How AstraZeneca created an automated DNA assembly framework to support rapid and cost-efficient construct generation

Fragment recycling significantly reduces DNA synthesis time and costs

AstraZeneca’s solution is an elegant integration of multiple softwares and automation, spearheaded by Associate Principal Scientist David Öling and his group in Sweden. This group provides most of the DNA constructs used by AstraZeneca’s teams across Sweden and the U.K. 

Öling’s team began by optimizing a golden gate assembly-based approach to building DNA constructs. They created and validated 19 different modules that are sufficient for most complex constructs. But the key to AstraZeneca’s success is in how the fragments that are plugged into those modules to create the complete construct are identified, generated, and pieced together. 

Traditionally, a significant amount of synthesis time and cost is spent on DNA sequences that are present multiple times across a construct, as those sequences have to be ordered individually as many times as they are present. To address this problem, Öling and his team worked with the computational biology team at AstraZeneca to create what he calls “a workaround for the whole DNA synthesis industry” — an algorithm for fragment recycling called FRAGLER (FRAGment recycLER).

Fragment recycling identifies shared coding sequence regions across the construct so that those sequences only need to be ordered once, reducing both the cost of DNA synthesis and the time it takes to generate DNA fragments. FRAGLER not only performs amino acid sequence alignment and codon optimization of desired sequences, but it also fragments those sequences to increase the success of DNA production. Fragmentation of long sequences into shorter fragments of 300-900 base pairs reduces synthesis time and ensures production success, says Öling. Moreover, fragmentation eliminates sequence complexities and minimizes cancellations, which are very common for longer sequences.

To illustrate how FRAGLER works, AstraZeneca used its software solution to rapidly generate complex SARS-CoV-2 spike protein constructs for expression optimization (Figure 1). The spike protein coding sequence was fragmented into four submodules, which could then be combined with various signal peptides and trimerization domains. The resulting constructs were expressed in HEK293 cells. FRAGLER was also used to fragment 30 full-length SARS-CoV-2 spike protein variants, generating 55 unique fragments and recycling 11 fragments 65 times — a base pair recycle rate of 55.3%. 

According to Öling, the savings afforded by this approach equates to at least a 50% reduction in costs, and sometimes even more, depending on the constructs. For example, most of the DNA can be reused when performing single point mutations over a hundred constructs, which affords cost reductions upwards of 80-90%. These long complex constructs were generated rapidly by the fragmentation approach.