RNA structure through multidimensional chemical mapping.

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Non-coding RNAs exhibit dynamic conformational flexibility, complicating structure determination.
• High-throughput chemical mapping provides transcriptome-wide data but often lacks resolution for de novo structure determination.
• Multidimensional chemical mapping (MCM) methods offer enhanced RNA structural insights.

Purpose of the Study:

• To address the challenge of determining RNA structures, especially in complex or heterogeneous states.
• To evaluate the accuracy and scope of existing MCM techniques.
• To propose an expansion of MCM methods for broader in vivo RNA structurome analysis.

Main Methods:

• Mutate-and-map (M2), RNA interaction groups by mutational profiling (RING-MaP, MaP-2D), and multiplexed •OH cleavage analysis (MOHCA) were employed.
• These MCM methods measure nucleotide reactivity changes in response to modifications at other sites.
• In vitro blind tests and compensatory mutation experiments validated structural predictions.

Main Results:

• MCM methods accurately determined secondary and tertiary structures for RNAs up to 200 nucleotides.
• MCM successfully resolved structurally heterogeneous RNA states, such as ligand-free riboswitches.
• Current MCM protocols have limitations in scalability due to quadratic sequencing requirements with RNA length.

Conclusions:

• MCM methods are powerful tools for RNA structure determination, particularly for dynamic and heterogeneous states.
• A novel modify-cross-link-map (MXM) method is proposed to overcome current MCM limitations.
• MXM aims to enable comprehensive in vivo RNA structurome resolution, including transcriptomes and viral genomes.