The Dual Nature of RNA Ligation and Degradation by T4 RNA Ligase 1: Insights Into an AMP‐Driven, Nucleobase‐Dependent Reverse Reaction

T4 RNA Ligase 1 (Rnl1) catalyzes the ligation of a 5′‐phosphate to a 3′‐hydroxyl in single‐stranded RNA and is widely used in RNA biotechnology for applications such as RNA library preparation and 3′‐adapter ligation. In addition to ligation reaction, Rnl1 catalyzes the reverse reaction, leading to RNA degradation. However, how RNA sequence affects susceptibility to this reverse reaction remains unclear, and practical strategies for controlling it through buffer conditions have not been established. Previous studies showed that AMP promotes and ATP suppresses the reverse reaction; we confirmed these effects by electrophoretic analysis of FAM‐labeled RNA homopolymers, a substrate not previously examined. Furthermore, using miRNA substrates, we demonstrated that the reverse reaction can occur under conditions that more closely resemble the practical conditions in which Rnl1 is used, thereby extending the relevance of previous findings. Additionally, we showed that the Rnl1‐mediated reverse reaction exhibits pronounced nucleobase preference. The reverse reaction is suppressed by elevated temperature and duplex formation. Furthermore, experimental results combined with stochastic simulations indicate that Rnl1‐mediated degradation proceeds via an endonucleolytic mechanism. These findings define how reaction conditions and nucleobase difference influence reverse reaction efficiency and provide practical guidance for minimizing RNA degradation while maintaining ligation efficiency.