Influences of chain length and conformation of guanidinylated linear synthetic polypeptides on nuclear delivery of siRNA with potential application in transcriptional gene silencing

Transcriptional gene silencing (TGS) mediated by siRNA holds promise for long-term silencing efficacy, determined by effective nuclear delivery of siRNA. However, non-viral vectors for this purpose are limited. In this work, we synthesized guanidinylated linear synthetic polypeptides (GLSPs) to explore how chain length and conformation impact siRNA delivery, especially nuclear entry. Results show that helical conformations, particularly right-handed ones, enhance siRNA loading and silencing efficiency compared to unordered structures. Increasing chain length also improves these aspects. The endocytic pathways of carrier/siRNA nanocomplexes (NCs) are mainly determined by conformation, regardless of length. Notably, some NCs derived from right-handed helices can enter cells via direct membrane penetration, like bioactivity of cell penetrating peptides (CPPs). When the peptide chain of GLSPs is long enough, all vectors can rapidly deliver siRNA to the nucleus, similar to bioactivity of nuclear localization signal peptides (NLSPs). Interestingly, helicity of the vectors aids endosomal escape of NCs. Moreover, delivering siRNA to the nucleus via GLSPs induces TGS associated with DNA promoter methylation or histone deacetylation. This study clarifies the structure-activity relationship of GLSPs in siRNA delivery, providing new insights for designing non-viral carriers for TGS.