The mutate-and-map protocol for inferring base pairs in structured RNA.

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• Chemical mapping is a key technique for nucleic acid structural analysis, quantifying nucleotide reactivity for structural modeling.
• Recent advancements have focused on high-throughput methods for chemical modification, readouts, and data analysis in structural biology.
• Coupling chemical mapping with high-throughput mutagenesis offers a powerful approach to probe nucleic acid interactions.

Purpose of the Study:

• To present an in-house protocol for the "mutate-and-map" (M2) strategy.
• To detail the application of M2 strategy for inferring nucleic acid structure.
• To provide practical guidance on interpreting M2 data for structural biology research.

Main Methods:

• Integration of high-throughput mutagenesis with chemical mapping techniques.
• Utilizing point mutations to reveal exposed nucleotides and their interaction partners.
• Implementation of a 96-well capillary electrophoresis-based system for the mutate-and-map strategy.

Main Results:

• Systematic mutation and mapping experiments generate an experimental approximation of a molecule's contact map.
• The M2 strategy effectively identifies nucleotide-nucleotide interactions within nucleic acid structures.
• The protocol facilitates the interpretation of chemical mapping data for structural elucidation.

Conclusions:

• The mutate-and-map (M2) strategy provides a robust method for experimental nucleic acid structural analysis.
• This approach enhances the resolution and accuracy of structural modeling by providing contact map data.
• The described protocol offers practical insights for researchers applying M2 to study nucleic acid structures.