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8 Synthesis of Viral Genomes Filippa Lentzos and Pamela Silver The emerging field of synthetic biology seeks to create a rational framework for manipulating the DNA of living organisms through the application of engineering principles.1 This chapter focuses on a key enabling technology for synthetic biology: the ability to synthesize strands of DNA from off-the-shelf chemicals and assemble them into genes and microbial genomes. When combined with improved capabilities for the design and assembly of genetic circuits that perform specific tasks, synthetic genomics has the potential for revolutionary advances. At the same time, it could permit the reconstruction of dangerous viruses from scratch, as well as genetic modifications designed to enhance the virulence and military utility of pathogens. The potential misuse of gene synthesis to re-create deadly viruses for biological warfare or terrorism would require the integration of three processes: the automated synthesis of DNA segments, the assembly of those segments into a viral genome, and the production and weaponization of the synthetic virus. Each of these steps differs with respect to the maturity of the technologies involved, the ease with which it could be performed by nonexperts, and the associated threat. Even with access to synthetic DNA, assembling the DNA segments into a synthetic virus and converting the virus into a deliverable weapon would pose significant technical hurdles. This chapter reviews the security concerns related to DNA synthesis technology and suggests some measures to limit the risk of misuse. Overview of the Technology DNA molecules consist of four fundamental building blocks: the nucleotide bases adenine (A), thymine (T), guanine (G), and cytosine (C), which can be linked together in any sequence to form a linear chain that encodes genetic information. A DNA molecule may consist of a single strand of nucleotide bases along a sugar backbone or two mirror-image strands that pair up to form a double helix, with adenine (A) always complementary to thymine (T) and guanine (G) complementary 134 F. Lentzos and P. Silver to cytosine (C). A second type of nucleic acid called RNA differs from DNA in the structure of its sugar backbone and the fact that one of the four nucleotide bases is uracil (U), which replaces thymine as the complementary base for adenine. An infectious virus consists of a long strand of single-stranded or double-stranded DNA or RNA, encased in a protein shell. There are at least three ways to acquire a synthetic viral genome. The first and most straightforward approach is to order the entire viral genome from a commercial gene-synthesis company by entering the DNA sequence on the company’s Web site. (A leading commercial supplier, Blue Heron Biotechnology in Bothell, Washington, has synthesized DNA molecules up to 52,000 base pairs long.) The genomic sequence would be synthesized in a specialized facility using proprietary technology that is not available for purchase, packaged in a living bacterial cell, and shipped back to the customer. The second option would be to order oligonucleotides (single-stranded DNA molecules less than 100 nucleotides in length) from one or more providers and then stitch them together in the correct order to create an entire viral genome. The advantage of this approach is that one can obtain more accurate DNA sequences, avoid purchasing expensive equipment, and outsource the necessary technical expertise. The third option would be to synthesize oligonucleotides with a standard desktop DNA synthesizer and then assemble the short fragments into a genome. This approach would require acquiring a DNA synthesizer (purchased or custom built) and a relatively small set of chemicals. Although the chemical synthesis of oligonucleotides up to 120 base pairs is now routine, accurately synthesizing DNA sequences greater than 180 base pairs remains somewhat of an art. For this reason, the de novo synthesis of most viruses is still more difficult than stealing a sample from a laboratory or isolating the agent from nature.2 It is just a matter of time, however, before technological advances further reduce costs and the frequency of errors, making genome synthesis readily affordable and accessible.3 History of the Technology The field of synthetic genomics dates back to 1979, when the first gene was synthesized by chemical means.4 The Indian American chemist Har Gobind Khorana and seventeen coworkers at the Massachusetts Institute of Technology took several years to produce a small gene made up of 207 DNA nucleotide base pairs. In the early 1980s, two technological developments facilitated the synthesis of DNA constructs: the invention of the...

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