Magnetic RNA building blocks (RBBs)-driven modular spheroid assembly

The development of physiologically relevant three-dimensional (3D) cell culture platforms is essential for advancing tissue engineering, disease modeling, and drug screening. However, conventional spheroid fabrication methods face persistent challenges in reproducibility, architectural control, and biocompatibility. Here, we present a modular spheroid assembly strategy based on magnetic RNA building blocks (RBBs) and azide-presenting cell building blocks (CBBs), enabling programmable and bioorthogonal construction of 3D multicellular architectures. RBBs were synthesized via rolling circle transcription in the presence of Mn²⁺, forming magnetically responsive RNA-based nanostructures. Incorporation of DBCO-modified nucleotides enabled strain-promoted azide-alkyne cycloaddition (SPAAC) with CBBs, yielding highly selective and rapid spheroid formation. The resulting spheroids exhibited uniform 3D organization, high viability, and robust biocompatibility across multiple cell types. Furthermore, modular assembly via secondary click chemistry allowed for heterotypic spheroid integration and magnetic spatial control, mimicking complex tissue microenvironments. Importantly, the magnetic scaffolds were fully degradable under physiological reductive conditions, permitting residue-free removal post-assembly. This versatile, tunable, and biodegradable RNA-based platform offers a powerful solution for constructing magnetically controllable, heterocellular spheroids, advancing next-generation 3D culture systems.