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- MRNA_display abstract "mRNA display is a display technique used for in vitro protein, and/or peptide evolution to create molecules that can bind to a desired target. The process results in translated peptides or proteins that are associated with their mRNA progenitor via a puromycin linkage. The complex then binds to an immobilized target in a selection step (affinity chromatography). The mRNA-protein fusions that bind well are then reverse transcribed to cDNA and their sequence amplified via a polymerase chain reaction. The result is a nucleotide sequence that encodes a peptide with high affinity for the molecule of interest.Puromycin is an analogue of the 3’ end of a tyrosyl-tRNA with a part of its structure mimics a molecule of adenosine, and the other part mimics a molecule of tyrosine. Compared to the cleavable ester bond in a tyrosyl-tRNA, puromycin has a non-hydrolysable amide bond. As a result, puromycin interferes with translation, and causes premature release of translation products. All mRNA templates used for mRNA display technology have puromycin at their 3’ end. As translation proceeds, ribosome moves along the mRNA template, and once it reaches the 3’ end of the template, the fused puromycin will enter ribosome’s A site and be incorporated into the nascent peptide. The mRNA-polypeptide fusion is then released from the ribosome (Figure 2).To synthesize an mRNA-polypeptide fusion, the fused puromycin is not the only modification to the mRNA template. Oligonucleotides and other spacers need to be recruited along with the puromycin to provide flexibility and proper length for the puromycin to enter the A site. Ideally, the linker between the 3’ end of an mRNA and the puromycin has to be flexible and long enough to allow the puromycin to enter the A site upon translation of the last codon. This enables the efficient production of high-quality, full-length mRNA-polypeptide fusion. Rihe Liu et al. optimized the 3’-puromycin oligonucleotide spacer. They reported that dA25 in combination with a Spacer 9 (Glen Research), and dAdCdCP at the 5’ terminus worked the best for the fusion reaction. They found that linkers longer than 40 nucleotides and shorter than 16 nucleotides showed greatly reduced efficiency of fusion formation. Also, when the sequence rUrUP presented adjacent to the puromycin, fusion did not form efficiently.In addition to providing flexibility and length, the poly dA portion of the linker also allows further purification of the mRNA-polypeptide fusion due to its high affinity for dT cellulose resin.The mRNA-polypeptide fusions can be selected over immobilized selection targets for several rounds with increasing stringency. After each round of selection, those library members that stay bound to the immobilized target are PCR amplified, and non-binders are washed off.".
- MRNA_display thumbnail Slide10.png?width=300.
- MRNA_display wikiPageExternalLink publications.html.
- MRNA_display wikiPageExternalLink liu_dw.htm.
- MRNA_display wikiPageExternalLink publications.
- MRNA_display wikiPageExternalLink entrez?Db=pubmed&Cmd=Search&Term=%22Hartman%20MC%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus.
- MRNA_display wikiPageID "7792469".
- MRNA_display wikiPageLength "16337".
- MRNA_display wikiPageOutDegree "27".
- MRNA_display wikiPageRevisionID "678955476".
- MRNA_display wikiPageWikiLink Adenosine.
- MRNA_display wikiPageWikiLink Affinity_chromatography.
- MRNA_display wikiPageWikiLink Bacterial_display.
- MRNA_display wikiPageWikiLink Category:Molecular_biology.
- MRNA_display wikiPageWikiLink Complementarity_determining_region.
- MRNA_display wikiPageWikiLink Complementarity_determining_regions.
- MRNA_display wikiPageWikiLink DNA_ligase.
- MRNA_display wikiPageWikiLink File:Slide10.png.
- MRNA_display wikiPageWikiLink File:Slide8.png.
- MRNA_display wikiPageWikiLink File:Slide9.png.
- MRNA_display wikiPageWikiLink MRNA.
- MRNA_display wikiPageWikiLink Messenger_RNA.
- MRNA_display wikiPageWikiLink Nucleotide.
- MRNA_display wikiPageWikiLink PCR.
- MRNA_display wikiPageWikiLink Peptide.
- MRNA_display wikiPageWikiLink Peptides.
- MRNA_display wikiPageWikiLink Phage_display.
- MRNA_display wikiPageWikiLink Polymerase_chain_reaction.
- MRNA_display wikiPageWikiLink Protein.
- MRNA_display wikiPageWikiLink Protein_engineering.
- MRNA_display wikiPageWikiLink Proteins.
- MRNA_display wikiPageWikiLink Protein–protein_interaction_screening.
- MRNA_display wikiPageWikiLink Puromycin.
- MRNA_display wikiPageWikiLink Reverse_transcriptase.
- MRNA_display wikiPageWikiLink Ribosome_display.
- MRNA_display wikiPageWikiLink Tyrosine.
- MRNA_display wikiPageWikiLink Yeast_display.
- MRNA_display wikiPageWikiLinkText "MRNA display".
- MRNA_display wikiPageWikiLinkText "mRNA display".
- MRNA_display hasPhotoCollection MRNA_display.
- MRNA_display wikiPageUsesTemplate Template:Colbegin.
- MRNA_display wikiPageUsesTemplate Template:Colend.
- MRNA_display wikiPageUsesTemplate Template:Lowercase.
- MRNA_display wikiPageUsesTemplate Template:Protein_methods.
- MRNA_display wikiPageUsesTemplate Template:Reflist.
- MRNA_display subject Category:Molecular_biology.
- MRNA_display hypernym Technique.
- MRNA_display type Software.
- MRNA_display comment "mRNA display is a display technique used for in vitro protein, and/or peptide evolution to create molecules that can bind to a desired target. The process results in translated peptides or proteins that are associated with their mRNA progenitor via a puromycin linkage. The complex then binds to an immobilized target in a selection step (affinity chromatography).".
- MRNA_display label "MRNA display".
- MRNA_display sameAs In_vitro_virus.
- MRNA_display sameAs m.026d5ss.
- MRNA_display sameAs Q6717517.
- MRNA_display sameAs Q6717517.
- MRNA_display wasDerivedFrom MRNA_display?oldid=678955476.
- MRNA_display depiction Slide10.png.
- MRNA_display isPrimaryTopicOf MRNA_display.