The PentatricoPeptide Repeat (PPR) proteins are a large group of RNA binding eukaryotic proteins particularly well represented in plants. Known to localize to both plastids and mitochondria where they serve roles in RNA stabilization, editing, maturation and post-transcriptional modifications, these proteins understandably represent an attractive potential tool for manipulation of eukaryotic organelles. Members of the alpha-solenoid superfamily, PPRs are composed of a degenerate 35 amino acid helix turn helix repeating domain that has been determined to drive the specificity of RNA binding in a semi-predictable manner. Decoding of the PPR proteins involves the 5th and last (35th) residues of each alpha helical repeat that are known to have the greatest influence on mode of binding to cognate RNA nucleotides, as determined through analysis of crystallographic PPR/RNA complexes. Recent work however has highlighted the non-obvious importance of other residues flanking these key positions. This work presents a mutagenic analysis of a 10-motif synthetic PPR that varies at positions 2, 5, and 35 in an attempt to elucidate the role of the second residue in effecting RNA binding. Through the use of microscale thermophoresis (MST) and isothermal titration calorimetry (ITC) to monitor binding of mutants to a designated RNA oligomer and attempt to provide greater accuracy in our ability to both predict RNA binding partners of known PPRs as well as design novel PPRs to target RNA sequences of interest.