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Article ; Online: Hybridization-based CpG methylation level detection using methyl-CpG-binding domain-fused luciferase.

Goto, Ayano / Yoshida, Wataru

2023 Volume 415, Issue 12, Page(s) 2329–2337

Abstract: Hypermethylation of tumor-suppressor genes and global hypomethylation, which is related to methylation level at the retroelement, have been recognized as features of the cancer genome. In this study, we developed a hybridization-based CpG methylation ... ...

Abstract	Hypermethylation of tumor-suppressor genes and global hypomethylation, which is related to methylation level at the retroelement, have been recognized as features of the cancer genome. In this study, we developed a hybridization-based CpG methylation level detection method using methyl-CpG-binding domain-fused firefly luciferase (MBD-Fluc). In this method, methylated probe oligonucleotides were used to capture target oligonucleotides. Fully methylated and hemimethylated double-stranded DNA (dsDNA) was formed by hybridization of the methylated captured oligonucleotides with methylated or unmethylated target oligonucleotides, respectively. MBD-Fluc specifically binds to fully methylated dsDNA but not to hemimethylated dsDNA; therefore, methylated target oligonucleotides can be detected by measuring the luciferase activity of the bound MBD-Fluc. Using the corresponding methylated probe oligonucleotides, the CpG methylation levels of SEPT9, BRCA1, and long interspersed nuclear element-1 (LINE-1) oligonucleotides were quantified. Moreover, we demonstrated that the emission detection signal was not affected by the methylation state of the overhang region of the target oligonucleotide, which was not hybridized to the probe oligonucleotide, indicating that methylated CpG of the target region could be accurately detected. Unmethylated-CpG-binding domain-fused luciferases and 5-hydroxymethyl-CpG-binding domain-fused luciferases have been constructed, suggesting that other modified bases can be detected by the same platform.
MeSH term(s)	DNA-Binding Proteins/metabolism ; CpG Islands ; DNA Methylation ; DNA/genetics ; Oligonucleotides ; Luciferases/genetics
Chemical Substances	DNA-Binding Proteins ; DNA (9007-49-2) ; Oligonucleotides ; Luciferases (EC 1.13.12.-)
Language	English
Publishing date	2023-03-24
Publishing country	Germany
Document type	Journal Article
ZDB-ID	201093-8
ISSN	1618-2650 ; 0016-1152 ; 0372-7920
ISSN (online)	1618-2650
ISSN	0016-1152 ; 0372-7920
DOI	10.1007/s00216-023-04657-z
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Article ; Online: Universal Design of Luciferase Fusion Proteins for Epigenetic Modifications Detection Based on Bioluminescence Resonance Energy Transfer

Miyata, Takamichi / Shimamura, Hazuki / Asano, Ryutaro / Yoshida, Wataru

Analytical Chemistry. 2023 Feb. 07, v. 95, no. 7 p.3799-3805

2023

Abstract: Global hypomethylation and promoter hypermethylation of tumor-suppressor genes are the hallmarks of cancer. We previously reported a global DNA methylation level sensing system based on dual-color bioluminescence resonance energy transfer (BRET) using ... ...

Abstract	Global hypomethylation and promoter hypermethylation of tumor-suppressor genes are the hallmarks of cancer. We previously reported a global DNA methylation level sensing system based on dual-color bioluminescence resonance energy transfer (BRET) using methyl-CpG binding domain (MBD)-fused firefly luciferase (Fluc) and unmethyl-CpG binding domain (CXXC)-fused Oplophorus luciferase (Oluc). Moreover, BRET-based hydroxymethylation and hemi-methylation level sensing systems have been developed using hydroxymethyl-CpG and hemi-methyl-CpG binding domain-fused Fluc. These studies suggest that target epigenetic modifications can be simultaneously quantified using target-modification-binding protein-fused luciferases. In this study, we focused on the SnoopTag (SnT)/SnoopCatcher (SnC) protein ligation system to establish a universal design for fusion protein construction for any combination. SnT spontaneously forms an isopeptide bond with SnC; therefore, any kind of fusion protein would be constructed by the SnT/SnC system. To establish the proof of concept, MBD-SnT, CXXC-SnT, and SnC-Oluc were prepared and ligated MBD-SnT or CXXC-SnT to SnC-Oluc. The ligation products of MBD-SnT-SnC-Oluc and CXXC-SnT-SnC-Oluc showed luciferase activity and specific binding activity to methyl-CpG and unmethyl-CpG, respectively. The BRET signal using MBD-SnT-SnC-Oluc and CXXC-SnT-SnC-Oluc increased the amount of methyl-CpG and unmethyl-CpG in genomic DNA, respectively. There was a significant negative correlation between the BRET signals; therefore, the global DNA methylation level was quantified using the BRET signals (R ² = 0.99, and R.S.D. <3.5%). These results indicate that the SnT/SnC protein ligation system can be utilized to construct target modification-binding protein-fused luciferases in any combination that detects target modifications in genomic DNA based on BRET.
Keywords	DNA ; DNA methylation ; analytical chemistry ; bioluminescence ; energy transfer ; epigenetics ; luciferase
Language	English
Dates of publication	2023-0207
Size	p. 3799-3805.
Publishing place	American Chemical Society
Document type	Article ; Online
ZDB-ID	1508-8
ISSN	1520-6882 ; 0003-2700
ISSN (online)	1520-6882
ISSN	0003-2700
DOI	10.1021/acs.analchem.2c05066
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Article ; Online: Rearing method for the endangered species Dineutus mellyi mellyi Régimbart, 1882 (Coleoptera: Gyrinidae)

Watanabe, Kohei / Saiki, Ryota / Sumikawa, Tomoki / Yoshida, Wataru

Aquatic Insects. 2023 July 3, v. 44, no. 3 p.195-204

2023

Abstract: Rearing research is being conducted to conserve many endangered aquatic Coleoptera species; however, studies on Gyrinidae species are scarce. Herein, we present an efficient rearing method for the endangered species Dineutus mellyi mellyi Régimbart, 1882 ...

Abstract	Rearing research is being conducted to conserve many endangered aquatic Coleoptera species; however, studies on Gyrinidae species are scarce. Herein, we present an efficient rearing method for the endangered species Dineutus mellyi mellyi Régimbart, 1882 (Coleoptera: Gyrinidae), practiced at the Ishikawa Insect Museum. The survival rate of the larvae reared using this method at different developmental stages was as follows: first-instar larvae, 100% (n = 26); second-instar larvae, 100% (n = 26); third-instar larvae, 92% (n = 24); landing to pupate, 73% (n = 19); pupa, 73% (n = 19); and emergence to escape from pupal chamber, 69% (n = 18). Therefore, we recommend this method for sustainable ex situ conservation of this species. This method can be generalised to other Gyrinidae and aquatic Coleoptera species.
Keywords	Dineutus ; endangered species ; ex situ conservation ; insects ; museums ; pupae ; survival rate ; Aquatic insects ; immature stage ; museum ; red list
Language	English
Dates of publication	2023-0703
Size	p. 195-204.
Publishing place	Taylor & Francis
Document type	Article ; Online
ISSN	1744-4152
DOI	10.1080/01650424.2022.2107676
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Article ; Online: Detection of CpG methylation level using methyl-CpG-binding domain-fused fluorescent protein.

Fujita, Marika / Goto, Masanori / Tanaka, Masayoshi / Yoshida, Wataru

Analytical methods : advancing methods and applications

2023 Volume 15, Issue 19, Page(s) 2294–2299

Abstract: Methylation of cytosine to 5-methylcytosine on CpG dinucleotides is the most frequently studied epigenetic modification involved in the regulation of gene expression. In normal tissues, tissue-specific CpG methylation patterns are established during ... ...

Abstract	Methylation of cytosine to 5-methylcytosine on CpG dinucleotides is the most frequently studied epigenetic modification involved in the regulation of gene expression. In normal tissues, tissue-specific CpG methylation patterns are established during development. In contrast, alterations in methylation patterns have been observed in abnormal cells, such as cancer cells. Cancer type-specific CpG methylation patterns have been identified and used as biomarkers for cancer diagnosis. In this study, we developed a hybridization-based CpG methylation level sensing system using a methyl-CpG-binding domain (MBD)-fused fluorescent protein. In this system, the target DNA is captured by a complementary methylated probe DNA. When the target DNA is methylated, a symmetrically methylated CpG is formed in the double-stranded DNA. MBD specifically recognizes symmetrical methyl-CpG on double-stranded DNA; therefore, the methylation level is quantified by measuring the fluorescence intensity of the bound MBD-fused fluorescent protein. We prepared MBD-fused AcGFP1 and quantified the CpG methylation levels of the target DNA against
MeSH term(s)	DNA-Binding Proteins/chemistry ; DNA-Binding Proteins/genetics ; DNA-Binding Proteins/metabolism ; CpG Islands/genetics ; DNA Methylation/genetics ; Cytosine/chemistry ; DNA/chemistry
Chemical Substances	DNA-Binding Proteins ; Cytosine (8J337D1HZY) ; DNA (9007-49-2)
Language	English
Publishing date	2023-05-18
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2515210-5
ISSN	1759-9679 ; 1759-9660
ISSN (online)	1759-9679
ISSN	1759-9660
DOI	10.1039/d3ay00227f
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Article ; Online: Universal Design of Luciferase Fusion Proteins for Epigenetic Modifications Detection Based on Bioluminescence Resonance Energy Transfer.

Miyata, Takamichi / Shimamura, Hazuki / Asano, Ryutaro / Yoshida, Wataru

Analytical chemistry

2023 Volume 95, Issue 7, Page(s) 3799–3805

Abstract	Global hypomethylation and promoter hypermethylation of tumor-suppressor genes are the hallmarks of cancer. We previously reported a global DNA methylation level sensing system based on dual-color bioluminescence resonance energy transfer (BRET) using methyl-CpG binding domain (MBD)-fused firefly luciferase (Fluc) and unmethyl-CpG binding domain (CXXC)-fused
MeSH term(s)	Universal Design ; Epigenesis, Genetic ; DNA Methylation ; DNA/genetics ; Luciferases/metabolism ; Energy Transfer
Chemical Substances	DNA (9007-49-2) ; Luciferases (EC 1.13.12.-)
Language	English
Publishing date	2023-02-07
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1508-8
ISSN	1520-6882 ; 0003-2700
ISSN (online)	1520-6882
ISSN	0003-2700
DOI	10.1021/acs.analchem.2c05066
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Article ; Online: Thermal Stability Changes in Telomeric G-Quadruplex Structures Due to

Wada, Ryohei / Yoshida, Wataru

Epigenomes

2021 Volume 5, Issue 1

Abstract: ... ...

Abstract	N
Language	English
Publishing date	2021-02-02
Publishing country	Switzerland
Document type	Journal Article
ISSN	2075-4655
ISSN (online)	2075-4655
DOI	10.3390/epigenomes5010005
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Article ; Online: Amide-functionalised phosphonium-based ionic liquids as ligands for rhodium(iii) extraction.

Yoshida, Wataru / Goto, Masahiro

RSC advances

2021 Volume 11, Issue 16, Page(s) 9386–9394

Abstract: Seven new amide-functionalised phosphonium-based ionic liquids (APILs) with chloride anions are synthesised and applied to extraction of rhodium(iii) from HCl solution. The effects of structural modification of the APILs on the extraction performance are ...

Abstract	Seven new amide-functionalised phosphonium-based ionic liquids (APILs) with chloride anions are synthesised and applied to extraction of rhodium(iii) from HCl solution. The effects of structural modification of the APILs on the extraction performance are examined by liquid-liquid extraction using toluene as a diluent and the results compared with those for trihexyltetradecylphosphonium chloride ([P
Language	English
Publishing date	2021-03-02
Publishing country	England
Document type	Journal Article
ISSN	2046-2069
ISSN (online)	2046-2069
DOI	10.1039/d1ra00489a
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Article: Thermal Stability Changes in Telomeric G-Quadruplex Structures Due to N6-Methyladenine Modification

Wada, Ryohei / Yoshida, Wataru

Epigenomes. 2021 Feb. 02, v. 5, no. 1

2021

Abstract: N⁶-methyladenine modification (m⁶dA) has recently been identified in eukaryote genomic DNA. The methylation destabilizes the duplex structure when the adenine forms a Watson–Crick base pair, whereas the methylation on a terminal unpaired adenine ... ...

Abstract	N⁶-methyladenine modification (m⁶dA) has recently been identified in eukaryote genomic DNA. The methylation destabilizes the duplex structure when the adenine forms a Watson–Crick base pair, whereas the methylation on a terminal unpaired adenine stabilizes the duplex structure by increasing the stacking interaction. In this study, the effects of m⁶dA modification on the thermal stability of four distinct telomeric G-quadruplex (G4) structures were investigated. The m⁶dA-modified telomeric oligonucleotide d[AGGG(TTAGGG)₃] that forms a basket-type G4 in Na⁺, d[(TTAGGG)₄TT] that forms a hybrid-type G4 in K⁺ (Form-2), d[AAAGGG(TTAGGG)₃AA] that forms a hybrid-type G4 in K⁺ (Form-1), and d[GGG(TTAGGG)₃T] that forms a basket-type G4 with two G-tetrads in K⁺ (Form-3) were analyzed. Circular dichroism melting analysis demonstrated that (1) A7- and A19-methylation destabilized the basket-type G4 structure that formed in Na⁺, whereas A13-methylation stabilized the structure; (2) A15-methylation stabilized the Form-2 G4 structure; (3) A15- and A21-methylations stabilized the Form-1 G4 structure; and (4) A12-methylation stabilized the Form-3 G4 structure. These results suggest that m⁶dA modifications may affect the thermal stability of human telomeric G4 structures in regulating the biological functions.
Keywords	DNA ; adenine ; circular dichroism spectroscopy ; eukaryotic cells ; humans ; methylation ; oligonucleotides ; telomeres ; thermal stability
Language	English
Dates of publication	2021-0202
Publishing place	Multidisciplinary Digital Publishing Institute
Document type	Article
Note	NAL-AP-2-clean
ISSN	2075-4655
DOI	10.3390/epigenomes5010005
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Article: Quantitative detection of CpG methylation level on G-quadruplex and i-motif-forming DNA by recombinase polymerase amplification

Goto, Masanori / Baba, Yuji / Yoshida, Wataru

Analytical and bioanalytical chemistry. 2022 Aug., v. 414, no. 20

2022

Abstract: Detection of CpG methylation levels holds immense potential for application in medical diagnosis of various diseases. In this study, we report the development of a recombinase polymerase amplification (RPA)-based CpG methylation level sensing system on G- ...

Abstract	Detection of CpG methylation levels holds immense potential for application in medical diagnosis of various diseases. In this study, we report the development of a recombinase polymerase amplification (RPA)-based CpG methylation level sensing system on G-quadruplex (G4) and intercalated motif (i-motif)-forming regions, which are stabilized by CpG methylation. This detection system is based on the principle that DNA polymerase is stalled at the methylated G4 and i-motif-forming region, which results in a decrease in the initial elongation efficiency of RPA. This reduction in turn affects the onset of amplification depending on the extent of CpG methylation; therefore, the methylation level is quantified by RPA. We demonstrate that the onset of amplification was delayed by CpG methylation when PCR products containing the vascular endothelial growth factor (VEGF) G4 and i-motif-forming region were used as the template. Furthermore, onset of amplification was delayed with the increase in CpG methylation of the VEGF region on genomic DNA. These results demonstrate that the sensing system is capable of directly detecting the methylation level at a constant temperature (39 °C) within 30 min without performing bisulfite conversion or affinity capture of methylated DNA.
Keywords	DNA ; DNA methylation ; DNA-directed DNA polymerase ; analytical chemistry ; bisulfites ; recombinase polymerase amplification ; temperature ; vascular endothelial growth factors
Language	English
Dates of publication	2022-08
Size	p. 6223-6231.
Publishing place	Springer Berlin Heidelberg
Document type	Article
ISSN	1618-2642
DOI	10.1007/s00216-022-04192-3
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Article: Stabilization of VEGF i-motif structure by CpG methylation

Kimura, Kosuke / Oshikawa, Daiki / Ikebukuro, Kazunori / Yoshida, Wataru

Biochemical and biophysical research communications. 2022 Feb. 26, v. 594

2022

Abstract: The intercalated motif (i-motif) is a non-canonical nucleic acid structure formed by intercalated hemi-protonated cytosine base pairs (C–C⁺) under acidic conditions. The i-motif structure formation is involved in biological processes such as ... ...

Abstract	The intercalated motif (i-motif) is a non-canonical nucleic acid structure formed by intercalated hemi-protonated cytosine base pairs (C–C⁺) under acidic conditions. The i-motif structure formation is involved in biological processes such as transcription regulation. Therefore, the identification of factors controlling i-motif formation is important in elucidating the cellular functions it controls. We previously reported that the VEGF G-quadruplex structure is stabilized by CpG methylation. In this study, the effect of CpG methylation on the stability of the VEGF i-motif structure was investigated. The VEGF i-motif-forming oligonucleotide contains four cytosines on CpG sites, and three of the four cytosines (C4, C15, and C20) are involved in C–C⁺ formation in the i-motif structure. Circular dichroism (CD) spectra analysis demonstrated that full CpG methylation increased the pH of mid transition (pHT) of the i-motif structure by 0.1, and the melting temperature (Tₘ) by 5.1 °C in 25 mM sodium cacodylate buffer at pH 5.0. Moreover, single methylation at C4, C15, and C20 increased Tₘ by 0.5, 1.7, and 2.0 °C in the buffer, respectively. These results demonstrated that CpG methylation stabilized the VEGF i-motif structure.
Keywords	DNA methylation ; circular dichroism spectroscopy ; cytosine ; nucleic acids ; oligonucleotides ; pH ; research ; sodium ; temperature ; transcription (genetics)
Language	English
Dates of publication	2022-0226
Size	p. 88-92.
Publishing place	Elsevier Inc.
Document type	Article
ZDB-ID	205723-2
ISSN	0006-291X ; 0006-291X
ISSN (online)	0006-291X
ISSN	0006-291X
DOI	10.1016/j.bbrc.2022.01.054
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