Programmable computational RNA droplets assembled via kissing-loop interaction

Programmable droplets, artificial liquid-like condensates of nucleic acids, are intelligent out-of-equilibrium systems that allow the critical aspects of their biological counterparts to be harnessed programmably, such as molecular sensing and phase-state change. While DNA has been central to the previous efforts, less attention has been paid to RNA. Besides DNA-like programmability, RNA plays a diverse range of protein-like roles (catalytic and regulatory ones), facilitated by its structural flexibility. Such multifaceted RNA has rich potential as a design material for creating novel programmable droplets. Here, we design and demonstrate programmable RNA droplets capable of two-input AND logic operations using a 6-way-branched RNA motif as a building block. It comprises six single-stranded RNAs. Four of them are involved in kissing-loop interaction that enables the self-assembly of network-like structures. To optimize the KL design, we numerically and experimentally show the programmability of KL interaction strength using various engineered sequences. The other two strands possess a toehold that mediates strand displacement reactions upon inputs of two target molecules, leading to a motif break-up. The designed sensing motif thus functions toward two opposite directions, composition or decomposition of the self-assembled structure, in an input-dependent manner. Experimentally, our RNA droplets show a drastic phase-state change from liquid to dispersed states upon inputs of the two miRNAs and no phase change otherwise. This observation strongly suggests that our multi-stranded sensing motif provides a flexible means to programming phase behavior. Unlike submicroscopic RNA-based logic operators, the macroscopic phase change functions as a readily detectable readout.