mRNA display enables the direct evolution of new proteins or peptides that bind specific targets, such as drugs or disease receptors. mRNA display allows scientists to screen trillions of different molecules at once and "read" the genetic instructions of the selected molecule(s). mRNA display links a protein to its genetic code.
The amplification of the linked genetic products generates a library enriched in functional sequences. Next, iterative selection cycles are performed until the desired level of function is achieved, at which time the identities of candidate peptides can be determined by sequencing the genetic material.
Each translated protein or peptide is covalently attached to its own mRNA. When the selected protein binds its target, the genetic sequence that encodes the protein or peptide can be determined.
Comparison of display methods:
| mRNA Display | Ribosome Display | Phage display | Cell-surface Display |
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Example: Fused mRNA-Protein construct | 
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Graphical depiction of a mRNA display protocol (adapted from Barendt et al. 2014). Barendt et al. developed a streamlined protocol for mRNA display that reduces the time for a single selection round from ~4 – 7 days to 2 days.
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| Protocol Steps [1] In vitro transcription (3 hr) [2] LiCl-based purification of mRNA (2.5 hr) [3] Phosphorylation of DNA-puromycin linker (2.5 hr) [4] Splinted ligation of DNA-puromycin linker and mRNA (1 hr) [5] Ultrafiltration of ligated mRNA (1.5 hr) [6] In vitro translation with PURExpress system (1 hr) [7] Selection (varies) [8] Reverse transcription (RT) (1.5 hr). ~ 2 days |
mRNA display enables a wide array of protein engineering applications; however, the technical difficulty of this method has so far precluded its widespread use.
The key to mRNA display is puromycin. Puromycin is an antibiotic that mimics the end of a tRNA molecule. Puromycin is a translation inhibitor that can enter the ribosome during translation, forming a stable covalent bond with the nascent protein and creating a stable linkage between the mRNA display template and the protein it encodes, resulting in an mRNA-displayed protein.
mRNA display involved the following steps. A DNA library is converted into an mRNA library. A puromycin linker is chemically attached to the "end" (the 3' end) of each mRNA strand. Ribosomes begin translating the mRNA into a protein or peptide. When the ribosome reaches the end of the mRNA, it encounters the puromycin. The ribosome "thinks" the puromycin is the next amino acid and tries to add it to the growing protein chain. Because puromycin is already attached to the mRNA, the protein becomes permanently linked to its own genetic code, forming a fused mRNA-protein construct.
A brief step by step description of a general protocol:
[1] Create mRNA library: Synthesize a large and diverse library of mRNA sequences with approximately 10¹² to 10¹⁴ variants. This library is larger than cell-based methods.
[2] Link to puromycin: Each mRNA has a puromycin attached to its 3′-end that mimics a tRNA and can enter the ribosome’s A site.
[3] In vitro translation: During translation, when the ribosome reaches the end of the mRNA puromycin enters the ribosome forming a covalent bond with the nascent peptide resulting in a protein–mRNA fusion product.
[4] Selection or panning: The fused molecules are exposed to a target such as a protein, receptor, small molecule, or others. During this selection step only peptides or proteins that bind are retained.
[5] Recovery and amplification: The bound mRNA is reverse-transcribed into cDNA, PCR-amplified, and used for additional selection rounds or sequencing.
Why Use mRNA Display?
mRNA Display has several advantages over other display techniques as a peptide and protein design tool:
(1) higher diversity,
(2) lack of context dependence,
(3) strictly monomeric libraries,
(4) the ability to use an expanded genetic code,
(5) ease of use with high throughput sequence analysis.
With a diversity of ~10,000 times greater than the primary immune repertoire, it allows the discovery of better ligands than with other methods such as phage display.
Common Applications for mRNA display:
Alternative scaffolds: Selection and design of disulfide-free antibody-like proteins that can be used to create general protein targeting tools.
Antibody development: Identification and selection of specific single-chain variable fragments (scFvs).
Development of macrocyclic or constrained peptides: Identify peptides for the design of therapeutic drugs.
Discovery of high-affinity peptide binders: Identify "macrocyclic" peptides that act like mini-antibodies to block protein-protein interactions.
Drug discovery and diagnostics: Identify high-affinity binders for use in biosensors or imaging.
Enzyme evolution: Create new enzymes that can catalyze specific chemical reactions.
Protein–protein interaction studies: Create new peptides or proteins useful for the study of protein-protein interactions.
mRNA Display versus Phage Display
mRNA display is often compared to Phage Display, but it has several distinct advantages (see table below).
Comparison of display methods
| Method | mRNA Display | Phage Display |
| Library Size | Huge (to variants) | Smaller (variants) |
| System | Entirely in vitro (test tube) | In vivo (requires living bacteria) |
| Chemical Diversity | Can use unnatural amino acids | Limited to the 20 natural amino acids |
| Speed | Faster; no need to wait for cells to grow | Slower; requires bacterial infection cycles |
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| Method | Library size | Linkage | In vivo? |
| mRNA display | 10¹²–10¹⁴ | Covalent | No |
| Phage display | 10⁹–10¹⁰ | Non-covalent | Yes |
| Ribosome display | 10¹²–10¹³ | Non-covalent | No |
A brief history of mRNA displays
1997: Development of methods for in vitro selection and evolution of functional proteins by using ribosome display and RNA-peptide fusions for in vitro selection of peptides and proteins. (Roberts et al.; Hanes & Pluecktun).
2000: Isolation of peptide aptamers that inhibit intracellular processes. (Blum et al.).
2001: Protein selection using mRNA display (Keefe). mRNA display to select high-affinity protein-binding peptides (Wilson et al.).
2004: In-vitro protein evolution by ribosome display and mRNA display (Lipovsek & Plückthun).
2006: Review of display technologies: application for the discovery of drug and gene delivery agents. Cell display, phage display, ribosome display, mRNA display, and DNA display. In vitro evolution of single-chain antibodies (Sergeeva et al.; Fukuda et al.).
2009: Display of fibronectin-based intrabodies that detect and inhibit severe acute respiratory syndrome coronavirus nucleocapsid protein (Liao et al.)
2010: A comprehensive resource of interacting protein regions for refining human transcription factor networks (Miyamoto-Sato et al.).
2011: Ribosome display: a technology for selecting and evolving proteins from large libraries. (Dreier & Plückthun). In vitro selection of peptide inhibitor of human IL-6 using mRNA display (Kobayashi et al.). mRNA display for the selection and evolution of enzymes from in vitro-translated protein libraries (Seelig).
2012: mRNA display for the selection of synthetic peptides and protein libraries (Cotton et al.).
2013: A streamlined protocol for mRNA display (Barendt et al.).
2019: In Vitro Selection of Peptides and Protein. Reviewing advantages of mRNA Display. (Newton et al.)
2020: Study of the effect of RNA sequence and stereochemistry on G-quadruplex-RHAU53 peptide binding. The RHAU53 peptide preferentially binds to 5′-G-quartet over 3′-G-quartet. RHAU53 tends to interact with D-rG4 rather than L-rG4.
Rhau53 peptide: SMHPGHLKGREIGMWYAKKQGQKNKEAERQERAVVHMDERREEQIVQLLNSVQAK (Mou & Kwok. PDB ID codes 2N16 and 2N21).
2021: Directing evolution of novel ligands by mRNA display (Kamalinia et al.)
2023: Tyrosine-catalyzed peptide macrocyclization for mRNA display (Fleming et al. Identification of Covalent Cyclic Peptide Inhibitors using mRNA Display. (Iskandar et al.). Peptides Selected by G4-mRNA Display-Seq Enable RNA G-Quadruplex Recognition and Gene Regulation (Mou & Kwok).
2024: mRNA display for protein biosensor construction (Ciu et al.). Screening libraries of >1012 cyclic peptides against a protein target, enabling the rapid discovery of high affinity ligands (Hurd et al.).
2025: Discovery and characterization of a high-affinity G-quadruplex binding peptide via mRNA display. Identification of a G-quadruplex (G4)-specific peptide, LP7, using mRNA-display screening. It was found that basic and aromatic amino acid residues in LP7 contribute to G4 binding. Dimerization of LP7 resulted in a 70-fold increase in binding affinity to hTERC G4 RNA (rG4), achieving a Kd of 7 nM. The LP7 peptide inhibits reverse transcription in a G4-dependent manner (Ida et al.). RNA G-quadruplex structure targeting and imaging: recent advances and future directions (Wu et al.).
References
Barendt PA, Ng DT, McQuade CN, Sarkar CA. Streamlined protocol for mRNA display. ACS Comb Sci. 2013 Feb 11;15(2):77-81. [PMC]
Blum JH, Dove SL, Hochschild A, Mekalanos JJ. Isolation of peptide aptamers that inhibit intracellular processes. Proc Natl Acad Sci U S A. 2000 Feb 29;97(5):2241-6. Erratum in: Proc Natl Acad Sci U S A 2001 Jun 19;98(13):7647. PMID: 10688899; PMCID: PMC15785. [PMC]
Cotten SW, Zou J, Wang R, Huang BC, Liu R. mRNA display-based selections using synthetic peptide and natural protein libraries. Methods Mol Biol. 2012;805:287-97. [PMC]
Cui Z, Ayva CE, Liew YJ, Guo Z, Mutschler R, Dreier B, Fiorito MM, Walden P, Howard CB, Ely F, Plückthun A, Pretorius C, Ungerer JP, Buckle AM, Alexandrov K. mRNA Display Pipeline for Protein Biosensor Construction. ACS Sens. 2024 Jun 28;9(6):2846-2857. [PMC]
Fleming MC, Bowler MM, Park R, Popov KI, Bowers AA. Tyrosinase-Catalyzed Peptide Macrocyclization for mRNA Display. J Am Chem Soc. 2023 May 17;145(19):10445-10450. [PMC]
Hurd CA, Bush JT, Powell AJ, Walport LJ. mRNA Display in Cell Lysates Enables Identification of Cyclic Peptides Targeting the BRD3 Extraterminal Domain. Angew. Chem. Int. Ed. Engl. 2024 Sep 16;63(38):e202406414. [Wiley] Screening libraries of >1012 cyclic peptides against a protein target, enabling the rapid discovery of high affinity ligands.
Ida N.K., Kawaguchi Y., Futaki S., Imanishi M., Discovery and characterization of a high-affinity G-quadruplex binding peptide via mRNA display. Bioorganic & Medicinal Chemistry, 2025, 118543. [SSRN]
Iskandar SE, Chiou LF, Leisner TM, Shell DJ, Norris-Drouin JL, Vaziri C, Pearce KH, Bowers AA. Identification of Covalent Cyclic Peptide Inhibitors in mRNA Display. J Am Chem Soc. 2023 Jul 19;145(28):15065-15070. [PMC]
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Liao HI, Olson CA, Hwang S, Deng H, Wong E, Baric RS, Roberts RW, Sun R. mRNA display design of fibronectin-based intrabodies that detect and inhibit severe acute respiratory syndrome coronavirus nucleocapsid protein. J. Biol. Chem. 2009;284:17512–17520. [PMC] [PubMed]
Lipovsek D, Plückthun A. In-vitro protein evolution by ribosome display and mRNA display. J Immunol Methods. 2004 Jul;290(1-2):51-67. Erratum in: J Immunol Methods. 2004 Nov;294(1-2):213. PMID: 15261571. [PubMed]
Mou X, Kwok CK. Effect of RNA sequence context and stereochemistry on G-quadruplex-RHAU53 interaction. Biochem Biophys Res Commun. 2020 Dec 17;533(4):1135-1141. [PubMed]
Mou, X., Kwok, C. K.; Peptides Selected by G4-mRNA Display-Seq Enable RNA G-Quadruplex Recognition and Gene Regulation. J. Am. Chem. Soc., 2023,145/34, 18693 – 18697. [sciencedirect]
mRNA_display
Newton MS, Cabezas-Perusse Y, Tong CL, Seelig B. In Vitro Selection of Peptides and Proteins-Advantages of mRNA Display. ACS Synth Biol. 2020 Feb 21;9(2):181-190. [PMC]
Roberts RW, Szostak J. RNA-peptide fusions for the in vitro selection of peptides and proteins. Proc. Natl. Acad. Sci. USA. 1997;94:12297–12302. [PMC] [PubMed]
Roberts Lab USC: mRNA display
Roberts RW, Szostak J. RNA-peptide fusions for the in vitro selection of peptides and proteins. Proc. Natl. Acad. Sci. USA. 1997;94:12297–12302. [PMC] [PubMed]
Sergeeva A, Kolonin MG, Molldrem JJ, Pasqualini R, Arap W. Display technologies: application for the discovery of drug and gene delivery agents. Adv Drug Deliv Rev. 2006 Dec 30;58(15):1622-54. [PMC]
Wilson DS, Keefe AD, Szostak JW. The use of mRNA display to select high-affinity protein-binding peptides. Proc Natl Acad Sci U S A. 2001 Mar 27;98(7):3750-5. [PMC]
Wu TY, Lau HL, Santoso RJ, Kwok CK. RNA G-quadruplex structure targeting and imaging: recent advances and future directions. RNA. 2025 Jul 16;31(8):1053-1080. [PMC]
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