Methyltransferase‐Directed Labeling of Biomolecules and its Applications

Deen, Jochem; Vranken, Charlotte; Leen, Volker; Neely, Robert K.; Janssen, Kris P. F.; Hofkens, Johan

doi:10.1002/anie.201608625

Cited by 62 publications

(67 citation statements)

References 141 publications

(121 reference statements)

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“…Thus this approach is not compatible with high-throughput DNA stretching in nanochannels that we employ. The second approach is covalent labeling of short motifs using methylation-specific proteins or methylases, to create a barcode-like pattern on DNA molecules [15]. Usually the flanking sequence of the CG site will strongly influence the specificity.…”

Section: Introductionmentioning

confidence: 99%

DNA looping by two 5-methylcytosine-binding proteins quantified using nanofluidic devices

Liu

Movahed

Dangi

et al. 2020

Epigenetics & Chromatin

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Background: MeCP2 and MBD2 are members of a family of proteins that possess a domain that selectively binds 5-methylcytosine in a CpG context. Members of the family interact with other proteins to modulate DNA packing. Stretching of DNA-protein complexes in nanofluidic channels with a cross-section of a few persistence lengths allows us to probe the degree of compaction by proteins. Results: We demonstrate DNA compaction by MeCP2 while MBD2 does not affect DNA configuration. By using atomic force microscopy (AFM), we determined that the mechanism for compaction by MeCP2 is the formation of bridges between distant DNA stretches and the formation of loops. Conclusions: Despite sharing a similar specific DNA-binding domain, the impact of full-length 5-methylcytosinebinding proteins can vary drastically between strong compaction of DNA and no discernable large-scale impact of protein binding. We demonstrate that ATTO 565-labeled MBD2 is a good candidate as a staining agent for epigenetic mapping.

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Section: Introductionmentioning

confidence: 99%

DNA looping by two 5-methylcytosine-binding proteins quantified using nanofluidic devices

Liu

Movahed

Dangi

et al. 2020

Epigenetics & Chromatin

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“…22 However, while the carrier designs used in present study have been implemented using DNA self-assembly from small fragments, similar structures may be created employing enzymatic modification. 56,57,58,37 The latter may be significantly more cost-effective and enable the preparation of larger amounts, both required for a viable sensor technology in the future. The incorporation of several larger features on the other hand may complicate the preparation of the carriers, which would be less desirable.…”

Section: Discussionmentioning

confidence: 99%

Electric Single-Molecule Hybridization Detector for Short DNA Fragments

et al. 2018

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In combining DNA nanotechnology and high-bandwidth single-molecule detection in nanopipettes, we demonstrate an all-electric, label-free hybridisation sensor for short DNA sequences (< 100 nt).Such short fragments are known to occur as circulating cell-free DNA in various bodily fluids, such as blood plasma and saliva, and have been identified as disease markers for cancer and infectious diseases. To this end, we use as a model system a 88-mer target from the RV1910c gene in Mycobacterium tuberculosis that is associated with antibiotic (isoniazid) resistance in TB. Upon binding to short probes attached to long carrier DNA, we show that resistive pulse sensing in nanopipettes is capable of identifying rather subtle structural differences, such as the hybridisation state of the probes, in a statistically robust manner. With significant potential towards multiplexing and highthroughput analysis, our study points towards a new, single-molecule DNA assay technology that is fast, easy to use and compatible with point of care environments. Nanopore devices are a new class of stochastic single-molecule sensors. As nanoscale analogues of the well-known Coulter counter, which is routinely used for cell counting in hospital environments, they have been developed towards fast and label-free DNA sequencing. 1 This feat has now largely been achieved with (modified) biological pores, such as -hemolysin. 2 However, resistive pulse sensing with solid-state nanopores and nanopipettes offers a range of other potential applications.These nanodevices are relatively easy to fabricate (especially nanopipettes 3,4 ) and there is usually considerable flexibility in their design, with regards to the pore dimensions (diameter, channel length,

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“…In the present study, a C5-DNA-MTase from P. arcticus 273-4, ParI, was characterized on the basis of its potential to possess features relevant for biotechnological applications, such as labeling of biopolymers, DNA mapping or epigenetic analysis [22][23][24].…”

Section: Introductionmentioning

confidence: 99%

Biochemical characterization of ParI, an orphan C5-DNA methyltransferase from Psychrobacter arcticus 273-4

Grgić

Williamson

Bjerga

et al. 2018

Protein Expression and Purification

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Cytosine-specific DNA methyltransferases are important enzymes in most living organisms. In prokaryotes, most DNA methyltransferases are members of the type II restriction-modification system where they methylate host DNA, thereby protecting it from digestion by the accompanying restriction endonucleases. DNA methyltransferases can also act as solitary enzymes having important roles in controlling gene expression, DNA replication, cell cycle and DNA post-replicative mismatch repair. They have potential applications in biotechnology, such as in labeling of biopolymers, DNA mapping or epigenetic analysis, as well as for general DNA-protein interaction studies. The parI gene from the psychrophilic bacterium Psychrobacter arcticus 273-4 encodes a cytosine-specific DNA methyltransferase. In this work, recombinant ParI was expressed and purified in fusion to either an N-terminal hexahistidine affinity tag, or a maltose binding protein following the hexahistidine affinity tag, for solubility improvement. After removal of the fusion partners, recombinant ParI was found to be monomeric by size exclusion chromatography, with its molecular mass estimated to be 54 kDa. The apparent melting temperature of the protein was 53 °C with no detectable secondary structures above 65 °C. Both recombinant and native ParI showed methyltransferase activity in vivo. In addition, MBP- and His-tagged ParI also demonstrated in vitro activity. Although the overall structure of ParI exhibits high thermal stability, the loss of in vitro activity upon removal of solubility tags or purification from the cellular milieu indicates that the catalytically active form is more labile. Horizontal gene transfer may explain the acquisition of a protein-encoding gene that does not display common cold-adapted features.

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Methyltransferase‐Directed Labeling of Biomolecules and its Applications

Cited by 62 publications

References 141 publications

DNA looping by two 5-methylcytosine-binding proteins quantified using nanofluidic devices

DNA looping by two 5-methylcytosine-binding proteins quantified using nanofluidic devices

Electric Single-Molecule Hybridization Detector for Short DNA Fragments

Biochemical characterization of ParI, an orphan C5-DNA methyltransferase from Psychrobacter arcticus 273-4

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