Quantitative in vitro assays to measure RNA methyltransferase and demethylase activity for epitranscriptomics research.
Understanding the functional role of RNA modifications such as m6A requires robust, quantitative methods to measure the activity of the writing and erasing enzymes. Our RNA Modification Enzyme Activity Assay service provides a standardized, customizable platform to assess methyltransferase and demethylase activity in vitro, using defined RNA substrates and sensitive detection via mass spectrometry.
Our service is designed for biotech startups and academic groups developing small-molecule inhibitors or studying the epitranscriptomic regulation of disease pathways. By delivering accurate kinetic parameters and relative activity data, we enable you to screen candidates, validate targets, and advance your mechanistic understanding with confidence.
Each project is scoped individually to match your specific enzyme, substrate, and buffer requirements. We provide end-to-end support from substrate design through data reporting, with rigorous quality controls at every step.
Key Features
Custom Substrate RNA Design
We design and synthesize short RNA substrates containing specific modification sites (e.g., GGACU motifs for m6A) or custom sequences to match your target enzyme. Substrates can be labeled or unlabeled depending on detection method.
LC-MS Detection and Quantification
Enzyme activity is quantified by liquid chromatography–mass spectrometry (LC-MS) for direct measurement of modified nucleoside products or by changes in substrate/product ratios. This label-free approach provides absolute quantification and high sensitivity.
Kinetic Parameter Analysis
We perform Michaelis-Menten kinetic analysis to determine Km and Vmax values for your enzyme under optimized buffer conditions. For inhibitor studies, IC50 values are calculated from dose-response curves.
Positive Control Validation
Each assay run includes known enzyme standards and negative controls to ensure reproducibility and accuracy. We validate assay conditions before proceeding with your test compounds or experimental samples.
Assay Options
Our platform supports the major classes of RNA modification enzymes. The following assay types are routinely performed:
m6A Methyltransferase Activity Assay: Measures incorporation of methyl groups onto adenosine residues in substrate RNA. Compatible with the METTL3/METTL14 complex, METTL16, and other writers. Detection via LC-MS or radiometric assays.
m6A Demethylase Activity Assay: Quantifies removal of methyl groups from m6A-containing RNA. Supports FTO and ALKBH5 demethylase studies. Typically uses LC-MS to monitor demethylation over time.
Inhibitor Screening: Dose-response experiments to determine IC50 values of small-molecule inhibitors against your target enzyme, with known-inhibitor positive control and DMSO vehicle control, typically in duplicate.
Time-Course Profiling: Additional time points are included for time-course studies to capture linear kinetics and reaction progression.
Workflow
Our standard workflow proceeds through five phases, from experimental design to final report.
Project Scoping and Substrate Design: We discuss your enzyme target, desired substrate sequence, modification site, and buffer conditions. Substrate RNA is chemically synthesized or in vitro transcribed and purified.
Enzyme Incubation Under Optimized Conditions: Your enzyme (provided by you or sourced) is incubated with the substrate RNA under defined temperature, pH, and cofactor conditions. Reaction time points are selected to capture linear kinetics.
Reaction Quench and Sample Cleanup: Reactions are quenched with organic solvent or heat. Samples are cleaned up by phenol-chloroform extraction or spin columns to remove proteins and salts, preparing for detection.
Detection by LC-MS or Alternative Method: Cleaned samples are analyzed by LC-MS to quantify modified and unmodified nucleosides. Alternatively, radiometric detection or HPLC-UV can be used as scoped.
Data Processing and Final Report: We process raw data to calculate kinetic parameters (Km, Vmax, kcat) or relative activity. A comprehensive report includes methods, raw data, and summary statistics with QC results.
Flexible Workflow
Workflow phases are adapted per project. For inhibitor screening, we incorporate dose-response curves and IC50 calculations. For time-course studies, additional time points are included.
Quality Assurance
All assays include positive controls (known active enzyme and standard substrate) and negative controls (no enzyme, heat-inactivated enzyme) to validate assay performance.
We perform intra-assay and inter-assay reproducibility assessments using control samples. Standard curves for quantification are run with each batch to maintain accuracy. Our LC-MS methods include internal standards to correct for matrix effects and injection variability.
Why Choose Us
Customizable Assay Design: Every project starts with a consultation to tailor substrate sequence, buffer, enzyme source, and detection method to your specific needs.
Experienced Scientific Team: Our team has extensive experience in RNA enzymology, epitranscriptomics, and LC-MS method development, ensuring reliable results.
Comprehensive Reporting: You receive a detailed report including raw data, kinetic parameters, QC statistics, and a clear summary to support publications or internal decisions.
High Sensitivity Detection: LC-MS methods achieve detection limits in the femtomole range, allowing measurement of low-activity enzymes or limited substrate amounts.
Backed by Creative Biogene: All projects are delivered under Creative Biogene's ISO-aligned quality system, with dedicated project management, milestone-based reporting, and full intellectual property protection for your data.
Sample Types and Enzyme Sources
Our assay is compatible with a variety of enzyme formats and sample types, as outlined below.
Recombinant Enzymes: Purified recombinant methyltransferases or demethylases (e.g., METTL3–METTL14, FTO) expressed in E. coli, insect cells, or mammalian systems.
Nuclear or Whole-Cell Extracts: Enzyme activity in native cellular extracts can be measured after optimization to reduce background and enhance specificity.
Immunoprecipitated Complexes: Endogenous enzyme complexes pulled down from cell lysates can be assayed for activity in vitro.
Purified Protein Domains: Truncated or mutant enzyme variants can be assayed to study structure-function relationships or domain requirements.
Contact Us
Whether you are screening small-molecule inhibitors, characterizing a newly identified writer or eraser enzyme, or building mechanistic data for a publication, the scientific team at Creative Biogene can help you design the most appropriate enzyme activity assay—from substrate design through kinetic interpretation. Reach out to discuss a tailored solution for your next project.
