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Published on: September 21, 2017
RNA-peptide fusions for the in vitro selection of peptides and proteins
1Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA.
Abstract:
Covalent fusions between an mRNA and the peptide or protein that it encodes can be generated by in vitro translation of synthetic mRNAs that carry puromycin, a peptidyl acceptor antibiotic, at their 3' end. The stable linkage between the informational (nucleic acid) and functional (peptide) domains of the resulting joint molecules allows a specific mRNA to be enriched from a complex mixture of mRNAs based on the properties of its encoded peptide. Fusions between a synthetic mRNA and its encoded myc epitope peptide have been enriched from a pool of random sequence mRNA-peptide fusions by immunoprecipitation. Covalent RNA-peptide fusions should provide an additional route to the in vitro selection and directed evolution of proteins.
Insights
Researchers created novel covalent RNA-peptide fusions using synthetic messenger RNAs (mRNAs) with puromycin. This technique enables the enrichment of specific mRNAs from complex mixtures, advancing in vitro protein evolution.
Area of Science:
- Biochemistry
- Molecular Biology
- Synthetic Biology
Background:
- Covalent fusion of messenger RNA (mRNA) and its encoded peptide or protein is a novel approach.
- Synthetic mRNAs can be modified to incorporate puromycin, a peptidyl acceptor antibiotic, at their 3' end.
Purpose of the Study:
- To generate and utilize covalent mRNA-peptide fusions for specific mRNA enrichment.
- To explore the potential of these fusions in in vitro selection and directed protein evolution.
Main Methods:
- In vitro translation of synthetic mRNAs carrying puromycin at the 3' end.
- Generation of stable, covalent linkages between mRNA and encoded peptides.
- Enrichment of specific mRNA-peptide fusions from complex mixtures using immunoprecipitation.
Main Results:
- Successful generation of covalent mRNA-peptide fusions.
- Demonstrated enrichment of synthetic mRNA-myc epitope peptide fusions from a pool of random sequences via immunoprecipitation.
- Established the principle of selecting mRNA based on peptide properties.
Conclusions:
- Covalent RNA-peptide fusions offer a new method for mRNA enrichment based on encoded peptide characteristics.
- This approach provides an additional avenue for in vitro selection and directed evolution of proteins.
- The technique has broad implications for protein engineering and synthetic biology.
