Programmable Computational RNA Droplets Assembled via Kissing-Loop Interaction

• Published in: ACS nano 2024-06, Vol.18 (24), p.15477-15486
• Main Authors: Udono, Hirotake, Fan, Minzhi, Saito, Yoko, Ohno, Hirohisa, Nomura, Shin-ichiro M., Shimizu, Yoshihiro, Saito, Hirohide, Takinoue, Masahiro
• Format: Article
• Language: English
• Subjects: MicroRNAs - chemistry; MicroRNAs - genetics; Nanostructures - chemistry; Nucleic Acid Conformation; RNA - chemistry
• ISSN: 1936-0851; 1936-086X; 1936-086X
• DOI: 10.1021/acsnano.3c12161

DNA droplets, artificial liquid-like condensates of well-engineered DNA sequences, allow the critical aspects of phase-separated biological condensates to be harnessed programmably, such as molecular sensing and phase-state regulation. In contrast, their RNA-based counterparts remain less explored despite more diverse molecular structures and functions ranging from DNA-like to protein-like features. Here, we design and demonstrate computational RNA droplets capable of two-input AND logic operations. We use a multibranched RNA nanostructure as a building block comprising multiple single-stranded RNAs. Its branches engaged in RNA-specific kissing-loop (KL) interaction enables the self-assembly into a network-like microstructure. Upon two inputs of target miRNAs, the nanostructure is programmed to break up into lower-valency structures that are interconnected in a chain-like manner. We optimize KL sequences adapted from viral sequences by numerically and experimentally studying the base-wise adjustability of the interaction strength. Only upon receiving cognate microRNAs, RNA droplets selectively show a drastic phase-state change from liquid to dispersed states due to dismantling of the network-like microstructure. This demonstration strongly suggests that the multistranded motif design offers a flexible means to bottom-up programming of condensate phase behavior. Unlike submicroscopic RNA-based logic operators, the macroscopic phase change provides a naked-eye-distinguishable readout of molecular sensing. Our computational RNA droplets can be applied to in situ programmable assembly of computational biomolecular devices and artificial cells from transcriptionally derived RNA within biological/artificial cells.