Site-Specific Covalent Labeling of DNA Substrates by an RNA Transglycosylase.
Site-Specific Covalent Labeling of DNA Substrates by an RNA Transglycosylase.
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DOI：
10.1021/jacs.3c00861
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发表时间：
2023-04-12
影响因子：
15
通讯作者：
Devaraj, Neal K.
中科院分区：
文献类型：
--
作者：
Tota, Ember M.;Devaraj, Neal K.
细菌tRNA鸟嘌呤转糖基酶（TGT）催化鸟嘌呤与7-脱氮鸟嘌呤鸟嘌呤前体前鸟苷1（preQ 1）的交换。虽然细菌TGT的天然核酸底物是丝氨酸同源tRNA的反密码子环，但该酶的最小识别序列是在“UGU”环基序中含有靶G核碱基的结构化发夹。先前的工作已经建立了RNA修饰系统，RNA-TAG，其中大肠杆菌TGT将目标RNA上的靶标G交换为与小分子报告分子（如生物素或荧光团）连接的化学修饰的preQ 1底物。虽然将RNA转糖基酶的底物范围扩展到包括DNA将使许多应用成为可能，但先前已报道TGT不能修饰天然DNA。在这里，我们证明了TGT实际上可以识别和标记特定的DNA底物。通过对合理突变的DNA发夹序列的反复测试，我们确定了E. coli TGT。控制DNA发夹中的空间限制显著影响标记效率，并且当优化时，可以导致接近定量的位点特异性修饰。我们证明了我们新开发的DNA-TAG系统的实用性，通过快速合成探针的荧光北方印迹剪接体U6 RNA和RNA FISH可视化的长非编码RNA，转移相关的肺腺癌转录1（MALAT 1）。DNA-TAG系统的简便性和便利性将为研究人员提供一种工具，用于访问各种各样的通用和负担得起的修饰DNA底物。
Bacterial tRNA guanine transglycosylases (TGTs) catalyze the exchange of guanine for the 7-deazaguanine queuine precursor, prequeuosine1 (preQ1). While the native nucleic acid substrate for bacterial TGTs is the anticodon loop of queuine-cognate tRNAs, the minimum recognition sequence for the enzyme is a structured hairpin containing the target G nucleobase in a “UGU” loop motif. Previous work has established an RNA modification system, RNA-TAG, in which Escherichia coli TGT exchanges the target G on an RNA of interest for chemically modified preQ1 substrates linked to a small-molecule reporter such as biotin or a fluorophore. While extending the substrate scope of RNA transglycosylases to include DNA would enable numerous applications, it has been previously reported that TGT is incapable of modifying native DNA. Here, we demonstrate that TGT can in fact recognize and label specific DNA substrates. Through iterative testing of rationally mutated DNA hairpin sequences, we determined the minimal sequence requirements for transglycosylation of unmodified DNA by E. coli TGT. Controlling steric constraint in the DNA hairpin dramatically affects labeling efficiency, and, when optimized, can lead to near-quantitative site-specific modification. We demonstrate the utility of our newly developed DNA-TAG system by rapidly synthesizing probes for fluorescent Northern blotting of spliceosomal U6 RNA and RNA FISH visualization of the long noncoding RNA, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). The ease and convenience of the DNA-TAG system will provide researchers with a tool for accessing a wide variety of versatile and affordable modified DNA substrates.
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DOI：
10.1261/rna.065136.117
发表时间：
2018-04
期刊：
RNA (New York, N.Y.)
影响因子：
--
作者：
Didychuk AL;Butcher SE;Brow DA
通讯作者：
Brow DA
影响因子：
15
作者：
Alexander, Seth C.;Busby, Kayla N.;Devaraj, Neal K.
通讯作者：
Devaraj, Neal K.
影响因子：
16.6
作者：
Hoevelmann, Felix;Gaspar, Imre;Seitz, Oliver
通讯作者：
Seitz, Oliver
影响因子：
4.8
作者：
Holstein, Josephin M.;Rentmeister, Andrea
通讯作者：
Rentmeister, Andrea
影响因子：
56.9
作者：
Femino, A;Fay, FS;Singer, RH
通讯作者：
Singer, RH