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February 15, 2018 (Vol. 38, No. 4)

Life’s Code Accepts New Characters

Tweaking Life’s Information-Processing Platform Could Simplify the Development of Biotech and Healthcare Apps

Schematic depicting the incorporation of a non-canonical amino acid (ncAA) into a protein via ribosomal decoding of an unnatural codon in a semi-synthetic organism. [Image created by Jodie Chin, Ph.D., [email protected]]

  • Although the DNA “alphabet” consists of four letters that can be arranged in countless sequences, providing programs for all living organisms, the code of life is just too constraining—if you’re a synthetic biologist, that is. Synthetic biologists refuse to accept the limitations that are built into life’s system for information storage and retrieval.

    Instead, they intend to create their own programming platforms, which will, in turn, facilitate the development of life-enhancing applications—improved cell lines for protein production, genetic firewalls against infectious agents, anticancer therapeutics, and more.

    Synthetic biologists have been creating modified versions of life’s most basic building blocks and introducing entirely new letters to DNA’s alphabet. To give themselves even more elbow room, synthetic biologists are also reworking the genetic code that relates messenger RNA nucleotide sequences to amino acid sequences. Finally, at yet another layer of life’s chemistry, synthetic biologists are getting proteins to incorporate alternative versions of natural amino acids, as well as “nonnatural” amino acids.

    Such efforts offer profound insight into how the code of life works and what changes it can and can’t handle. Besides advancing scientific understanding, such efforts are giving developers the ability to tweak life’s code on command.

    According to Abhishek Chatterjee, Ph.D., a professor of chemistry at Boston College, this kind of code modification has a lot of potential. He offered this observation as a presenter at Biology by Design, a recent Gordon Research Conference focused on the redesign of multiscale systems at the gene, network, and whole-genome levels. “It’s an exciting time,” commented another presenter, Peter Carr, Ph.D., a senior scientist at MIT’s Synthetic Biology Center.

  • Adding X and Y

    Click Image To Enlarge +
    Floyd E. Romesberg, Ph.D., (right) professor in the department of chemistry at the Scripps Research Institute, and Yorke Zhang graduate student working in Dr. Romesberg’s lab. [Madeline McCurry-Schmidt, [email protected]]

    The alphabet of life has four letters: A for adenine, C for cytosine, G for guanine, and T for thymine. They are key to the code for 20 amino acids, which link together to form the proteins essential for life. It’s been that way for millions of years, maybe even longer.

    Recently, researchers added two nonnatural letters, dubbed X and Y. When researchers introduced X and Y to the genetic alphabet of Escherichia coli, they showed it was possible for the nonnatural bases to code for a nonnatural amino acid that was then incorporated into a protein.

    The feat demonstrated that information that natural organisms lack, and for which they lack coding capacity, can be accepted and used effectively inside a cell, said Yorke Zhang, a graduate student working in the laboratory of Floyd E. Romesberg, Ph.D., a professor of chemistry at The Scripps Research Institute in La Jolla, CA. In experiments carried out at the Romesberg laboratory, Zhang and colleagues created new codons—combinations of three genetic letters that typically correspond to a particular amino acid or a signal that stops protein synthesis.

  • Click Image To Enlarge +
    Semi-synthetic Escherichia coli producing green fluorescent protein via decoding of an unnatural codon. [Adapted from an image taken by William B. Kiosses, Ph.D., [email protected]]

    Organisms typically work with 64 codons. But as Zhang noted, natural organisms have never encountered codons that contain unnatural bases. “Thus,” he asserted, “these codons are blank, free of any natural function and free to be dedicated for unnatural amino acid incorporation.”

    In an article published November 30, 2017 in the journal Nature,1 Zhang and colleagues demonstrated that two of the unnatural codons work in the cell. The article’s authors also indicated that 152 blank codons might be available to help with synthesis of nonnatural proteins.

    “It’s likely that not all of [the new codons] work,” Zhang admitted, “but I’m pretty confident that we will have more codons that work that could ever be practically used.” The Romesberg lab, he continued, has been working on unnatural base pairs and their implications for nearly two decades. “In a sense, we’ve just gotten started,” he maintained.

  • Universal Expansion

    While Zhang and colleagues have tackled the challenge of creating codons by adding nonnatural base pairs to E. coli, others are taking a different approach. They’re working with molecules already operating in the cell, particularly an enzyme called aminoacyl tRNA synthetase (aaRS), which attaches to tRNA that suppresses stop codons.

    Right now, with this approach, each non-natural amino acid must be genetically encoded using two distinct platforms, one for bacterial cells and the other for eukaryotic cells. Accommodating two platforms is laborious, Dr. Chatterjee complained.

    Dr. Chatterjee and colleagues have been working on an easier way. Their idea is to take advantage of an E. coli strain in which one of the native agars–tRNA pairs is functionally replaced with a eukaryotic–archaeal counterpart. The replaced pair can then be reintroduced into E. coli to get its genetic code to produce nonnatural amino acids.

    Because the pair originated in bacteria, it can also be used to expand the genetic code of eukaryotes, the team reported in a paper published February 13, 2017 in the journal Nature Chemical Biology.2

    “Our strategy will enable the development of additional ‘universal’ aaRS–tRNA pairs,” the scientists reported. They also noted that their technique should enable genome engineers to incorporate multiple, distinct nonnatural amino acids into proteins in both eukaryotes and bacteria. That opens the door for using synthetic biology to accomplish a variety of tasks, including the probing of protein-protein interactions and the development of viruses that can home in on and help kill cancer cells, explained Dr. Chatterjee.

  • Chromosome by Chromosome

    Inserting unnatural base pairs into cells and tinkering with tRNA enzymes is not the only way to transform codons. Another way to do it is to completely rewrite the genetic code of an organism. Harvard geneticist George Church, Ph.D., and colleagues have been doing just that with E. coli; they are now generating a code for the bacterium that uses 57 codons, rather than the standard 64. With seven codons removed from E. coli’s code, the researchers can reintegrate the string of letters into the cell so that they introduce nonnatural amino acids into proteins instead.

    Something similar is being done with brewer’s yeast (Saccharomyces cerevisiae). So far, scientists have stripped the yeast genome to its essentials, making the eukaryote’s code more stable and easier to engineer.

    “By building the genome from scratch, we’re testing the very foundation of biological knowledge,” stated Leslie Mitchell, Ph.D., a geneticist who works in the laboratory of Jef D. Boeke, Ph.D., director of the Institute for Systems Genetics at New York University’s Langone Medical Center.

    Dr. Mitchell and colleagues have been designing the new yeast genome chromosome by chromosome. In each chromosome, the scientists have removed repetitive code and introduced loxPsym sites to the ends of all nonessential genes. This allows for a random shuffling of genes, the way a dealer shuffles cards, Dr. Mitchell explained. The addition of loxPsym markers, she continued, could lead to the design of yeast strains that make more ethanol, endure high temperatures, or survive in other extreme environments.

    So far, the scientists have synthesized 6.5 of the yeast’s 16 chromosomes, and they are working on creating the remaining ones. Once all 16 chromosomes are ready, the researchers will integrate them into a single cell. By doing so, “we’re changing what is known to nature,” Dr. Mitchell emphasized, “and testing the rules of genetics to see if what we understand is actually true.”

  • Testing New Codes

    Genomically recoded organisms (GROs) is a term of art used to designate E. coli and yeast that contain altered genetic codes, noted Dr. Carr. Several years ago, he worked on the initial stages of a project alongside Dr. Church and others to free up one of E. coli’s codons.

    Although he is no longer directly involved in the project, which is currently focused on freeing up more of the bacterium’s codons, Dr. Carr said that he has been using what he learned for new work on designing effective genetic codes that don’t take 10 years of experiments to get right.

    Instead, he and colleagues are working to predict, computationally, what codes will be successful, then testing the protein synthesis of those in test tubes. The team corrects code mistakes back on the computer and then retests corrected codes in test tubes in a reiterative process, over and over, until a code is ready to be tried in a cell.

    This design process is important, Dr. Carr explained, as researchers strive to build genetic codes that live up to all of the promising opportunities of synthetic biology. Among Dr. Carr’s favorites: Engineering resistance to viral infection into cells, while producing “genetic firewalls” to block the flow of genes to and from these organisms. For this to happen, the genetic code of an organism must be altered to allow it to live, but different enough from a virus or bacterium to prevent them and their infiltrating genetic material from being translated into proteins in the host.

    The language must be different even though the blueprint remains the same, Dr. Carr insisted. How different is different enough? No one knows yet, but it’s something Dr. Carr and others want to figure out. If they succeed, agricultural animal cells could be recoded to stop the spread of disease. Human cells could be rewritten, too. They could be engineered so that a virus would never infect them again.

    “It is a long road,” concluded Dr. Carr, “but there are some amazing opportunities ahead.”

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