Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093, United States.
J Am Chem Soc. 2023 Apr 12;145(14):8099-8106. doi: 10.1021/jacs.3c00861. Epub 2023 Mar 29.
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 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 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.
细菌 tRNA 鸟嘌呤转移酶（TGTs）催化鸟嘌呤与 7-脱氮鸟嘌呤 queuine 前体 preQ1 的交换。虽然细菌 TGTs 的天然核酸底物是 queuine 识别 tRNA 的反密码环，但该酶的最小识别序列是一个含有目标 G 核苷酸的结构发夹，在“UGU”环基序中。先前的工作已经建立了一个 RNA 修饰系统 RNA-TAG，其中 TGT 将感兴趣的 RNA 上的靶 G 用化学修饰的 preQ1 底物交换，这些底物与小分子报告物（如生物素或荧光团）相连。虽然将 RNA 转糖基酶的底物范围扩展到包括 DNA 将能够实现许多应用，但先前的报道表明 TGT 不能修饰天然 DNA。在这里，我们证明 TGT 实际上可以识别和标记特定的 DNA 底物。通过对理性突变的 DNA 发夹序列进行迭代测试，我们确定了 TGT 对未修饰 DNA 进行转糖基化的最小序列要求。控制 DNA 发夹中的空间位阻极大地影响标记效率，并且在优化时，可以导致近乎定量的特异性修饰。我们通过快速合成用于 spliceosomal U6 RNA 的荧光 northern 印迹和长非编码 RNA MALAT1 的 RNA FISH 可视化的探针，展示了我们新开发的 DNA-TAG 系统的实用性。DNA-TAG 系统的简便性将为研究人员提供一种工具，用于获取各种用途广泛且价格合理的修饰 DNA 底物。
