Pentatricopeptide repeat proteins (PPR) are a large family of modular single-stranded RNA-binding proteins, whereby each module can be modified to bind to a specific ssRNA nucleobase. As such, there is interest in developing ‘designer’ PPRs (dPPRs) for a range of biotechnology applications, including diagnostics or in vivo localisation of ssRNA species; however, the mechanistic details regarding how PPRs search for and bind to target sequences is unclear. Studies of wild-type, and consensus PPR proteins demonstrate a conformational change on RNA binding: structures of idealised “designer” PPR (dPPR) proteins in the presence and absence of RNA show that they have a superhelical structure of 9, or 10 repeats per superhelical turn. The conformational change on RNA binding results in a contraction of the superhelical pitch from 85 Å to 43 Å, a change that is compatible with the Foerster distance of commonly used FRET fluorophores. We thus built a protein-based RNA FRET sensor by introducing two cysteine residues at appropriate spacing in the structure, and chemically labelling them fluorophores. We used two- and three-colour single-molecule FRET to interrogate the mechanism of ssRNA binding to individual dPPRs in real time. We demonstrate that dPPRs are slower to bind longer ssRNA sequences (or could not bind at all) and that this is, in part, due to their propensity to form stable secondary structures that sequester the target sequence from dPPR. Importantly, dPPR binds only to its target sequence (i.e., it does not associate with non-target ssRNA sequences) and does not ‘scan’ longer ssRNA oligonucleotides for the target sequence. The kinetic constraints imposed by random three-dimensional diffusion may explain the long-standing conundrum of why PPR proteins are abundant in organelles, but almost unknown outside them (i.e. in the cytosol and nucleus).