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

Abstract

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. Paenibacillus, a hard-to-transform bacterial genus, was used as a model. Two hypothetical genes showing amino acid sequence similarity to ι-carrageenases, termed cgiA and cgiB, were identified from the draft genome of Paenibacillus sp. strain YYML68, and CPP–PNA probes targeting the mRNA of the acyl carrier protein gene, acpP, and the two ι-carrageenase candidate genes were synthesized. Upon direct incubation of CPP–PNA targeting the mRNA of the acpP 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.

Optimization of Dendritic Polypeptide Delivery System for Antisense Antibacterial Agents Targeting ftsZ

“…However, the relatively low drug loading capacity, as well as the absence of the antisense effect in vivo and the safety study, limited the further study and application of this vehicle. − Cell-penetrating peptides attracted enormous interest as an ASOs delivery system as it could combine ASOs by both covalent and noncovalent methods. However, only neutral ASOs such as peptide nucleotide (PNA) could be delivered by the covalent method, while the noncovalent method was limited by the poor stability and lower cellular uptake efficiency in bacteria . In summary, the lack of a delivery system deeply hampered the further research and application of ASOs in bacteria.…”

Rationally Designed Self-Assembling Nanovaccines Elicit Robust Mucosal and Systemic Immunity against Rhabdovirus

“…28 Moreover, the comprehensive understanding of adaptive immunity has often benefited from studies in invertebrates and lower vertebrates. 29 Teleost fish represent the most ancient bony vertebrates with a classical adaptive immune system. 30 Similar to mammals, the adaptive immunity of teleost fish includes systemic immunity and mucosal adaptive immunity.…”

“…Understanding the regulation mechanism of vaccines can provide a basis for constructing vaccines . Moreover, the comprehensive understanding of adaptive immunity has often benefited from studies in invertebrates and lower vertebrates . Teleost fish represent the most ancient bony vertebrates with a classical adaptive immune system .…”