Targeted Protein Translation Suppression in the Dinoflagellate, Prorocentrum lima, Using Cell-Penetrating Peptides

Dinoflagellates remain among the most challenging eukaryotic systems for functional molecular studies due to their complex genetic architecture, which limits the availability of tools for targeted translational regulation. This study therefore aimed to evaluate whether cell-penetrating peptide–conjugated peptide nucleic acids (CPP-PNAs) could provide an alternative approach for targeted translational regulation in dinoflagellates, using the okadaic acid (OA)-producing Prorocentrum lima as a model species. Fluorescence-labeled CPPs were screened for intracellular delivery using fluorescence and confocal microscopy, and CPP-PNAs were designed to target three ATP-binding cassette (ABC) transporters associated with OA export: ABCB1, ABCG2, and ABCC1. Extracellular and intracellular OA levels were quantified using a protein phosphatase inhibition assay (PPIA). Among the CPPs screened, (DabFF)4Dab-K-FAM showed effective intracellular delivery and was selected as the scaffold for PNA conjugation. Whereas individual targeting of ABCB1, ABCG2, or ABCC1 produced only limited effects, simultaneous targeting of all three transporters significantly reduced extracellular OA. The accompanying increase in intracellular OA was consistent with reduced toxin release rather than reduced toxin production. Extracellular OA subsequently recovered toward control levels by day 10, indicating that the CPP-PNA-mediated effect was transient and reversible. Together, these findings establish the feasibility of CPP-PNA-mediated translational regulation in P. lima and provide a basis for exploring its application as a functional tool in other dinoflagellates.