Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

Gisela Maria Hanz; Britta Jung; Anna Giesbertz; Matyas Juhasz; Elmar Weinhold

doi:10.3791/52014

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Method Article

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

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DOI:

10.3791/52014

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November 22nd, 2014

Gisela Maria Hanz^*¹ , Britta Jung^*¹ , Anna Giesbertz¹ , Matyas Juhasz¹ , Elmar Weinhold¹

¹Institute of Organic Chemistry, Department of Chemistry, RWTH Aachen University

^* These authors contributed equally

Summary

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

Abstract

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.

Introduction

Specific labeling of nucleic acids^1,2 and proteins^3,4 is of major interest for functional characterizations, medical diagnosis and (nano)biotechnology. Here we present an enzymatic labeling method for these biopolymers which is based on S-adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTases). This class of enzymes (EC 2.1.1.) targets individual nucleophilic positions (nitrogen, oxygen, sulfur and carbon atoms) within specific residues of nucleic acids and proteins and naturally transfers the activated methyl group of the cofactor AdoMet (Figure 1A)⁵. In addition, MTases can utilize synthe....