In a groundbreaking development that promises to revolutionize the field of protein engineering, researchers at Peking University have unveiled a pioneering strategy that dramatically expands the genetic code in mammalian cells. This novel approach circumvents a long-standing hurdle faced by scientists attempting to incorporate noncanonical amino acids (ncAAs) into proteins — the challenge of translation termination interference caused by stop codon reassignment. By harnessing post-transcriptionally modified RNA codons, chemically distinct from the canonical set of 64 standard triplet codons, this innovative methodology establishes an unprecedented level of precision in genetic code manipulation, enabling the incorporation of diverse ncAAs in a manner that does not disrupt native cellular processes.
Traditional genetic code expansion (GCE) techniques have largely depended on reassigning stop codons — sequences that normally signal the termination of protein synthesis — as blank codons to incorporate ncAAs. While this strategy has yielded important advances, it inherently suffers from limited orthogonality, meaning that reassigned stop codons can still be recognized by the natural translation termination machinery. This overlap jeopardizes precise ncAA insertion and restricts the complexity of engineered protein modifications achievable within a living mammalian system. The Peking University team’s breakthrough lies in the creation of what they term an RNA codon expansion (RCE) platform, which leverages the installation of pseudouridine-modified RNA codons (ΨCodons) on targeted mRNA transcripts as new, bioorthogonal codons.
Central to this elegant system is the programmable installation of pseudouridine — a naturally occurring RNA nucleoside known for its structural and functional versatility — into messenger RNA at specified sites, thus converting standard codons into ΨCodons that are distinctly recognized during translation. These modified codons, specifically ΨGA, ΨAA, and ΨAG, do not match any of the conventional genetic codons, effectively creating a separate coding language that can be exploited without cross-reactivity with native translational machinery. This advance circumvents the termination interference issue by separating ncAA incorporation from conventional stop codon decoding, enabling the mammalian translation system to read these new codons as encoding ncAAs with high fidelity.
The sophisticated RCE platform comprises three pivotal components working in harmony. First, a programmable guide RNA directs the enzymatic installation of pseudouridine at desired codon positions on target mRNA transcripts. Second, distinct, engineered decoder tRNAs are tailored for each ΨCodon variant — each tRNA selectively pairs with its corresponding ΨCodon, ensuring exclusive recognition and minimizing unintended decoding of native codons. Third, custom aminoacyl-tRNA synthetases charge these decoder tRNAs with selected ncAAs, coupling the genetic code expansion machinery with chemical specificity. Importantly, these synthetic tRNA-aminoacylation systems maintain strict orthogonality relative to endogenous tRNAs and synthetases, a critical feature that prevents perturbations in the host cell’s native protein synthesis landscape.
Detailed ribosome profiling studies substantiate the system’s high specificity, revealing that incorporation of ncAAs by the RCE platform occurs predominantly at ΨCodon sites without compromising the decoding fidelity of the canonical stop codon UGA. This is particularly notable given that UGA accounts for approximately 52% of all stop codons in the human genome, underscoring the method’s ability to selectively engineer the translational code without compromising termination efficiency. The combination of biochemical assays and proteomic analyses further demonstrates that each decoder tRNA-ΨCodon pair operates orthogonally not only against natural codons but also in relation to one another, enabling simultaneous and independent incorporation of multiple, chemically diverse ncAAs within single proteins in mammalian cells.
Beyond expanding the repertoire of codons available for genetic encoding, the RCE platform integrates with traditional GCE methodologies. This compatibility allows for dual ncAA incorporation strategies — a previously elusive capability — permitting a higher order of complexity in engineered protein architectures. Such a multiplexed system paves the way for intricate site-specific modifications with novel side chains that could enable tailored control over protein folding, function, and interactions, essentially creating a new dimension for protein design in eukaryotic biological contexts.
This work carries profound implications for the broader fields of synthetic biology and molecular therapeutics. By vastly improving the precision and orthogonality of ncAA incorporation in mammalian cells, it opens avenues to map protein functions with unprecedented resolution, engineer enzymes with novel catalytic capabilities, and design protein-based therapeutics with enhanced stability or targeted bioactivities. Moreover, the use of pseudouridine as a post-transcriptional “letter” introduces a versatile biochemical handle that may be further exploited for dynamic and reversible modulation of genetic code expression, adding an additional layer of regulatory control.
Technically, this strategy represents a conceptual departure from the existing paradigm of genetic code manipulation. Rather than altering the canonical DNA template or relying solely on stop codon reassignment, the approach leverages RNA post-transcriptional modifications to engineer the transcriptome itself. This opens exciting possibilities for temporal and spatial control over ncAA incorporation, provided that guide RNA programming can be finely tuned to target mRNAs based on cellular conditions, subcellular localization, or developmental stages.
Furthermore, the demonstrated mutual orthogonality among the three ΨCodon-decoder tRNA systems is a foundational advance that supports the future development of expanded chemical genetic libraries. Scientists can now envisage simultaneously introducing multiple distinct chemical functionalities into proteins within live mammalian cells, vastly expanding the toolkit for probing complex biological processes such as signal transduction, protein-protein interactions, and cellular responses to environmental stimuli.
This achievement from Peking University, published in Nature, represents not only a milestone in basic molecular biology but also a platform technology poised to accelerate discoveries and innovations across biomedical research. The study’s authors — led by Chen Peng and Yi Chengqi — emphasize the potential of RCE to facilitate advanced functional investigations of proteins adapted to natural eukaryotic contexts, a vitality that transcends limitations imposed by conventional genetic code constraints.
Looking ahead, further refinement of the RCE platform could incorporate natural or synthetic RNA modification enzymes, expanding the diversity of RNA codons beyond pseudouridine modifications to include other epitranscriptomic marks. Coupled with advances in RNA-guided editing and synthetic biology, this could create a versatile suite of RNA-based tools to program translation with extraordinary precision. The technological implications extend towards design of novel gene therapies, cell-based biosensors, and programmable biomanufacturing systems capable of producing proteins with tailored properties on demand.
The successful integration of this post-transcriptional modification-based codon expansion within mammalian cells represents a tour de force in overcoming classical challenges faced by genetic code engineering. By decoupling ncAA incorporation from native translation termination and establishing mutual orthogonality among engineered tRNA systems, this work rewrites the rules of translational engineering. It offers a robust and flexible strategy to systematically interrogate and manipulate protein function, expanding the canvas upon which molecular biologists and bioengineers can paint new solutions to biomedical and biotechnological challenges.
In summary, the RNA codon expansion platform pioneered by Peking University scientists introduces a paradigm shift in genetic code engineering for mammalian biology. Employing programmable RNA pseudouridine modifications in combination with bespoke decoder tRNAs and aminoacyl-tRNA synthetases, the platform achieves precise and highly orthogonal incorporation of noncanonical amino acids. This breakthrough holds transformative potential for expanding the functional diversity of proteins, enabling new frontiers in synthetic biology, therapeutic protein design, and fundamental studies of complex cellular systems. As researchers continue to explore and refine this approach, it promises to become an indispensable tool for next-generation bioengineering.
Subject of Research: Genetic code expansion via RNA pseudouridine modification for ncAA incorporation in mammalian cells
Article Title: RNA codon expansion via programmable pseudouridine editing and decoding
News Publication Date: June 27, 2025
References: Nature, June 25, 2025
Image Credits: Peking University
