Synthetic molecular evolution of hybrid cell penetrating peptides

Abstract

Peptides and analogs such as peptide nucleic acids (PNA) are promising tools and therapeutics, but the cell membrane remains a barrier to intracellular targets. Conjugation to classical cell penetrating peptides (CPPs) such as pTat48–60 (tat) and pAntp43–68 (penetratin) facilitates delivery; however, efficiencies are low. Lack of explicit design principles hinders rational improvement. Here, we use synthetic molecular evolution (SME) to identify gain-of-function CPPs with dramatically improved ability to deliver cargoes to cells at low concentration. A CPP library containing 8192 tat/penetratin hybrid peptides coupled to an 18-residue PNA is screened using the HeLa pTRE-LucIVS2 splice correction reporter system. The daughter CPPs identified are one to two orders of magnitude more efficient than the parent sequences at delivery of PNA, and also deliver a dye cargo and an anionic peptide cargo. The significant increase in performance following a single iteration of SME demonstrates the power of this approach to peptide sequence optimization.

Synthetic molecular evolution of host cell-compatible, antimicrobial peptides effective against drug-resistant, biofilm-forming bacteria

“…The library was synthesized as a one-bead one-sequence library ( Fig. 1B) and was validated as described in detail previously (29)(30)(31)(32). Upon irradiation of the UV-cleavable linker, each bead released about 0.5 nmol of a single peptide sequence, sufficient to perform the multiple, parallel assays used here for selection.…”

Cytosolic delivery of inhibitory antibodies with cationic lipids

“…Endosome escape is the major bottleneck for cytosolic delivery, resulting in large false-positive rates for assays that measure total cellular uptake of cargo proteins (22,23). Accordingly, we used a stringent self-assembling splitGFP (24) reporter system in which one half of the splitGFP, the S11 peptide, is fused to pAbBD-ApP, whereas the other half, splitGFP(1-10), is expressed in reporter cells (25)(26)(27)(28). Only once IgG-(pAbBD-ApP-S11) 2 is successfully delivered into the cytosol does splitGFP complementation and turn-on fluorescence occur (Fig.…”

“…When compared to CPP-mediated delivery of small peptides or proteins, our approach enables cytosolic delivery of a much larger IgG cargo with similar or greater efficiencies at an ∼100fold lower concentration (25)(26)(27)(28). It is difficult to directly compare our technology to previous carrier-mediated approaches due to the use of different reporter systems.…”

Routing Nanomolar Protein Cargoes to Lipid Raft‐Mediated/Caveolar Endocytosis through a Ganglioside GM1‐Specific Recognition Tag

“…We set out to find a short peptide tag, a non‐toxic minimal motif that is able to specifically route its macromolecular cargo to an entry pathway that mimics the ganglioside‐mediated internalization of viruses and bacterial toxins through the major lipid raft/caveolar component GM1. By focusing on a structurally well‐defined receptor and conducting a thorough biophysical characterization of the interaction, we aimed to open the way to structure‐based design, which is rare in protein delivery approaches . Here, we show that a minimal palindromic pentapeptide sequence (WYKYW) specifically binds the carbohydrate moiety of GM1 with low nanomolar affinity.…”