Using mRNA Display to Find Cyclic Peptides That Can Penetrate Cell Membranes | Research

The takeaway

mRNA display identified a cyclic peptide that penetrates both embryonic and mammalian cell membranes — outperforming its linear counterpart and all known cell-penetrating peptides tested.

First to cross both membrane types
No previously known peptide could penetrate both cytoplasmic and outer embryonic membranes — this cyclic peptide does both

What the researchers found

Researchers used mRNA display — a technique that screens trillions of peptide variants simultaneously — to discover cyclic peptides capable of penetrating biological membranes. The top hit, cyclo[Glut-MRKRHASRRE-K*], successfully penetrated both fruit fly embryos and mammalian cells (human embryonic stem cells and mouse fibroblasts). This is notable because no previously known peptide could cross both cytoplasmic membranes and the tough outer embryonic membrane. The cyclic version significantly outperformed its linear (non-cyclized) counterpart.

Why it matters

One of the biggest challenges in drug delivery is getting therapeutic molecules inside cells. Cell-penetrating peptides are promising delivery vehicles, but most have limited ability to cross different types of membranes. This study introduces a high-throughput method to discover cyclic peptides with unusually broad membrane-penetrating ability — which could be used to deliver drugs, gene therapies, or research tools into cells that were previously difficult to access.

How the study worked

The researchers constructed large libraries of cyclic peptides using mRNA display, a technique where each peptide is physically linked to its own genetic code, allowing rapid identification of hits. They screened these libraries against fruit fly embryos at different developmental stages to select peptides that could penetrate tough embryonic membranes. Top candidates were then tested against mammalian cells. Permeation was measured using confocal microscopy (visual confirmation) and flow cytometry (quantitative measurement of how much peptide entered cells).

Who was studied

In vitro study using Drosophila embryos, human embryonic stem cells, and mouse fibroblast cell lines

What this study cannot tell us

This is an in vitro proof-of-concept study. The peptides were tested in cell lines and embryos, not in living organisms. Whether they can deliver actual drug cargo — not just themselves — across membranes hasn't been demonstrated. The fruit fly embryo screen may select for properties that don't perfectly translate to all mammalian cell types. Toxicity at therapeutic doses in vivo is unknown.

How to read the evidence

This is a proof-of-concept laboratory study demonstrating a new screening method and its outputs. While the results are well-characterized with multiple validation techniques, the work is entirely in vitro with no animal or human data.

When this study was published

Published in 2020, this study introduced a method that has had several years to influence the field. mRNA display for peptide discovery has continued to advance, and this work may have inspired subsequent efforts to find therapeutic cell-penetrating peptides.

The bigger picture

mRNA display and related techniques (like phage display) are revolutionizing how researchers discover peptides with specific functions. The ability to screen billions of candidates at once means scientists can find needles in molecular haystacks. Cyclic peptides are especially interesting because their ring structure makes them more stable and often more potent than linear peptides. This study connects two hot areas of peptide science — high-throughput discovery and cell-penetrating delivery — and could accelerate the development of peptide-based drug delivery systems.

Questions still open

Can these cyclic peptides carry drug molecules or gene therapy cargo across membranes, or only themselves?
What is the mechanism by which these cyclic peptides cross membranes — do they punch through or get taken up by cells?
Would these peptides be toxic when administered systemically in animal models?

Common questions

What is mRNA display and how does it find useful peptides?

mRNA display is a technique that creates trillions of different peptides at once, with each peptide physically attached to the RNA molecule that encodes it. Scientists then expose this massive library to a target — in this case, embryos — and collect only the peptides that successfully penetrate. Because each peptide carries its own genetic barcode, researchers can quickly identify the winners.

Why are cyclic peptides better than linear ones at crossing membranes?

Cyclic (ring-shaped) peptides are more rigid and compact than their linear counterparts, which helps them resist enzymatic breakdown and maintain the shape needed to interact with membranes. This study directly showed that the same amino acid sequence in cyclic form penetrated membranes much better than in linear form — the ring structure itself confers an advantage.

Read the original research

Discovery of Membrane-Permeating Cyclic Peptides via mRNA Display.

Bioconjugate chemistry, 31(10), 2325-2338

View on PubMed ↗Publisher’s paper ↗

Citation

Bowen, John; Schloop, Allison E; Reeves, Gregory T; Menegatti, Stefano; Rao, Balaji M. (2020). Discovery of Membrane-Permeating Cyclic Peptides via mRNA Display.. Bioconjugate chemistry, 31(10), 2325-2338. https://doi.org/10.1021/acs.bioconjchem.0c00413