Rational Optimization of a Modular Cell-Penetrating Peptide for Efficient Nuclear Delivery of Genetic Cargo

Abstract

Background: Getting genetic material safely and efficiently into the nucleus of a cell is a monumental hurdle in gene therapy. While viruses are efficient carriers, their potential risks have pushed us to engineer smarter, non-viral alternatives. Our inspiration came from cell-penetrating peptides (CPPs), but we know that a single-function peptide is not enough; it needs to be a multi-tool capable of navigating every step of the complex delivery. Objectives: This study aimed to rationally design and optimize a modular CPP that integrates distinct functional domains to coordinate all critical stages of gene delivery: DNA binding, cellular uptake, endosomal escape, and nuclear import. Methods: In the present study, we designed a library of 30 chimeric peptides by combining functional motifs including an H1 histone-derived DNA-binding domain, the TAT membrane-translocating sequence, endosomal escape motifs such as gp41FP and H5WYG, and the SV40 nuclear localization signal (NLS) connected by flexible linkers. Candidates were screened computationally for key properties. The lead peptide was expressed in Escherichia coli, purified, and evaluated for DNA-binding activity via gel retardation assays. Results: Computational screening identified seven top-ranked candidates, among which Peptide_24 exhibited favorable predicted stability, solubility, and pH-responsive charge characteristics. Successful recombinant expression and DNA-binding ability of Peptide_24 were confirmed experimentally for further investigations. Conclusions: This study demonstrates a rational, modular integration rather than de novo motif discovery to create a unified, multi-domain CPP architecture. Our lead candidate, Peptide_24, embodies this synergistic design, mimicking a coordinated, viral-like delivery pathway. Although computational analyses and preliminary experimental results support the potential of Peptide_24, further biological evaluation is required to assess its delivery performance in cellular systems.