Oligonucleotides come in many different flavors depending on their sequence, length, and purity. Served up in a variety of custom combinations and formats, these nucleic acid oligomers are consumed in research, diagnostic, and therapeutic applications. As lab and industrial tools, oligos are used in sequencing, editing, and constructing genes. They can even store data.
Thanks to new applications in fields such as synthetic biology and gene editing, demand for oligos is growing by as much as 30% per year. New suppliers are forming, and entrenched players are expanding their synthesis services.
Many of these firms are bringing new technologies and advanced engineering to bear on well-established chemistry. They crow about throughput, parallel synthesis, automation, and miniaturization. And they aren’t shy about claiming what their technologies can do in terms of speed, price, quality, and scale.
Beneath the bravado, though, they are all scrambling to stand out as leaders in a volatile, fast-changing field. Companies are starting up, acquiring, partnering, and even suing each other. Because no one approach does everything, the market will have room for many players. But it won’t necessarily have room for them all.
Making oligos of up to about 100 nucleotide bases with high fidelity is relatively easy, according to suppliers. Most use phosphoramidite synthesis—in which oligonucleotides grow while attached to a solid support through a cycle of deprotection, base coupling, capping, and oxidation steps.
Synthesizing longer oligos is more challenging because errors in the sequence of bases increase with length. Precise control over chemistry and process can reduce the number of downstream steps required and enable oligo synthesis of up to about 200 bases. To make even larger constructs, such as genes, shorter oligos are typically pieced together.
One of the first firms to commercially realize phosphoramidite chemistry was Applied Biosystems in the 1980s. At the company, J. William Efcavitch and Curt Becker played key roles in R&D and commercialization, respectively, in the development of its early DNA synthesizers.
The pair has now formed a new company, Molecular Assemblies, to make long oligos using enzymes. They are trying to advance an enzymatic process that uses aqueous reagents and, compared with traditional synthesis, requires fewer steps, takes less time, and is not as error prone.
|AREA||APPLICATION||NUMBER OF BASES|
|Research||PCR primers and probes||Tens|
|DNA sequencing||Tens to hundreds|
|Gene editing (CRISPR)||Few hundred|
|Fluorescence in situ hybridization||Tens|
|Synthetic biology||Gene synthesis and engineering||Thousands|
|Nucleic acid aptamers||Tens|
|Gene and cell therapy||Thousands|
In December 2016, the company completed a seed-funding round that included Agilent Technologies as an investor. It expects to close a series A round this year.
Molecular Assemblies promises to generate DNA that is up to 50 times as long as that made by existing methods and to avoid the downstream step of stitching oligos together. “The enzymatic process will allow us to make very long strands of sequence-specific DNA reliably and affordably,” Chief Executive Officer Michael J. Kamdar says. “We think that there will be a strong demand for the type of DNA we can provide.”
For now, demand for very long oligos is being met through assembly methods, and the big challenge is to scale up these methods to make thousands of genes at a time. For example, instead of using traditional 96-well plates, one prominent player, Twist Bioscience, synthesizes oligos simultaneously in 9,600-nanowell silicon plates. By spatially controlling the reactions, it can make 100 different oligos of about 100 bases each per nanowell. Enzymes then stitch the oligos together to make multiple genes.
As of February, the four-year-old company had shipped genes as big as 3,200 base pairs to the synthetic biology company Ginkgo Bioworks. Although Ginkgo was Twist’s first customer and most rapidly took advantage of its production capacity, “others are emulating their approach and scaling up as well,” says Emily Leproust, Twist’s founder and CEO.
In addition to synthetic biology, Twist’s customers are in academia and industries such as agbiotech and the life sciences. Pharma and biotech firms have become the firm’s top market, Leproust says. Twist shipped 10,000 genes in December 2016 alone and recently doubled its production space in the San Francisco Bay Area.
“The big driver of demand for oligos, whether they are short or long chain, is rapid prototyping,” says Victor Oh, an analyst at the market research firm Lux Research. Longer oligos are needed for genes used to design and engineer new pathways, strains, and enzymes, as well as for CRISPR-based gene editing. Shorter oligos are popular for gene sequencing and polymerase chain reaction uses.
With increased demand comes growing competition. “Everyone is looking for a cheaper option and a faster option,” Oh says. “Those are the two bottlenecks right now for DNA synthesis.” Commercial prices generally range from a few cents to about 30 cents per base. Turnaround times on orders for genes, typically about 100 different genes, average a few weeks.
In the quest to make gene engineering cheaper and faster for its own customers, Ginkgo acquired the DNA supplier Gen9 in January. Ginkgo had been buying DNA on the outside, contracting last June with Twist and Gen9 for each to deliver 300 million base pairs of DNA. Now Ginkgo has moved Gen9’s BioFab manufacturing system into its automated organism-engineering facilities.
Investors put about $50 million into Gen9 to develop an automated, high-throughput chip-based system for producing DNA. Using engineered proteins to detect and eliminate mistakes during synthesis, Gen9 claimed it could make high-quality 10,000-base-pair-long stretches of DNA. In early 2016, it revealed a multiplex assembly method with which it could build 50 gene-length DNA constructs simultaneously in a single reaction.
In 2013, to help Gen9 get its business going, Agilent invested $21 million in the then-four-year-old company and provided oligonucleotide libraries for building genes. Agilent’s own manufacturing technology “allows for synthesis of long oligos exceeding 200 nucleotides in length” with high accuracy, the company tells C&EN.
Agilent has developed an industrial inkjet printing process to deposit nucleotides one by one onto glass slides. It can make up to 1 million oligos per slide. As a result, Agilent competes in the market for oligo libraries and related products. It’s also a major producer of oligos for pharmaceutical ingredients.
Seeing similarities between its technology and Twist’s, Agilent sued Twist and Leproust in 2016. Agilent alleges Leproust, a former Agilent chemistry R&D director, breached her employment contract, stole trade secrets and technology, and used Agilent resources to help create Twist. Only by doing do, Agilent suggests, could Twist have found investors, hired key personnel, filed for patents, and gotten its synthesis process running as fast as it did.
Early this year, Twist filed a response in which it denied the allegations. It contends that the idea for the company came from engineers Bill Banyai and Bill Peck while they were at Complete Genomics. Whereas Agilent claims that Twist was built on the back of its decades of effort and investment, Twist asserts that the lawsuit is trying to stifle legitimate competition from an entrepreneurial firm that has raised $166 million from investors. Twist tried and failed to get the case thrown out.
In the meantime, Eurofins Genomics has been trying to find a sweet spot between traditional and chip-based methods. Traditional synthesizers make high-quality DNA, but often more of it than scientists need, which means a lot of waste, says Corey Williams, the company’s vice president of sales. Chip-based methods can make many oligos cheaply, but slowly and in tiny quantities with more errors.
Eurofins spent about five years, even consulting with aerospace engineers, to develop a new synthesis machine, Williams says. A critical feature is the machine’s ability to dispense small amounts of reagents quickly and accurately while operating continuously. The result is a 90% reduction in reagent use and waste and a 50% decrease in price for high-throughput, low-volume short oligos.
By optimizing coupling reaction efficiency, Eurofins claims to reduce the error rate when quickly making high-quality oligos. “We get about a 20–25% increase in purity on the new synthesizer,” which for many applications avoids having to do error correction or chromatographic steps to remove faulty oligos and impurities, Williams says. “Customers are requiring high-quality synthesis because today’s applications are getting much more demanding.”
To lower costs and speed turnaround, Eurofins set up a lab and production facility in Louisville, the home of UPS. Eurofins now offers oligos of up to about 50 nucleotides in 10 nanomolar quantities for overnight delivery without premium pricing, Williams says. Another new offering is nanomole amounts of high-fidelity oligos of up to 200 nucleotides.
Eurofins’s core customers are researchers in academic, clinical, diagnostic, and drug labs needing the short oligos for polymerase chain reaction and next-generation sequencing. But by also supplying longer oligos, Williams says, the firm can serve the hot areas of gene construction, gene-capture panels, and guide RNAs for CRISPR-based gene editing.
In fact, it’s hard to find an oligo supplier, small or large, that hasn’t recently launched a service to provide guide RNAs. These RNAs are what direct gene-cutting proteins to the desired location in a genome.
For example, in February 2016, oligo maker Integrated DNA Technologies obtained a nonexclusive license to intellectual property held by the CRISPR technology firm Caribou Biosciences to commercialize reagents.
Then in January, CRISPR pioneer Jennifer Doudna was among individuals and venture firms investing $41 million in Synthego (see Q&A with Doudna on page 28). The five-year-old firm focuses on supplying custom and chemically modified guide RNAs. Customers can request these for specific targets or in predesigned libraries for screening.
Synthego’s founders, brothers Paul and Michael Dabrowski, set out to create automated systems for biological research after having been software engineers at the rocket designer SpaceX and the now defunct gene-sequencing firm Halcyon Molecular. To build Synthego’s system, an interdisciplinary team focused on liquid handling, automated online monitoring and analysis, and smart manufacturing with the help of software.
“We are not chemists by training or nature, and so we haven’t tried to innovate on the chemistry,” CEO Paul Dabrowski says. The company produces RNAs of about 100 or more bases via a synthetic method typically used for making 20- to 30-base-long oligos, but “the purity is even higher than what you’d expect at the short lengths,” he adds.
“Quality of guide RNA is essential” for making consistent and precise CRISPR gene edits and avoiding off-target effects, Dabrowski explains. And he says Synthego has demonstrated gene-editing efficiencies of more than 90%, compared with 10–30% from competitors’ products.
Synthego entered the public eye in August 2016, but it already claims to be shipping CRISPR RNAs to universities and research institutions in more than 30 countries. “There was a lot of pent-up demand,” Dabrowski says. Many researchers are looking for an alternative to generating their own guide RNAs through in vitro transcription or plasmid cloning techniques.
Along with trying to make gene editing easier and more accurate, “we have made it very accessible both from time and cost perspectives,” Dabrowski says. Synthego’s plan is to ship 1-nanomole quantities of material within a week at one-fifth of competitors’ prices.
Scale is important too. Using high-throughput parallel synthesis, Synthego, Twist, and others are generating whole libraries of RNA. “We are seeing demand in the market to be able to look for multiple locations in the genome at the same time,” Twist’s Leproust says. For example, genomewide experiments are of interest for drug target discovery.
Synthetic oligos are nothing new, but the way in which they are now being created makes complex experiments possible in a practical sense, and maybe even affordable. Researchers who are able to afford DNA for the first time are both stretching their budgets further and speeding up the scale and rate of experimentation.
These factors ring especially true for researchers who were painstakingly cloning individual genes in-house. “The intent is to enable people to stop doing the low-value tedious things and instead focus on what moves the needle,” Leproust says.
Scientists today attempt things they previously avoided or didn’t consider trying because “it would have been so expensive and so slow,” she says. “It was very, very rare to have people looking at thousands of genes at the same time, and now that is routine.”