[P007] Poster

12:10 PM - 1:00 PM JST(3:10 AM - 4:00 AM UTC)

[P04-577] Cell-Penetrating Peptides as Tools for Post-Transcriptional Regulation in Prorocentrum lima

○Qisya M. Amirul¹, Tetsushi Mori¹ (1. Tokyo University of Agriculture and Technology (Japan))

Keywords:

Cell-Penetrating Peptide (CPP),Dinoflagellates,Protein Translation Regulation,Prorocentrum

Dinoflagellates are important unicellular aquatic microorganisms that function as primary producers in marine ecosystems. Despite their ecological importance, certain dinoflagellate species are capable of forming harmful algal blooms (HABs) that release potent toxins into surrounding water. These toxic blooms pose significant threats to marine biodiversity, fisheries, aquaculture, and human health through seafood contamination. The frequency and severity of such blooms are expected to increase due to climate change, rising sea surface temperatures, and nutrient enrichment in coastal waters. However, molecular studies of dinoflagellates remain limited due to lack of efficient genetic manipulation tools. The large and complex genomes of dinoflagellates, combined with their unusual chromosomal organisation and thick thecal plates, have made conventional genetic transformation techniques difficult to implement. To address these limitations, this study explores the use of cell-penetrating peptide-peptide nucleic acid conjugates (CPP-PNAs) as a novel molecular approach to investigate and manipulate protein translation in dinoflagellates. CPPs are short peptides capable of crossing cellular membranes and delivering molecular cargo into cells, while PNAs are synthetic nucleic acid analogues that bind specifically to complementary RNA sequences, thereby inhibiting gene translation. By combining these molecules, CPP-PNA conjugates provide a potential strategy for targeted gene suppression without the need for permanent genetic modification. The marine dinoflagellate Prorocentrum lima, a known producer of the diarrhetic shellfish poisoning toxin, okadaic acid (OA), was selected as the model organism. Initially, fluorescence-labelled CPPs were evaluated to determine their ability to penetrate P. lima cells using fluorescence microscopy and confocal laser scanning microscopy. The fluorescence-labelled CPP, (DabFF)₄DabK-FAM, successfully entered P. lima cells despite the presence of rigid thecal plates, demonstrating the capability of CPPs to overcome structural barriers that typically hinder molecular delivery. Importantly, minimal cytotoxic effects were observed, indicating that the peptide delivery system is compatible with dinoflagellate cell viability. Subsequently, targeted CPP-PNA conjugates were designed to suppress the expression of ATP-binding cassette (ABC) transporters, specifically ABCB1, ABCG2, and ABCC1, which play a role in the export of OA from the cell. OA concentrations were quantified using a protein phosphatase inhibition assay (PPIA), while cell viability was monitored microscopically. A significant reduction in extracellular OA levels was observed after four days of treatment, indicating successful suppression of ABC transporter translation. Transporter activity recovered after approximately ten days, suggesting that the CPP-PNA effect is temporary and reversible. Increased intracellular OA levels accompanied by decreased extracellular OA confirmed that CPP-PNA treatment selectively inhibited toxin release rather than toxin biosynthesis. This study demonstrates a simple yet effective molecular strategy for regulating protein translation in dinoflagellates and provides a valuable tool for investigating gene function and developing new strategies for understanding and managing harmful algal bloom-forming species.