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Poster

Session: Session 1 Presenting Authors at Posters (Evens)

P-324 - High Precision Quantification of small RNA Slicing Activity - Native Index Ligation-based Targeted Degradome Sequencing (NIL-TDS)

Tuesday, July 15, 2025
09:55 - 11:30  
Location: Confex Hall 3
RNA on the move: Advances in Cross-Species communiation &Translational Research

    Presenting Author(s)

  • SN

    Sabrine Nasfi

    Institute for Phytopathology, Justus Liebig University
    Giessen, Hessen, Germany

    • Author(s)

  • BW

    Bernhard Timo Werner

    Postdoctoral researcher
    Justus Liebig University Giessen, Institute of Phytopathology
    Giessen, Hessen, Germany

  • JS

    Jens Steinbrenner

    IFZ- Phytopathology institute
    Giessen, Hessen, Germany

  • LS

    Lienhard Schmitz

    Institute of biochemistry, Medical Faculty
    Giessen, Hessen, Germany

  • MM

    Manar Makhoul

    Departement of Plant breeding, Research center for biosystems, land use and nutrition
    Giessen, Hessen, Germany

  • PS

    Patrick Schäfer

    Justus Liebig University Giessen, Institute of Phytopathology
    Giessen, Hessen, Germany

RNA interference (RNAi) regulates gene expression through small regulatory RNA (sRNA)-guided cleavage of target mRNAs. Targeting pathogen mRNA with sRNAs provides a promising strategy for plant disease control; however, accurate degradome analysis remains challenging. Existing methods (e.g. PARE, RLM-RACE) are labor-intensive, low in sensitivity, and lack quantitative precision. We therefore developed Native Index Ligation-based Targeted Degradome Sequencing (NIL-TDS), a novel, cost-effective Oxford Nanopore sequencing (ONT)-based method for direct, high-resolution detection and quantification of sRNA-guided cleavage events. As a proof of concept, NIL-TDS was applied on heat and salt-stressed plants, analysing Ath-miR400 and its target PENTATRICOPEPTIDE REPEAT 1 (PPR1). Quantitative data confirmed the presumed stress-mediated reduction in PPR1 cleavage for the first time, demonstrating NIL-TDS´s suitability for accurate prediction and selection of small RNAs. This approach refines degradome analyses thereby streamlining the discovery and validation of naturally occurring silencing events in plant-microbe-interactions and facilitating the development of RNA-based crop protection strategies. NIL-TDS application extends beyond plants and is demonstrated by detecting rare slicing events in mammals, such as miR-196-HOXB8 interactions in lung cancer cells. These findings highlight NIL-TDS’s potential to uncover novel regulatory mechanisms in gene expression and disease progression.