Spontaneous membrane translocating peptides: characterization of common motifs and design of novel screens for delivery of bioactive cargoes
Cell penetrating peptides (CPPs) are a unique family of peptides capable of crossing the selectively-permeable lipid bilayer membrane of cells. The mechanism of entry remains an important characterization for CPPs if they are to be used for therapeutic and biotechnological tools. Two often non-exclusive processes are involved with CPPs crossing the lipid bilayer; energy-dependent or energy-independent mechanisms of entry. CPPs that rely heavily on energy-dependent mechanisms of entry are unfavorable for the advancement of CPPs as therapeutic and biotechnological applications because of the problems faced with endosomal sequestration and lysosomal degradation of a CPP-cargo conjugate. Thus, developing CPPs reliant on energy-independent mechanisms of entry increases the usefulness of CPPs to deliver cargoes directly across the lipid bilayer and into the cytosol of cells. Recently, our lab developed a high-throughput orthogonal screen capable of identifying spontaneous membrane translocating peptides (SMTPs) from a 10,000-peptide member library. The sequences identified in the screen, which were observed to cross lipid bilayers of synthetic vesicles using energy-independent mechanisms, shared a common LRLLR sequence motif. The readily occurring LRLLR motif prompted further investigation if the common sequence motif was necessary and sufficient for spontaneous membrane translocation. In addition, the LRLLR motif was used to rationally design arginine spacing variants of the motif to test the effects that arginine positioning had on spontaneous translocation. Elucidation of the mechanisms involved during spontaneous translocation for the motif and arginine spacing variants were also conducted. The results gained from the guided rational design of motif variants and the mechanisms responsible for spontaneous translocation will inform designs of future peptide libraries ,which will undergo iterative screenings to expand the repertoire of SMTPs.