Enhancing small-molecule-mediated translational control through multivalent RNA aptamers.

The precise translational control of gene expression by small molecules through RNA-based switches holds considerable promise for both research and therapeutic developments. However, current high-performance RNA switches remain limited in adaptability, with strong responses typically constrained to a narrow set of specific ligand-aptamer pairs. To address this limitation, we introduce a robust and generalizable RNA platform based on a multivalent aptamer design, which significantly enhances ligand-responsive protein expression through alternative splicing regulation. We have demonstrated that the inherently weak aptamers, such as those for theophylline or tetracycline, can be dramatically improved through this multivalency circuit, elevating the induction levels from modest (<10-fold) to over 100-fold, an increase of more than an order of magnitude. Leveraging these improved switches, we achieve multiplex and orthogonal control over distinct protein outputs with these suboptimal aptamers. Furthermore, we implement precise manipulation of cellular phenotypes through the ligand-controlled expression of functional proteins, including the pro-apoptotic effector BAX and the adhesion protein E-cadherin. This work establishes a general and adaptable RNA platform for expanding the toolbox of small-molecule regulators of protein expression, with potential applications across synthetic biology and therapeutic applications.