Structural inference of native and partially folded RNA by high-throughput contact mapping

Area of Science:

• Structural Biology
• RNA Biology
• Biochemistry

Background:

• Functional RNA molecules like ribozymes and riboswitches rely on complex tertiary structures for their biological activity.
• Characterizing the heterogeneous and partially folded states of large RNAs is challenging with traditional high-resolution methods.

Purpose of the Study:

• To introduce a novel method for rapidly inferring the tertiary helical arrangements of large RNA molecules in solution.
• To characterize previously intractable non-native RNA states.

Main Methods:

• Multiplexed Hydroxyl Radical Cleavage Analysis (MOHCA) was developed for high-throughput detection of residue contacts.
• The method involves random incorporation of radical cleavage agents followed by two-dimensional gel electrophoresis.
• MOHCA was validated on the P4-P6 domain of the Tetrahymena ribozyme and applied to a salt-stabilized non-native RNA state.

Main Results:

• MOHCA successfully recapitulated known states of the P4-P6 RNA domain at subhelical resolution.
• The method provided a three-dimensional structural portrait of a compact, non-native RNA state.
• This non-native state exhibited ordered tertiary contacts, differing from protein molten globule states.

Conclusions:

• MOHCA is a powerful tool for determining the structures of large RNAs in various solution states, including non-native ones.
• The technique is applicable to diverse RNA molecules and RNA/protein complexes, advancing the understanding of RNA structure-function relationships.