Novel tools for in silico design of RNA biosensors are required in order to reduce time and cost to production for the development of diagnostic advances. We developed MoiRNAiFold, a versatile and user-friendly tool for de novo synthetic RNA design. It is based on Constraint Programming and includes novel variable types, heuristics, design constraints, quality measures and restart strategies for Large Neighbourhood Search improving features for RNA regulation control of gene expression. Using this best-in-class software, we generated novel translational regulators called Plug&Play RNAs, which rely on the sequestration of the RBS, disallowing the binding of the ribosome. We designed fluorescent reporter biosensors capable of detecting E.coli 16S rRNA and S.aureus mecA gene sequences. The designs were prioritised and experimentally validated to detect the presence of <10⁴ CFU/mL in a cell-free system. These are useful tools for future projects requiring ultrasensitive bacterial or viral detection, as they are easily adaptable and fast and cost-effective to generate.