Hanz Gisela Maria, Jung Britta, Giesbertz Anna, Juhasz Matyas, Weinhold Elmar
Institute of Organic Chemistry, Department of Chemistry, RWTH Aachen University.
Institute of Organic Chemistry, Department of Chemistry, RWTH Aachen University;
J Vis Exp. 2014 Nov 22(93):e52014. doi: 10.3791/52014.
S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5'-ATCGAT-3' sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.
S-腺苷-L-甲硫氨酸（AdoMet或SAM）依赖性甲基转移酶（MTase）催化活性甲基基团从AdoMet转移至DNA、RNA、蛋白质和小生物分子中的特定位置。利用合成辅因子类似物，这种天然甲基化反应可扩展为各种各样的烷基化反应。用氮丙啶环取代AdoMet的活性锍中心会产生可被各种DNA MTase与DNA偶联的辅因子。这些氮丙啶辅因子可在腺嘌呤部分的不同位置配备报告基团，并用于DNA的序列特异性甲基转移酶诱导标记（SMILing DNA）。作为一个典型例子，我们给出了一个使用DNA MTase M.BseCI和氮丙啶辅因子6BAz一步法在5'-ATCGAT-3'序列处对pBR322质粒DNA进行生物素化的方案。用不饱和烷基扩展活性甲基基团会产生另一类AdoMet类似物，它们用于甲基转移酶导向的活性基团转移（mTAG）。由于扩展的侧链被锍中心和不饱和键激活，这些辅因子被称为双激活AdoMet类似物。这些类似物不仅像氮丙啶辅因子一样作为DNA MTase的辅因子，也作为RNA、蛋白质和小分子MTase的辅因子。它们通常用于用独特官能团对MTase底物进行酶促修饰，这些官能团在第二步化学步骤中用报告基团标记。这在组蛋白H3蛋白荧光标记的方案中得到了体现。一个小的炔丙基通过组蛋白H3赖氨酸4（H3K4）MTase Set7/9从辅因子类似物SeAdoYn转移至蛋白质，随后用TAMRA叠氮化物对炔基化的组蛋白H3进行点击标记。用辅因子类似物进行MTase介导的标记是一项赋能技术，可用于许多令人兴奋的应用，包括MTase底物的鉴定和功能研究以及DNA基因分型和甲基化检测。
