Methyltransferase-Directed Labeling of Biomolecules and its Applications

Abstract

Methyltransferases (MTases) form a large family of enzymes that methylate a diverse set of targets, ranging from the three major biopolymers to small molecules. Most of these MTases use the cofactor S-adenosyl-l-Methionine (AdoMet) as a methyl source. In recent years, there have been significant efforts toward the development of AdoMet analogues with the aim of transferring moieties other than simple methyl groups. Two major classes of AdoMet analogues currently exist: doubly-activated molecules and aziridine based molecules, each of which employs a different approach to achieve transalkylation rather than transmethylation. In this review, we discuss the various strategies for labelling and functionalizing biomolecules using AdoMet-dependent MTases and AdoMet analogues. We cover the synthetic routes to AdoMet analogues, their stability in biological environments and their application in transalkylation reactions. Finally, some perspectives are presented for the potential use of AdoMet analogues in biology research, (epi)genetics and nanotechnology.

Keywords: DNA functionalization; S-adenosyl methionine; methyltransferases; protein modification; transalkylation.

Scheme 1

Scheme showing the transfer of a methyl group from the cofactor AdoMet (1) to the substrate by the MTase.

Scheme 2

Reaction mechanism of 5‐C methylation by M.HhaI

Scheme 3

DNA labeling using aziridine‐based (top) or doubly activated AdoMet analogues (bottom).

Scheme 4

Aziridoadenosine‐based AdoMet analogues.

Scheme 5

Stabilization of the S_N2 transition state.17b.

Scheme 6

Doubly activated AdoMet analogues.

Scheme 7

Decomposition pathways of (S,S)‐AdoMet (and its analogues). a) Inversion at the sulfonium center of (S,S)‐AdoMet results in (R,S)‐AdoMet. b) Deprotonation at the C‐5′ and subsequent elimination of adenine base to give S‐ribosylmethionine. c) Nucleophilic attack by the α‐carboxylate on the γ‐carbon of methionine delivers HSL and MTA. d) Nucleophilic addition at 1′′‐C. e) Addition of water to the 2′′‐C or 4′′‐C position.

Figure 1

Chalcogen‐containing AdoMet analogues.

Scheme 8

Synthesis of the N‐Adenosylaziridine AdoMet analogue. Step 1: Nucleophilic substitution of the tosylate (TsO) group in 5′‐deoxy‐5′‐tosyladenosine with aziridine.

Scheme 9

Synthesis of doubly activated AdoMet analogues. FA=formic Acid, Ns=nosyl.

Scheme 10

Enzymatic AdoMet synthesis using SalL and MAT.

Figure 2

The base‐flipping mechanism of MTase‐mediated DNA methylation. Most of the work on DNA MTases with AdoMet analogues is based on M.TaqI, a DNA MTase from the thermophilic bacterium Thermus aquaticus.

Figure 3

Use of an AdoMet analogue as a reporter of protein methylation. The AdoMet analogue can be utilized by endogenous methyltransferases to label cellular proteins with an alkyne moiety. The modified proteins can then be coupled to a fluorescent tag using CuAAC for further characterization. Reproduced from Ref. 21.

Figure 4

The two main methods for reading the distribution of fluorophores on DNA molecules. A) For high‐resolution localization of fluorophores on the DNA, the DNA needs to be rigidly attached to the surface. B) For rapid imaging of DNA, the DNA molecules can be passed through nanochannels where they are elongated and subsequently, the intensity pattern can be recorded.

Figure 5

In the highly multiplexed RNA profiling method of the Zhuang group81 hundreds of individual mRNA can be correctly be identified and localized in situ.

Figure 6

General principle for capture‐based detection of the unmethylated part of the genome, illustrated with a quantitative PCR assay.