Archaeal RNA ligase from thermoccocus kodakarensis for template dependent ligation

Abstract

ABSTRACTNicking-sealing RNA ligases play a significant biological role in host defense and cellular repair, and have become an important molecular tool in biomedical engineering. Due to the propensity for RNA to form secondary structures, RNA modifying enzymes with elevated optimum temperatures are highly desired. Current characterized double stranded RNA ligases, such as the bacteriophage T4 RNA ligase 2, while possessing good template dependency, are not active at elevated temperatures. The few characterized RNA ligases from thermophiles exhibit high template independency. We synthesize and characterize here, KOD RNA ligase (KOD1Rnl), a thermostable and template dependent RNA ligase from the archaeon, Thermoccocus Kodakarensis. We disclose that a 13 time reduction in template independent ligation can be achieved with the addition of a single stranded DNase, such as RecJ. We also elucidate the effects of the presence of blood proteins on the activity of KOD1Rnl. Template dependent and thermostable RNA ligases, such as KOD RNA ligase, can be utilized in RNA detection, modification and sequencing.