Journal: bioRxiv

Article Title: SUPREM: an engineered non-site-specific m 6 A RNA methyltransferase with highly improved efficiency

doi: 10.1101/2023.08.23.554385

Figure Lengend Snippet: (A) Schematic maximum likelihood tree. The node values showed ultrafast bootstrap values calculated by IQ-TREE. (B-C) MTase-Glo assay of M.EcoGII and its ancestral proteins for (B) DNA and (C) RNA at a single time point. (B) The DNA methylation reaction was performed using 100 nM of methyltransferases, 50 nM DNA substrate (3.5 kbp containing 1844 adenine bases, Supplementary Table S2) and 10 μM SAM in 1× CutSmart buffer (NEB) at 37 °C for 10 min. (C) The RNA methylation reaction was performed using 100 nM of the corresponding methyltransferases, 25 nM RNA substrate (1.7 kb containing 544 adenine bases, sequence in Supplementary Table S2), and 10 μM SAM in 1× CutSmart buffer (NEB) at 37 °C for 60 min. Three independent experiments were performed, and each point showed an average of technical duplicates. P values were determined by the one-way ANOVA test with the Dunnett test. (D) Time course experiments for RNA substrates. The condition of RNA methylation reaction was the same as (C) except for reaction time. Three independent experiments were performed, and each point showed an average of technical duplicates. SAH production amount was calculated by SAH standard curve. The lines represent simple linear regression analysis on the data from 0 to 120 min (SUPREM) or to 240 min (M.EcoGII, Anc291). (E) The fraction of m 6 A methylation in total adenine by UPLC analysis. The RNA methylation reaction was performed using 5 μg of the corresponding methyltransferases, 1 μg RNA substrate (294 nt containing 80 adenine bases, sequence in Supplementary Table S2) and 320 μM SAM (NEB) in 1× CutSmart buffer (NEB) at 37 °C for 120 min. Each point showed three independent methylation reactions. Error bars represent standard error of the mean (SEM).

Article Snippet: To investigate the presence of non-site-specific RNA methylation activity by M.EcoGII and ancestral proteins, we performed Oxford Nanopore direct RNA sequencing (DRS) to characterize the site specificity of RNA methylation in M.EcoGII, Anc291, and SUPREM.

Techniques: Glo Assay, DNA Methylation Assay, Methylation, Sequencing

Journal: bioRxiv

Article Title: SUPREM: an engineered non-site-specific m 6 A RNA methyltransferase with highly improved efficiency

doi: 10.1101/2023.08.23.554385

Figure Lengend Snippet: (A) Mapping detected RNA methylation sites on the 18 knt Fluc RNA sequence by MinION DRS and Nanocompore pipeline. (B) Mean RNA base frequency in modified 5-mer motifs detected by DRS. The “Expectation” represents the mean frequency of each base in 5-mers that contain at least one adenine within the RNA substrate.

Article Snippet: To investigate the presence of non-site-specific RNA methylation activity by M.EcoGII and ancestral proteins, we performed Oxford Nanopore direct RNA sequencing (DRS) to characterize the site specificity of RNA methylation in M.EcoGII, Anc291, and SUPREM.

Techniques: Methylation, Sequencing, Modification

Journal: Cold Spring Harbor Perspectives in Biology

Article Title: Chromatin Remodeling in Mammary Gland Differentiation and Breast Tumorigenesis

doi: 10.1101/cshperspect.a004515

Figure Lengend Snippet: Summary of candidate gene and genome-wide techniques for DNA methylation analysis a

Article Snippet: Microarray-based approaches , DMH (differential methylation hybridization): MTA (methylation tissue array); MSO (methylation-specific oligonucleotide); HELP ( Hpa II tiny fragment enrichment by ligation-mediated PCR); AIMS (amplification of intermethylated sites); MSNP (methylation single nucleotide polymorphism chip-based method); MMASS (microarray-based methylation assessment of single sample); PMAD (promoter-associated methylated DNA amplification); MSDK (methylation-specific digital karyotyping) MIAMI (microarray-based integrated analysis of methylation); MCAM (methylated CpG island amplification and microarray); MeDIP-chip (methylated DNA immunoprecipitation on microarray); MIRA-chip (methylated-CpG island recovery assay on microarray); Mcr BC ; MethylScope ; Pharmacologic unmasking analysis ; and Infinium BeadArray.

Techniques: Genome Wide, DNA Methylation Assay, Cloning, Methylation Sequencing, Methylation, Combined Bisulfite Restriction Analysis Assay, Microarray, Hybridization, Ligation, Amplification, DNA Amplification, Methylated DNA Immunoprecipitation, Immunoprecipitation, Next-Generation Sequencing, Methylated DNA Immunoprecipitation Sequencing

Journal: Cold Spring Harbor Perspectives in Biology

Article Title: Chromatin Remodeling in Mammary Gland Differentiation and Breast Tumorigenesis

doi: 10.1101/cshperspect.a004515

Figure Lengend Snippet: Summary of candidate gene and genome-wide techniques for DNA methylation analysis a

Article Snippet: Microarray-based approaches , DMH (differential methylation hybridization): MTA (methylation tissue array); MSO (methylation-specific oligonucleotide); HELP ( Hpa II tiny fragment enrichment by ligation-mediated PCR); AIMS (amplification of intermethylated sites); MSNP (methylation single nucleotide polymorphism chip-based method); MMASS (microarray-based methylation assessment of single sample); PMAD (promoter-associated methylated DNA amplification); MSDK (methylation-specific digital karyotyping) MIAMI (microarray-based integrated analysis of methylation); MCAM (methylated CpG island amplification and microarray); MeDIP-chip (methylated DNA immunoprecipitation on microarray); MIRA-chip (methylated-CpG island recovery assay on microarray); Mcr BC ; MethylScope ; Pharmacologic unmasking analysis ; and Infinium BeadArray.

Techniques: Genome Wide, DNA Methylation Assay, Cloning, Methylation Sequencing, Methylation, Combined Bisulfite Restriction Analysis Assay, Microarray, Hybridization, Ligation, Amplification, DNA Amplification, Methylated DNA Immunoprecipitation, Immunoprecipitation, Next-Generation Sequencing, Methylated DNA Immunoprecipitation Sequencing