The gold standard for DNA writing, phosphoramidite chemistry, works incredibly well on short, uncomplicated, oligos. For longer stretches of DNA, which may contain DNA with complex features such as secondary structure and high GC content, the workhorse method is being challenged by the method of enzymatic DNA synthesis.
Some argue that enzymatic synthesis has the ability to make longer oligos, with more accuracy. One of those companies, Ansa Biotechnologies, is working to make its case by announcing the successful de novo synthesis of the world’s longest DNA oligonucleotide ever reported to be produced in a single synthesis.
The 1,005 base sequence encodes a portion of an AAV vector that could be used for gene therapy development. The DNA is not only long, but it also contains complex features that pose a challenge to conventional methods that require assembly of shorter oligonucleotides.
When the oligo was cloned, it was found to contain approximately 28% sequence-perfect molecules, indicating an average stepwise yield of approximately 99.9% during its synthesis.
Ansa’s process starts with DNA on a solid support (like a chip). Then, the DNA is exposed to a terminal deoxynucleotidyl transferase (TdT)-nucleotide conjugate, whereupon the tethered nucleotide becomes covalently attached to the end of the primer. At this point, the nucleotide-extended primer is still attached to the whole enzyme, which blocks any other additions from being made. After a wash step, a second enzyme is added to cleave the linker, and the Tdt is released. Now the primer is not just one base longer, it is also ready for another base to be added. This method was first described in a 2018 Nature Biotechnology paper by Dan Lin-Arlow, PhD, and Sebastian Palluk, PhD, co-founders and CEO and CTO (respectively) of Ansa Biotechnologies.
There is increasing demand from scientists for gene-length synthetic DNA for applications such as cell and gene therapies, protein engineering, biomanufacturing, and for fundamental life sciences research. Longer sequences are currently made by “stitching together” shorter oligos, but the process struggles with sequences that contain certain features such as secondary structures, repeats, and high or low GC content.
“Synthesis of a 1005mer represents a major milestone for the field and is beyond what many thought was possible,” said Lin-Arlow.
Although these long oligos are not commercially available now, the Emeryville-based Ansa says that they plan to initiate an Early Access Program for clonal synthetic genes in April.
Lin-Arlow told GEN last year that he hopes to develop a method to print out whatever DNA constructs scientists need for an experiment. He wants to create something like Amazon Prime for DNA. “You don’t even think about it,” he explained. “It just comes the next day.”