Sequence-specific Methyltransferase-Induced Labelling (SMILing) of plasmid DNA for studying cell transfection

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Abstract

Plasmid DNA (pUC19 and pBR322) was sequence-specifically, covalently labelled with Cy3 fluorophores using a newly synthesised N-adenosylaziridine cofactor and the DNA methyltransferase M.TaqI. The fluorescently labelled plasmids were used for transfection of mammalian cells and their intracellular distribution was visualised by epifluorescence and confocal fluorescence microscopy. Although these prokaryotic plasmids do not contain nuclear import sequences, translocation into the nuclei was observed.

Graphical abstract

SMILing of plasmid pUC19 DNA with a fluorescent aziridine cofactor and a DNA methyltransferase (MTase) (A), transfection (B) and translocation into the nucleus (C). Cy3 fluorescence of labelled pUC19 inside the nucleus (right).

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Introduction

Despite huge progress in bioconjugation chemistry sequence-specific labelling of native DNA remains a challenging task. Methods for producing, for example, sequence-specific fluorescently labelled plasmid DNA are of great importance to study DNA transport and localisation processes inside cells using fluorescence microscopic techniques.1 Depending on the context of the study, DNA can be delivered by transfection (non-viral vectors), transduction (viral vectors) or microinjection into different compartments of the cell (cytoplasm or nucleus). Especially, the process of cell transfection using non-viral vectors is still poorly understood and DNA import into the nucleus of non-dividing cells is a matter of curiosity and intensive debate.2, 3, 4, 5, 6, 7

So far, mainly two DNA labelling approaches have been applied to follow the intracellular pathway of transfected or microinjected plasmid DNA by fluorescence microscopy. The first approach is based on fluorescently labelled triple helix-forming peptide nucleic acids (PNA clamps).8, 9, 10, 11 PNA clamps form non-covalent triplex invasion complexes (two PNA strands bind to one strand of double-stranded DNA) with high specificity and stability.12 However, complex formation is not efficient and the use of a 50-fold excess was necessary to achieve quantitative labelling.10 In addition, it is often required to introduce a suitable target sequence into the plasmids.

In the second approach DNA is labelled covalently which avoids any doubts about label dissociation from the DNA under physiological conditions. This approach is based on reactive chemicals able to modify DNA components and to deliver fluorophores or other reporter groups of interest to the DNA.13, 14, 15, 16, 17, 18, 19 However, labelling with such reactive chemicals is random allowing no control of the position and number of the labels on individual plasmids. Indeed, it was demonstrated that the average number of labels per plasmid plays an important role in transfection and that increasing label density reduces the transfection efficiency.14, 18 In particular, in vitro transcription/translation experiments using randomly labelled DNA indicated that a high labelling density inhibits transcription which might explain the lower transfection efficiency observed.18

To circumvent potential problems associated with non-covalent or random labelling we employed an enzymatic method for sequence-specific and covalent labelling of DNA with fluorophores. This method makes use of the ability of DNA methyltransferases (MTases) to couple sequence-specifically the synthetic cofactor N-adenosylaziridine with their DNA recognition sequences.20 By attaching reporter21, 22, 23 or functional groups24, 25 to N-adenosylaziridine at the adenine 8-position cofactors are obtained which can be used in combination with DNA MTases having different recognition sequences for Sequence-specific Methyltransferase-Induced Labelling of DNA (SMILing DNA).

Herein, we report the synthesis of the novel N-adenosylaziridine cofactor derivative 6Cy3Az (1) containing a Cy3 fluorophore attached via a linker at the 6-position of the adenine ring and its successful quantitative coupling with plasmid DNA by the DNA MTase M.TaqI. In addition, we demonstrate that the fluorescently labelled DNA is suitable for intracellular tracking after transfection of CHO-K1 cells.

Section snippets

Synthesis

The rationale for attaching the reporter group at the 6-position instead of the 8-position of N-adenosylaziridine was that bulky C8-substituents in adenosine analogues have a strong tendency to alter the rotation around the glycosidic bond from the naturally preferred anti into the syn conformation.26, 27, 28, 29, 30 Such a syn conformation should have two disadvantages: first, DNA MTases bind their natural cofactor S-adenosyl-l-methionine (AdoMet) in the anti conformation as observed in

Discussion

SMILing DNA is an attractive method for site-specific covalent labelling of DNA. By virtue of the sequence specificity of the applied DNA MTase, the method enables full control over the position and number of labels on the DNA substrate. In addition, a variety of fluorophores or other reporter groups can be delivered to the DNA due to the versatility of the cofactor preparation that permits introduction of the label in the last step of the synthesis. Here, we have shown that this method can be

General

6-Chloro-2′,3′-O-isopropylideneadenosine (2)40 and aziridine48, 49 were prepared according to literature procedures. N-Ethyldiisopropylamine (EDIA) was purchased from Fluka. 4-(Dimethylamino)-pyridine (DMAP) and methanesulfonyl chloride (MesCl) were purchased from Merck. All reagents were of p.a. grade. Dry solvents were either purchased or obtained by drying using common laboratory techniques.50 Proteinase K was obtained from Qiagen. The plasmids and the restriction endonuclease R.TaqI were

Acknowledgments

The authors gratefully acknowledge the assistance of Hervé Chneiweiss and Eric Etienne (Neuropharmacology Department, Collège de France, Paris) in cell culture, transfection and laser scanning microscopy experiments. In addition, we thank Marcel Ruiters (Synvolux therapeutics BV, Groningen, Netherlands) and Marie-Pierre Junier for fruitful discussions. This research was supported by a joint grant from the German Science Foundation (WE 1453/4-1 and 4-2) and the CNRS (fellowship to F.H.-G.S.).

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