Engineering of a DNA Polymerase for Direct m⁶ A Sequencing

Area of Science:

• Molecular Biology
• Genomics
• Biochemistry

Background:

• RNA modifications, particularly N⁶ -methyladenosine (m⁶ A), are crucial for gene expression regulation in mammals.
• Current m⁶ A detection methods are indirect, relying on antibody enrichment and often losing information during reverse transcription.

Purpose of the Study:

• To develop novel DNA polymerase variants for direct sequencing of m⁶ A modifications.
• To overcome the limitations of current antibody-based RNA modification detection techniques.

Main Methods:

• Evolved a reverse transcriptase-active KlenTaq DNA polymerase variant using a specialized screening method.
• Identified a polymerase mutant demonstrating increased misincorporation opposite m⁶ A compared to unmodified adenosine.
• Applied the engineered DNA polymerase in next-generation sequencing workflows.

Main Results:

• Successfully generated a DNA polymerase variant capable of distinguishing m⁶ A modifications.
• The engineered polymerase exhibits altered base incorporation opposite m⁶ A.
• Direct identification of m⁶ A sites was achieved using sequencing data from untreated RNA samples.

Conclusions:

• The novel DNA polymerase facilitates direct m⁶ A detection, advancing epitranscriptomics research.
• This method offers a more efficient and informative alternative to existing antibody-dependent techniques.
• Enables direct sequencing of m⁶ A sites without prior RNA treatment or enrichment.