The development of orthogonal fluorogenic RNA aptamer pairs

Fluorogenic RNA aptamers are important fluorescent tools that enable the direct investigation of RNA biology using optical and RNA-protein pull down methods. RNA Mango is a two-component system comprising an RNA aptamer with nanomolar affinity for its fluorogenic green ligand (TO1-B). Upon binding, it subsequently becomes highly fluorescent allowing for high contrast detection. I have characterized and reselected the red Peach:TO3-B fluorogenic system and demonstrated its utility for in vitro two-colour quantification when combined with the Mango system. Further, the Unrau and Britton laboratories have engineered improved binding orthogonality via chemical redesign of TO1-B and reselection into the Mango II and III aptamers. Notably, this has resulted in a Mango II- derived fluorogenic aptamer with very high picomolar binding affinity. Together, these new Peach and Mango aptamers display robust binding and fluorescent orthogonality that promises to allow for a broad range of in vitro and in vivo methods.