Pentatricopeptide repeat proteins (PPR) are a large family of modular RNA-binding proteins that bind to specific ssRNA target sequences. Their specificity is ultimately determined by repeating modular motifs, whereby each consecutive module is responsible for the specific recognition of a discrete RNA nucleotide. As such, there is significant interest in developing ‘designer’ PPRs for use in diagnostics and as a tool to detect and localize target RNA sequences in vivo; however, current models to predict PPR binding sites in vivo struggle to reconcile the effects that RNA mismatches and secondary structure have on PPR binding. To address this, we determined the structure of a designer PPR bound to its target sequence and used two- and three-colour single-molecule FRET to interrogate the mechanism of ssRNA binding to individual PPR proteins in real time. We observe that PPR binds to its target sequence with high affinity (~ 10 nM), however, the introduction of even a single mismatch to the target ssRNA sequence can significantly decrease PPR association rates and prevent stable binding. Notably, longer RNA sequences were significantly slower to bind to PPR (or could not bind at all) due to their propensity to form secondary structures, which restrict the accessibility of PPR to the target sequence. Importantly, PPR binding to its target sequence within longer ssRNA species occurs specifically (i.e., PPR does not associate with the non-target flanking sequences). These findings will be used to better predict PPR binding sites for the development of designer PPRs with improved specificity and affinity.