2025/06/02 by Jonathan Franke, Jan Vincent V. Arafiles, Christian Leis +1 · 2 voices · 1 citation
Biochemistry, Genetics and Molecular Biology · #RNA Interference and Gene Delivery #Virus-based gene therapy research #Viral Infectious Diseases and Gene Expression in Insects
paper · pdf · doi:10.1002/anie.202506802
openalex publication_date 2025/06/02 · openalex created_date 2025/10/10 · openalex updated_date 2026/06/26
Protein-based tools are emerging as innovative solutions to interfere with biological pathways in molecular biology and medicine. They offer advantages over traditional small molecules due to their adaptable structural diversity and their ability to engage previously inaccessible cellular targets. However, most proteins do not penetrate the lipid bilayer of mammalian cells and are therefore restricted to extracellular targets. Despite recent advances, a general method for the delivery of functional proteins into human cells remains a significant challenge. In this study, we present a bioreversible protein modification strategy of amines using short arginine-containing peptides (termed BioRAM) that enables cytosolic delivery starting from genetically non-engineered proteins. We optimized the bioconjugation strategy to achieve fast intracellular cleavage and complete recovery of the native protein. In combination with our previously established cell-penetrating peptide (CPP)-additive protocol, we show superior delivery of fluorescent protein and functional RNase A into the cytosol, achieving physiological response. Moreover, we are able to demonstrate the excellent performance of BioRAM in the presence of serum, thereby broadening the scope for intracellular applications of functional proteins.