Detection Of Methyltransferase Activity Research Articles

DNA methyltransferase activity detection based on fluorescent silver nanocluster hairpin-shaped DNA probe with 5’-C-rich/G-rich-3’ tails

DNA methylation has received a large amount of attention due to its close relationship to a wide range of biological phenomena, such as gene activation, gene imprinting, and chromatin stability. Herein, we have designed a hairpin-shaped DNA probe with 5’-C-rich/G-rich-3’ tails and developed a simple and reliable fluorescence turn-off assay for DNA adenine methylation (Dam) methyltransferase (MTase) detection combining site recognition and the fluorescence enhancement of DNA-templated silver nanoclusters (DNA–AgNCs) by guanine-rich DNA sequences. A designed hairpin probe with 5’ CCCTTACCCC and 3’ GGGTGGGGTGGGGTGGGG displays a bright red emission after reacting with AgNO3 and NaBH4. In the presence of Dam MTase, the methylation-sensitive restriction endonuclease Dpn I which has the same recognition site with the Dam MTase can split the probe, freeing the G-rich sequence from the C-rich sequence, thus quenching the fluorescence of DNA–AgNCs. Compared to traditional fluorescent-based methods, this strategy is simple and inexpensive. A linear response to concentrations of Dam MTase which range from 1U/mL to 100U/mL and a detection limit of 1U/mL are obtained without any amplification steps. In addition, we also demonstrate the method can be used for evaluation and screening of inhibitors for Dam MTase.

MoS2 nanosheets as an effective fluorescence quencher for DNA methyltransferase activity detection.

As one of the inorganic graphene analogues, two dimensional MoS2 nanosheets have been drawing extensive attention in the past few years due to their remarkable structural and electronic properties. Herein, a simple signal-on fluorescence DNA methyltransferase (MTase) activity assay using a MoS2 nanosheet mediated fluorescence quenching strategy is described. Briefly, substrate DNA is designed to possess a double-stranded DNA (ds-DNA) segment containing the recognition sequence of DNA adenosine methyltransferase (Dam) and a single-stranded DNA (ss-DNA) segment for anchoring the substrate DNA to MoS2 nanosheets via van der Waals interactions. Once the substrate DNA is absorbed onto MoS2 nanosheets, the fluorescence of the fluorophores labeled to the end of the ds-DNA region is quenched. When the substrate DNA is methylated by Dam, fluorescence is recovered resulting from the release of the fluorophore labeled segment cleaved by the methylation sensitive restriction endonuclease DpnI. Since the fluorescence recovery directly reflects the methylation level, the Dam MTase activity can be quantified accordingly. Based on this assay, a linear range of 0.2-20 U mL(-1) is achieved with high sensitivity and selectivity. Furthermore, the inhibitor screening ability is well demonstrated.

A label-free electrochemical assay for methyltransferase activity detection based on the controllable assembly of single wall carbon nanotubes

A sensitive label-free “signal-on” electrochemical approach for detection of methyltransferases (MTase) activity is developed based on the signal transduction and amplification of single wall carbon nanotubes (SWCNTs). In this method, the oligonucleotide I is first self-assembled on the electrode via Au–S bonding. After hybridization with its complement ssDNA (oligonucleotide II), duplex strand DNA (dsDNA) probes containing specific recognition sequence of Dam MTase and methylation-sensitive restriction endonuclease Dpn I is then formed on the electrode. In the presence of Dam MTase and Dpn I, the dsDNA probes are methylated and subsequently cleaved into two dsDNA fragments. After heating, the remained dsDNA fragments on the electrode melted into ssDNA fragments. Then the SWCNTs can be controllably assembled on the ssDNA fragments remained on the electrode, mediating efficient electron transfer between the electrode and electroactive species. It generates measurable current signal (eT ON), which is related to the concentration of the Dam MTase. The resulting change in electron transfer efficiency is readily measured by differential pulse voltammetry at Dam MTase concentrations as low as 0.04U/mL. This method does not need electroactive molecules labeling on the methylation-responsive DNA probes. The linear response of the developed facile signal-on electrochemical sensing system for Dam MTase is in the range of 0.1–1.0U/mL. In addition, such a SWCNTs based electrochemical assay also has the ability to screen inhibitors for Dam MTase.

A universal competitive fluorescence polarization activity assay for S-adenosylmethionine utilizing methyltransferases

A high-throughput, competitive fluorescence polarization immunoassay has been developed for the detection of methyltransferase activity. The assay was designed to detect S-adenosylhomocysteine (AdoHcy), a product of all S-adenosylmethionine (AdoMet)-utilizing methyltransferase reactions. We employed commercially available anti-AdoHcy antibody and fluorescein–AdoHcy conjugate tracer to measure AdoHcy generated as a result of methyltransferase activity. AdoHcy competes with tracer in the antibody/tracer complex. The release of tracer results in a decrease in fluorescence polarization. Under optimized conditions, AdoHcy and AdoMet titrations demonstrated that the antibody had more than a 150-fold preference for binding AdoHcy relative to AdoMet. Mock methyltransferase reactions using both AdoHcy and AdoMet indicated that the assay tolerated 1 to 3 μM AdoMet. The limit of detection was approximately 5 nM (0.15 pmol) AdoHcy in the presence of 3 μM AdoMet. To validate the assay’s ability to quantitate methyltransferase activity, the methyltransferase catechol- O-methyltransferase (COMT) and a known selective inhibitor of COMT activity were used in proof-of-principle experiments. A time- and enzyme concentration-dependent decrease in fluorescence polarization was observed in the COMT assay that was developed. The IC 50 value obtained using a selective COMT inhibitor was consistent with previously published data. Thus, this sensitive and homogeneous assay is amenable for screening compounds for inhibitors of methyltransferase activity.

Kinetin-induced caffeic acid O-methyltransferases in cell suspension cultures of Vanilla planifolia Andr. and isolation of caffeic acid O-methyltransferase cDNAs

Two different S-adenosyl-L-methionine:caffeic acid methyltransferases are induced in kinetin-treated cell cultures of Vanilla planifolia. Upon addition of kinetin to the culture, maximum caffeic acid 4-O-methyltransferase and caffeic acid 3-O-methyltransferase activity is obtained after around 2 and 40 h, respectively. The former enzyme is involved in the biosynthesis of vanillic acid and the latter enzyme is co-induced with other enzymes of the general phenylpropanoid pathway and is involved in the biosynthesis of lignin precursors. A number of cDNA clones encoding S-adenosyl-L-methionine:caffeic acid-methyltransferases were isolated by heterologous probe screening of a λZapII cDNA library constructed from mRNA of a kinetin-treated cell suspension culture of V. planifolia. A full-length cDNA clone, Vpomt35, contains a 1 089-bp open reading frame coding for 363 amino acid residues, a 25-bp 5′-end sequence and a 214-bp 3′-end non-coding sequence. The deduced amino acid sequence of Vpomt35 revealed 56 to 80 % sequence identity when compared to those of other plant caffeic acid O-methyltransferases. Detection of methyltransferase activity in extracts of Escherichia coli XL-1 Blue cells transformed with a pBluescript phagemid construct containing Vpomt35 confirmed that this cDNA clone encodes a caffeic acid 3-O-methyltransferase. The enzyme also exhibits a low caffeic acid 4-O-methyltransferase activity (2 % of the caffeic acid 3-O-methyltransferase activity). A number of other cDNA clones encoding caffeic acid O-methyltransferases was also sequenced. Comparison of nucleotide and deduced amino acid sequences of these cDNAs indicated two types of caffeic acid O-methyltransferase cDNAs which showed 94 % sequence identity in the coding region, but only 55 % homology with several gaps in the 3′ untranslated end. Results from Southern blot analysis suggest that the caffeic acid O-methyltransferase gene is organized as a small family with at least two genes, which are expressed in Vanilla cell suspension cultures.