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Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System
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RNA codon expansion via programmable pseudouridine editing and decoding.

Jiangle Liu1,2,3, Xueqing Yan1, Hao Wu1,3

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|June 25, 2025
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Summary
This summary is machine-generated.

Researchers developed a novel RNA codon-expansion (RCE) strategy using pseudouridine (Ψ) codons for precise non-canonical amino acid (ncAA) incorporation in mammalian cells. This method enhances specificity and offers a new route for genetic alphabet expansion.

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Identifying Amino Acid Overproducers Using Rare-Codon-Rich Markers
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Area of Science:

  • Synthetic Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Non-canonical amino acids (ncAAs) allow for tailored protein functions through custom chemistry.
  • Genetic code expansion (GCE) uses stop codon reassignment for ncAA incorporation but lacks complete orthogonality.
  • A need exists for more specific and orthogonal methods for ncAA encoding in cellular systems.

Purpose of the Study:

  • To develop a novel RNA codon-expansion (RCE) strategy for site-specific ncAA incorporation.
  • To create bioorthogonal 'blank' codons using pseudouridine (Ψ) for enhanced translation specificity.
  • To establish a method for expanding the genetic alphabet in eukaryotic cells.

Main Methods:

  • Developed an RCE strategy involving a programmable guide RNA, engineered tRNA, and aminoacyl-tRNA synthetase.
  • Introduced and decoded pseudouridine (Ψ) codons (ΨGA, ΨAA, ΨAG) on specific mRNA transcripts.
  • Tested the orthogonality and specificity of the RCE systems in mammalian cells.

Main Results:

  • The RCE(ΨGA) system demonstrated higher translatome-wide and proteomic specificity than GCE.
  • Established RCE(ΨAA) and RCE(ΨAG) systems, showing mutual orthogonality among all three Ψ codon pairs.
  • Demonstrated compatible cooperation between RCE and GCE for dual ncAA encoding.

Conclusions:

  • The RCE strategy effectively uses Ψ as a post-transcriptional element for encoding specific RNA codons.
  • This method provides a new, highly specific route for site-specific ncAA incorporation in eukaryotic cells.
  • RCE expands the possibilities for genetic alphabet expansion and custom protein engineering.