Sequencing and Structure Probing of Long RNAs Using MarathonRT: A Next-Generation Reverse Transcriptase.

Area of Science:

• Molecular Biology
• Biochemistry
• Genomics

Background:

• Reverse transcriptase (RT) enzymes are crucial for RNA analysis.
• Existing low-processivity RT enzymes struggle with long, complex RNA molecules.
• There is a need for RTs capable of high processivity for accurate RNA studies.

Purpose of the Study:

• To systematically characterize MarathonRT, a eubacterial group II intron-encoded RT.
• To optimize MarathonRT performance for various RNA research applications.
• To enhance tools for analyzing RNA metabolism and transcriptome composition.

Main Methods:

• Characterization of MarathonRT enzyme activity.
• Optimization of reaction protocols for MarathonRT.
• Application of MarathonRT in single-cycle reverse transcription of long RNAs.
• Integration of MarathonRT with dimethyl sulfate mutational profiling (DMS-MaP) and selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP).
• Utilizing MarathonRT for ultra-long amplicon generation and detection of natural RNA base modifications.

Main Results:

• MarathonRT demonstrates efficient copying of highly structured long RNA molecules in a single pass.
• Optimized protocols expand MarathonRT's utility across diverse RNA analysis techniques.
• Successful application in single-cycle reverse transcription, DMS-MaP, SHAPE-MaP, and ultra-long amplicon sequencing.
• Demonstrated capability for detecting natural RNA base modifications.

Conclusions:

• MarathonRT is a powerful tool enzyme for high-fidelity reverse transcription of challenging RNA substrates.
• Optimized MarathonRT protocols provide an upgraded toolkit for advanced RNA research.
• This enzyme and its associated methods hold significant potential for clinical applications in RNA analysis.