Addition-Elimination Mechanism-Activated Nucleotide Transition Sequencing for RNA Dynamics Profiling

Area of Science:

• Molecular Biology
• Genomics
• Biochemistry

Background:

• Understanding intracellular transcript dynamics is key to elucidating gene functions.
• Conventional RNA sequencing (RNA-seq) provides only steady-state information, lacking dynamic insights.
• There is a need for methods that capture the dynamic nature of RNA species.

Purpose of the Study:

• To develop a novel method, addition-elimination mechanism-activated nucleotide transition sequencing (AENT-seq), for transcriptome-wide RNA dynamics profiling.
• To enable the study of nascent RNA transcripts and their dynamic behavior.
• To investigate the roles of specific genes in cancer progression and metastasis through RNA dynamics.

Main Methods:

• Metabolically labeling nascent transcripts with 4-thiouridine (4sU).
• Treating total RNA with hydrazine (N₂H₄·H₂O) in aqueous solution.
• Utilizing an addition-elimination chemical reaction to convert 4sU to 4-hydrazino cytosine (C*), which is recognized as cytosine (C) during DNA extension, thereby marking nascent transcripts for sequencing.

Main Results:

• Successfully developed and implemented AENT-seq for RNA dynamics profiling.
• Demonstrated the conversion of 4sU to C* via a simple aqueous chemical reaction.
• Applied AENT-seq to analyze dynamic information of genes implicated in cancer progression and metastasis.

Conclusions:

• AENT-seq provides a powerful tool for transcriptome-wide RNA dynamics analysis.
• The method's reliance on a simple chemical reaction facilitates its widespread adoption and application.
• AENT-seq offers significant potential for advancing research in gene regulation and disease mechanisms, particularly in cancer.