Nano-DMS-MaP – isoform-specific RNA structure determination

RNA, the versatile molecule essential for various biological functions, holds many mysteries within its structure. Understanding how RNA works requires deciphering its intricate folds, especially considering the variations among RNA isoforms. Researchers at the Helmholtz Centre for Infection Research have developed a groundbreaking method called Nanopore Dimethyl Sulfate Mutational Profiling (Nano-DMS-MaP) offers a key to unlock these secrets.

Unlike traditional methods limited by short sequencing reads, Nano-DMS-MaP utilizes nanopore sequencing to explore the structures of long and closely related RNA molecules. By detecting subtle differences in these structures, researchers can uncover the unique characteristics of each RNA isoform.

This Protocol outlines the development and applications of Nano-DMS-MaP, providing a roadmap for molecular biologists to conduct in situ isoform-specific RNA structure determination experiments. From bench setup to data analysis, the Protocol offers step-by-step guidance to ensure the success of the experiment.

In cell probing experiments, which can be completed in 3-4 days by experienced molecular biologists, researchers use dimethyl sulfate to mark RNA structures, followed by nanopore sequencing to analyze the changes. However, data analysis requires familiarity with command line tools and Python scripts, taking an additional 3-5 days.

By shedding light on the structural differences among RNA isoforms, Nano-DMS-MaP opens new avenues for understanding RNA’s diverse biological functions. From unraveling the mechanisms of alternative splicing to deciphering the complexities of RNA processing, this method holds immense potential for advancing our knowledge of RNA biology.

As researchers continue to delve deeper into the world of RNA, tools like Nano-DMS-MaP will play a crucial role in uncovering its secrets. With each discovery, we move closer to unlocking the full potential of RNA and harnessing its power for various applications in biology and medicine.

Nano-DMS-MaP workflow

Fig. 1

a, Sample preparation (Steps 1–25). RNA can be probed with DMS either in situ or in vitro. DMS leads to methylation of nitrogens on A, C and U residues (red circles). Modified RNA is extracted (Steps 26–48) and then reverse transcribed with MarathonRT (Steps 49–54). Modified nucleotides (red diamonds) are converted into mutations (red stars). Full-length molecules are selected using PCR amplification (Steps 55–77). Samples are prepared for nanopore sequencing using an indexing kit to add adaptors (green bar) and barcodes (blue and brown bar) (Steps 78–132). b, Sequencing and analysis. Samples are sequenced on a nanopore flowcell (Q20+) (Step 133). Sequencing data are basecalled in super high accuracy mode, either on device or offline (Steps 134–138). Long reads are then assigned to isoforms using IsoQuant (Steps 139–144) and mutations counted using RNAframework (Steps 145–149). DMS reactivities can be plotted as heat maps or used to predict isoform-specific RNA structures (Steps 150–153). Heat maps represent DMS reactivities in red, white and blue, where red is 1, white is 0.5 and blue is 0.

Availability – Code used for the Nano-DMS-MaP analysis is accessible via the Smyth lab Github (https://github.com/smyth-lab/Nano-DMS-MaP)

Gribling-Burrer AS, Bohn P, Smyth RP. (2024) Isoform-specific RNA structure determination using Nano-DMS-MaP. Nat Protoc [Epub ahead of print], [abstract]

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