Conventionally, oligonucleotides have been synthesized using phosphoramidite chemistry—a technique developed in 1981 and still used widely in basic and applied research.1 However, this approach has several limitations. The efficiency of phosphoramidite synthesis decreases beyond ~200-mers. The manufacturing, processing, and purification workflow requires considerable manual labor, which added to transportation times and fluctuations in manufacturing competencies and logistics, can result in lengthy turnaround times and delays in researchers receiving their oligos. Furthermore, organic phosphoramidite chemistry generates hazardous wastes and creates environmental risks.

Researchers can now use enzymatic DNA synthesis (EDS) technology to synthesize custom-designed oligonucleotides at the lab bench without specialized training, handling harsh organic chemicals, or extended turnaround times. The SYNTAX platform2, developed by DNA Script, a disruptive DNA synthesis company based in South San Francisco, enables custom DNA printing on demand using EDS technology.

In an exclusive interview with GEN, Thomas Ybert, PhD, CEO and co-founder of DNA Script, says: “Like nature, EDS uses enzymes—not harsh organic chemistry. Think of these enzymes as nanorobots that work to build DNA molecules. Our EDS makes for sustainable, greener technology with reduced environmental impact. In practice, it is safer for the environment and easily handled by standard waste streams in most molecular biology labs.”

The system consists of three parts: a benchtop DNA-On-Demand instrument, SYNTAX console software, and a reagent consumables kit including four inks that represent the building blocks of DNA: A, T, C, and G. This system is capable of performing automated synthesis, purification, quantification, and normalization in a single machine. The technique is a simple, fast, and reliable alternative to the decades-old chemical synthesis technique.3

Ybert adds, “By creating a solution for companies to control their workflows and turnaround time by printing DNA on demand in-house, we enable researchers to accelerate the design-print-test iterative cycle and eliminate the time-intensive process of outsourcing production that ultimately results in slowed innovation.”

This article will cover the synthesis workflow of the EDS technology and the three core innovations that make up its biochemistry.

The solid support

The solid support of the EDS platform has two key components: a resin-based solid DNA support and a cleavable linker DNA molecule attached to it, known as “initiator DNA” (iDNA). The function of this complex is to initiate the synthesis while ensuring that the chemical and physical properties are optimal for enzymatic reaction. The iDNA can be optimized by changing its length to minimize steric hindrance during enzymatic elongation. It includes a specific cleavable site to ensure its release after completing the synthesis.

Synthesis enzyme: terminal deoxynucleotidyl-transferase (TdT)

The TdT enzyme is a specialized DNA polymerase found in immature, pre-B, pre-T lymphoid cells, and acute lymphoblastic leukemia or lymphoma cells. This enzyme is unique because it can introduce extra nucleotides to the 3’ terminus of a DNA molecule, and unlike most DNA polymerases, it does not require a template.4,5 TdT belongs to the X-family of DNA polymerases that introduces genetic diversity during immunity maturation gene recombination. It does so specifically by adding nucleotides to the 3′-termini of variable (V), diversity (D), and joining (J) gene segments of antibodies in a random, template-independent manner.

The TdT enzyme has been engineered in vitro by DNA Script to catalyze rapid and selective polynucleotide additions to the iDNA with high fidelity and coupling efficiency.

“This unique ability of TdT to create genomic material de novo has long been leveraged for labeling oligonucleotides in a wide range of applications. It also renders TdT, engineered as we have, as the enzyme of choice for EDS,” says Ybert. “Our proprietary EDS enzymes have been engineered to rapidly and selectively add our reversibly-terminated nucleotides to the 3′-end of any single DNA strand—the sequence context does not matter. In addition, our TdT allows for longer oligo length. Internally, we have achieved up to 360 nucleotides length. The benefits are clear.”

Reversibly-terminated nucleotides 

In the template-less EDS process for oligonucleotide synthesis, strategies must be employed to ensure that the intended base and only one molecule of the intended base is added to the growing oligonucleotide at every cycle of synthesis. DNA Script’s SYNTAX system accomplishes this through the use of modified building blocks (deoxy-nucleotide triphosphates, dNTPs).

“We developed a set of nucleotides that control the EDS process by effectively pausing synthesis after a single base addition and then restarting the cycle to add a new base. Like other applications, for example Illumina sequencing, we do this by using nucleotides with a modified 3′-hydroxyl group—or reversibly terminated dNTPs–that prevent extension of the growing DNA chain,” explains Ybert.

The modification is removed at each cycle using a mildly acidic reaction buffer, permitting DNA synthesis to continue. Since the dNTPs are only modified at the 3’ end, they are deprotected and do not leave any trace of the modification in the synthesized product. They can be directly used in experts without any further treatment and are identical to natural DNA.

2 step EDS process
Enzymatic DNA synthesis (EDS) is a two-step process [DNA Script]

Synthesis workflow

Oligos are synthesized in a two-step process. In the first step the TdT enzyme elongates the iDNA by adding a single nucleotide. This is followed by removal of the reversible terminator of the added nucleotide, leaving the growing strand ready for another elongation step. These two steps are repeated until the desired length is achieved. The resulting oligos are desalted, quantified, normalized, and ready for use in different applications.

Oligonucleotides are needed for almost every study design in molecular biology, genomics, and life science applications. The EDS technology is expected to revolutionize research in these areas, including targeted NGS, gene assembly, and production of nucleic acid-based vaccines.6 With EDS technology, DNA synthesis has entered a new stage that will transform life science research and reward researchers with significant independence and control over turnaround times and synthesis efficiency.

 

References

  1. Beaucage SL, Caruthers MH. Deoxynucleoside phosphoramidites—A new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedron Lett. 1981;22(20):1859-1862. doi:10.1016/S0040-4039(01)90461-7
  2. Eimerman P, Jeddeloh J, Champion E, et al. Development of a simple and versatile enzymatic DNA synthesis system that enables accurate, fast, and long oligos on demand. J Biomol Tech JBT. 2020;31(Suppl):S10.
  3. Technology. DNA Script. Accessed June 20, 2022.
  4. Delarue M, Boulé JB, Lescar J, et al. Crystal structures of a template-independent DNA polymerase: murine terminal deoxynucleotidyltransferase. EMBO J. 2002;21(3):427-439. doi:10.1093/emboj/21.3.427
  5. Motea EA, Berdis AJ. Terminal Deoxynucleotidyl Transferase: The Story of a Misguided DNA Polymerase. Biochim Biophys Acta. 2010;1804(5):1151-1166. doi:10.1016/j.bbapap.2009.06.030
  6. Applications. DNA Script. Accessed June 20, 2022.