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Updated: May 14, 2026
Antibody-Free Assay for RNA Methyltransferase Activity Analysis
Published on: July 9, 2019
An Investigation of RNA Methylations with Biophysical Approaches in a Cervical Cancer Cell Model
Buket Sağlam1, Onur Akkuş2, Azime Akçaöz-Alasar1
1Noncoding RNA Laboratory, Department of Molecular Biology and Genetics, İzmir Institute of Technology, 35430 Izmir, Türkiye.
Abstract:
RNA methylation adds a second layer of genetic information that dictates the post-transcriptional fate of RNAs. Although various methods exist that enable the analysis of RNA methylation in a site-specific or transcriptome-wide manner, whether biophysical approaches can be employed to such analyses is unexplored. In this study, Fourier-transform infrared (FT-IR) and circular dichroism (CD) spectroscopy are employed to examine the methylation status of both synthetic and cellular RNAs. The results show that FT-IR spectroscopy is perfectly capable of quantitatively distinguishing synthetic m6A-methylated RNAs from un-methylated ones. Subsequently, FT-IR spectroscopy is successfully employed to assess the changes in the extent of total RNA methylation upon the knockdown of the m6A writer, METTL3, in HeLa cells. In addition, the same approach is shown to accurately detect reduction in total RNA methylation upon the treatment of HeLa cells with tumor necrosis factor alpha (TNF-α). It is also demonstrated that m1A and m6A methylation induce quite a distinct secondary structure on RNAs, as evident from CD spectra. These results strongly suggest that both FT-IR and CD spectroscopy methods can be exploited to uncover biophysical properties impinged on RNAs by methyl moieties, providing a fast, convenient and cheap alternative to the existing methods.
Insights
Fourier-transform infrared and circular dichroism spectroscopy can quantitatively analyze RNA methylation. These biophysical methods offer a fast, cheap alternative for studying RNA modifications and their impact on RNA structure.
Area of Science:
- Biophysics
- Molecular Biology
- Spectroscopy
Background:
- RNA methylation, particularly N6-methyladenosine (m6A), is a crucial epigenetic modification regulating RNA function.
- Existing methods for analyzing RNA methylation are often complex or limited in scope.
- The potential of biophysical techniques for RNA methylation analysis remains largely unexplored.
Purpose of the Study:
- To investigate the utility of Fourier-transform infrared (FT-IR) and circular dichroism (CD) spectroscopy for analyzing RNA methylation.
- To determine if these methods can quantitatively distinguish methylated from unmethylated RNAs.
- To assess changes in RNA methylation levels in cellular contexts.
Main Methods:
- Fourier-transform infrared (FT-IR) spectroscopy was used to analyze synthetic and cellular RNAs.
- Circular dichroism (CD) spectroscopy was employed to examine RNA secondary structures.
- RNA methylation was modulated in HeLa cells via METTL3 knockdown and TNF-α treatment.
Main Results:
- FT-IR spectroscopy accurately and quantitatively differentiated between m6A-methylated and unmethylated synthetic RNAs.
- FT-IR successfully detected changes in total RNA methylation levels in HeLa cells following METTL3 knockdown and TNF-α treatment.
- CD spectra revealed distinct secondary structure alterations induced by m1A and m6A methylation.
Conclusions:
- FT-IR and CD spectroscopy are effective biophysical tools for analyzing RNA methylation status and its structural consequences.
- These spectroscopic methods provide a rapid, cost-effective, and convenient alternative to current RNA methylation analysis techniques.
- The findings open new avenues for exploring the biophysical properties of RNA modifications.
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