR
通讯作者：
Jaffrey SR
影响因子：
64.8
作者：
EADIE, JS;CONRAD, M;TOPAL, MD
通讯作者：
TOPAL, MD
影响因子：
48
作者：
Cozen AE;Quartley E;Holmes AD;Hrabeta-Robinson E;Phizicky EM;Lowe TM
通讯作者：
Lowe TM
DOI：
10.1073/pnas.0812938106
发表时间：
2009-08-25
影响因子：
11.1
作者：
Yu, Bomina;Hunt, John F.
通讯作者：
Hunt, John F.