The versatility of naturally-occurring RNAs that perform central cellular functions has generated intense interest in designing engineered RNAs to control gene expression. Mechanistic studies of natural RNA systems that sense and respond to changes in temperature, small molecule or metabolite concentration, and the expression of other RNA and protein species in the cell have been combined with advances in computational nucleic acid design to enable the de novo design of synthetic RNA regulators. While progress has been remarkable, challenges remain. In this work, we aim to close the gaps which exist in the design and characterization of RNA regulators to respond to cellular and environmental cues to regulate gene expression in predictable ways. Specifically, we present design strategies for synthetic RNAs to control multiple steps in gene synthesis in response to the expression of trans-acting RNAs, specialized ribosomes, or temperature changes. Moreover, we show how RNA chemical probing can be used alongside in silico predictions of RNA structure to both characterize and troubleshoot design flaws in synthetic regulatory schemes. Together, this work provides a framework for the use of experimental and computational tools to characterize natural RNA-based systems, distill their functions into a coherent and adaptable set of design rules, and apply these rules for the de novo design of regulatory RNAs to address unmet needs in cellular engineering and synthetic biology.