biostudies-literatureSingh AS.S.P.Latin American Fellow in Biomedical SciencesNIH HHSgkag008https://www.ebi.ac.uk/biostudies/studies/S-EPMC12820530biostudies-literatureUnknown54(2)Twinkle is the sole replicative helicase in human mitochondria, essential for mitochondrial DNA replication. Beyond its canonical unwinding activity, Twinkle has non-canonical activities, including DNA annealing and strand-exchange. Here, we show that these non-canonical activities extend to RNA. Twinkle binds RNA and catalyzes RNA:DNA hybrid formation through annealing, strand-exchange, and toehold-mediated strand displacement. Twinkle can unwind RNA:DNA forks when loaded onto the DNA tail but not the RNA tail. Although the physiological role of these RNA-related activities remains unclear, we show that Twinkle can strand-exchange an RNA downstream of a stalled replication fork to restart replication. The annealing/strand-exchange activity can be involved in DNA replication initiation andNucleic acids researchHuman mitochondrial helicase Twinkle has RNA binding, annealing, and strand-exchange activities.PMC12820530R01GM145925R35GM118086Yin YWSingh AJohnson LCBaruch-Torres NPatel SSfalseHuman mitochondrial helicase Twinkle has RNA binding, annealing, and strand-exchange activities.Twinkle is the sole replicative helicase in human mitochondria, essential for mitochondrial DNA replication. Beyond its canonical unwinding activity, Twinkle has non-canonical activities, including DNA annealing and strand-exchange. Here, we show that these non-canonical activities extend to RNA. Twinkle binds RNA and catalyzes RNA:DNA hybrid formation through annealing, strand-exchange, and toehold-mediated strand displacement. Twinkle can unwind RNA:DNA forks when loaded onto the DNA tail but not the RNA tail. Although the physiological role of these RNA-related activities remains unclear, we show that Twinkle can strand-exchange an RNA downstream of a stalled replication fork to restart replication. The annealing/strand-exchange activity can be involved in DNA replication initiation and2026-01-01T00:00:00Z2026 Jan2026-06-06T21:37:01.565Z2026-06-05T03:11:33.5ZS-EPMC128205304156225310.1093/nar/gkag008