AGENTEX Platform Expands Protein Engineering to 34 Amino Acids Without Genome Recoding
核心洞察
Harvard and Wyss Institute researchers unveiled AGENTEX (搜索), a cell-free platform that builds synthetic proteins using up to 34 custom amino acids instead of nature's 20.
The tool works in test-tube lysate solutions without living cells or genome editing, making protein production faster, safer and scalable, per the Nature study.
A surprise finding overturned decades of tRNA dogma: tRNAs lacking the standard CCA tail can still receive amino acids, enabling orthogonal translation systems.
Researchers at Harvard Medical School and the Wyss Institute for Biologically Inspired Engineering (搜索) have developed a cell-free platform that allows scientists to design and produce synthetic proteins using up to 34 different amino acids — far beyond the 20 that occur naturally — without recoding any organism's genome. The tool, called AGENTEX (搜索), was reported Aug. 26 in Nature and arose from an unexpected discovery that overturns decades of understanding of how transfer RNAs (tRNAs) help translate the genetic code into proteins.
"AGENTEX (搜索) enables researchers to generate entirely new genetic codes on demand in test tubes and use them at scale to build proteins far beyond what nature has evolved," said first author Felix Radford, Ph.D., HMS research fellow in genetics in the Church Lab, who spearheaded the study at HMS and the Wyss Institute. "This is more rapid and safe than existing methods, as it does not rely on handling living cells or altering their genomes."
Two decades of codon reprogramming
For more than 20 years, scientists have sought to instruct biological systems such as E. coli to produce proteins they do not naturally make. The prevailing approach — reprogramming an organism's DNA — proved difficult, time-consuming and produced limited results. After nine years of effort, the lab of geneticist George Church, Ph.D., demonstrated in 2013 that a codon could be freed up in E. coli so the bacteria could incorporate a new amino acid. It took another decade to free up a second codon, allowing cells to make proteins containing up to 22 different amino acids.
Progress worldwide remained slow, hampered by the fact that codon reprogramming interferes with normal cell functions and that engineered organisms must be kept safely walled off from natural life. AGENTEX (搜索) sidesteps these constraints by making 34 codons customizable and operating in a standard lab concoction containing cell components but no actual cells.
"The breakthrough turns protein engineering into something closer to a molecular design and discovery platform," Radford said. "Thousands of unique molecules can be built, tested, and evolved in parallel, without the years of genome rewriting in living cells that was previously required to add each new amino acid."
A tRNA tail that defies dogma
Underpinning the work was a finding that challenges long-held assumptions about tRNAs, the molecules that add amino acids one by one into a chain to build a protein. Every tRNA carries the genetic sequence CCA on its tail end, and dogma has held that any other sequence there flags the tRNA as defective: enzymes would not load it with an amino acid, and ribosomes would not admit it.
Contrary to previous evidence, Radford and colleagues found that the first part of that assumption is false — enzymes do allow some tRNAs with alternative tail-end sequences to receive amino acids. A tool built as part of AGENTEX (搜索), dubbed tSCAN (搜索), identified nonstandard sequences that work best, including CGA.
"We've shown that we can alter one of the most fundamental portions of one of the most fundamental systems found in nature, the protein-synthesis system that has existed pretty much unchanged for billions of years across all organisms, and it's functional," Radford said. "The CCA end is much more flexible than people assumed."
The discovery matters because non-CCA tRNAs are excluded from natural ribosomes. Scientists can therefore engineer ribosomes that work only with tRNAs bearing a specific alternative sequence such as CGA, and assign those tRNAs to carry any amino acid — natural or new — creating a parallel, side-by-side protein synthesis system that does not interfere with natural protein-making machinery.
How the workflow operates
AGENTEX (搜索), short for automated genetic tRNA expansion, provides an end-to-end workflow. It includes software written by Church's team, available for free, that runs on an open-source robot sold by the company Opentrons (搜索). Researchers can use the platform to design and produce tRNAs with different non-CCA end sequences and any of 34 amino acids, then batch-test them in cell-component soups known as lysate solutions to report which end-sequence variations most successfully attach amino acids.
In the next stage, AGENTEX (搜索) takes the most successful tRNAs, designs and produces matching ribosomes, and adds everything to more lysate solutions, where the components churn out brand-new proteins. For applications beyond protein synthesis, researchers can run quick tRNA screens before moving into more complex models. Radford noted it is not yet clear what else may be needed for the system to work optimally in cells or organisms. The team envisions integrating artificial intelligence into AGENTEX to further optimize protein design.
Therapeutic and diagnostic promise
Among many potential applications, the results hold promise for developing new therapeutics to combat disease. The authors also noted that tSCAN (搜索) offers a new way to study tRNAs in general, including mutations known to cause diseases such as diabetes (搜索) and hearing loss (搜索), which could lead to better understanding and improvement of human health.
"Trying to understand life at the molecular level and using that knowledge to develop new technologies like AGENTEX (搜索) is really amazing because you can make a positive impact on the world," Radford said. "You can use the power of evolution to transform the development of therapeutics, materials, food, pretty much anything."
Church, the Robert Winthrop Professor of Genetics in the Blavatnik Institute at HMS and Founding Core Faculty member and lead of Synthetic Biology at the Wyss Institute, is senior author of the study. Additional authors are Nayan Sapers, Hana M. Burgess, Lucy Ort and Bogdan Budnik. The work was funded by the National Science Foundation (grant 2123243), the Department of Energy (搜索) (grant DE-FG02-02ER63445) and an HMS Dean's Innovation Award for the Use of Artificial Intelligence in Education, Research, and Administration.