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bioRxiv · 10.64898/2026.10.12.758436

Chimeric archaeal BHB-human pre-tRNA scaffold HARBOR system enables versatile production of functional circular RNA modalities

Cao, S.-M.·Feng, X.·Zhang, J. T.-C.·Wu, H.·Wang, X.·Ye, K.·Chen, L.-L.
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Abstract

Circular RNAs (circRNAs) exhibit superior intrinsic stability and lower immunogenicity compared with linear RNAs, rendering them promising modalities for biomedical and biotechnological applications. Here, we establish the HARBOR (Human-Archaeal RNA Backbone for Overexpression of circular RNA) system, an engineered endogenous circRNA expression platform integrating a human precursor transfer RNA (pre-tRNA) scaffold with an archaeal Bulge-Helix-Bulge (BHB) motif. HARBOR repurposes the endogenous tRNA splicing machinery to generate chimeric pre-tRNA intermediates, which are rapidly degraded following processing, while producing circRNAs ranging from hundreds of nucleotides to 8 knt in cells and in vivo. Notably, it enables robust production of large-sized circRNAs with yields nearly 80-fold than the intronic complementary sequence-mediated back-splicing (3D5) system and can drive protein expression from translatable circRNAs at an efficiency close to 20-fold than the Tornado system. Such prominent properties support diverse applications of HARBOR, including producing therapeutic aptamers targeting PKR-associated neuroinflammation and pathological TDP-43 condensation, as well as protein-coding circRNAs encoding adenine base editors for genome editing and MYOD1 for fibroblast-to-myocyte trans-differentiation. Collectively, HARBOR represents a versatile, scalable, and user-friendly platform that unifies circRNA-based aptamer and protein expression applications, advancing circRNA-enabled engineering and biomedical applications.

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Cao, S.-M., Feng, X., Zhang, J. T.-C., Wu, H., Wang, X., Ye, K., Chen, L.-L.. 2026-10-13. Chimeric archaeal BHB-human pre-tRNA scaffold HARBOR system enables versatile production of functional circular RNA modalities. https://doi.org/10.64898/2026.10.12.758436

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