Cell-Penetrating Peptide-Peptide Nucleic Acid Conjugates as a Tool for Protein Functional Elucidation in the Native Bacterium

Approximately 30% or more of the total proteins annotated from sequenced bacteria genomes are annotated as hypothetical or uncharacterized proteins. However, elucidation on the function of these proteins is hindered by the lack of simple and rapid screening methods, particularly with novel or hard-to-transform bacteria. In this report, we employed cell-penetrating peptide (CPP) -peptide nucleotide acid (PNA) conjugates to elucidate the function of such uncharacterized proteins in vivo within the native bacterium. , a hard-to-transform bacterial genus, was used as a model. Two hypothetical genes showing amino acid sequence similarity to ι-carrageenases, termed A and B, were identified from the draft genome of sp. strain YYML68, and CPP-PNA probes targeting the mRNA of the acyl carrier protein gene, P, and the two ι-carrageenase candidate genes were synthesized. Upon direct incubation of CPP-PNA targeting the mRNA of the P gene, we successfully observed growth inhibition of strain YYML68 in a concentration-dependent manner. Similarly, both the function of the candidate ι-carrageenases were also inhibited using our CPP-PNA probes allowing for the confirmation and characterization of these hypothetical proteins. In summary, we believe that CPP-PNA conjugates can serve as a simple and efficient alternative approach to characterize proteins in the native bacterium.