To deliver on the promise of potent new biopharmaceuticals, synthetic biology critically relies on advances in DNA synthesis for easy, affordable and fast access to accurate nucleic acid sequences.

Innovative new methods and discoveries in next generation DNA sequencing (NGS) and molecular biology have fundamentally redefined the opportunities and challenges in reading and editing DNA, but advances in writing DNA have been incremental by comparison.

Standard DNA synthesis, including the current state-of-the-art silicon-chip based platform technology, continues to rely on a 40-year-old phosphoramidite chemistry. This technology falls short on delivering long (> 150 nucleotides) DNA of sufficient quality and in the required quantity to meet the needs of emerging applications in biopharmaceuticals and synthetic biology. In this article, we reflect on the state-of-the-art methods in writing DNA and leading directions for future DNA synthesis technology development.

The history of chemical DNA synthesis

Nucleoside phosphoramidite precursors and solid support methodologies, developed in the early 1980s at the University of Colorado in Boulder, have enabled automation and commercialization of the DNA synthesis process, leading to the widespread availability of synthetic oligonucleotides. Oligonucleotides and synthetic DNA are now an essential part of almost every aspect of recent biopharmaceutical development and its close cousin synthetic biology.

The success of the chemical DNA synthesis method has been phenomenal, and advances have led to higher throughput, less reagent consumption and a reduced cost. However, DNA synthesis via phosphoramidites is inherently limited by its chemistry. Even though reactions routinely proceed with >99.2% coupling efficiency per building block addition, the iterative process delivers DNA in quickly diminishing yields and high compounded error rates at sequence lengths of > 100 nucleotides. These limitations, combined with the harsh reagents and reaction conditions used, highlight the need for enabling DNA synthesis methods.

An enzymatic approach

Fortunately, template-independent DNA synthesis is well established in nature. As part of the vertebrate immune response, terminal deoxynucleotidyl transferase (TdT) polymerases drive the highly efficient random polymerization of deoxyribonucleoside triphosphate building blocks on a “first come, first serve” basis. For controlled synthesis of specific DNA sequences in the laboratory, the same TdTs can be used as catalysts in combination with four reversible terminator deoxyribonucleotide triphosphate (dNTP) analogs. In a process somewhat analogous to sequencing by synthesis (SBS) used in several NGS platforms, the reversible terminator dNTP analogs enable the controlled stepwise extension of an initiating primer in a user-defined sequence specific fashion. Unlike next generation SBS, the proposed method does not require a template nucleic acid and is capable of de novo synthesis. After multiple cycles of extension and terminator removal, the full length, single strand polydeoxynucleotide is cleaved from the solid support and isolated for subsequent use. Since the DNA is synthesized through an enzymatic process, it exists as a fully natural, biologically active molecule; thus eliminating some of the time-consuming and modification-inducing chemical manipulations that are required with DNA synthesized by the phosphoramidite method.

Challenges and solutions in enzymatic DNA synthesis

Aside from the obvious desire to move to a faster, cheaper and greener approach, longer length and higher purity oligonucleotides are the goals of a wave of new technology development that is sweeping the scientific community. Although it is technically feasible to assemble shorter oligonucleotides (i.e., < 100 nucleotides) into double strand constructs of gene length, TdT-driven synthesis of longer DNA sequences accelerate assembly processes. Future advances of the methods may even facilitate the direct synthesis of entire genes and gene clusters. Simplifying the post-synthesis process will lead to lower costs and a faster turn-around time. Additionally, there are applications for higher purity single-stranded DNA (ssDNA) molecules longer than what are currently economically feasible, perhaps the most exciting application being the use of ssDNA in homology directed repair in CRISPR-Cas genomic editing.

The success of enzymatic DNA synthesis depends on two distinct yet interdependent elements: an effective polymerization (bio)catalyst and the right reversible dNTPs. Native TdTs offer many of the necessary qualities as engines of enzymatic synthesis based on the combination of several well documented properties of that enzyme. First, the ability to extend primers in a near quantitative manner resulting in the addition of hundreds to thousands of nucleotides and second, the acceptance of a wide variety of modified and substituted dNTP analogs as efficient substrates.

The efficient nature of TdT-driven DNA synthesis also brings forward a key challenge: the controlled addition of one, and only one, nucleotide per cycle. The classic solution to this problem is the use of a reversible terminator at the 3’-OH of a dNTP to control the addition. Unfortunately, the extensive tolerance of TdT to variation in the nucleobase does not extend to modifications in the sugar portion of the reversible terminator dNTPs. As such, the new challenge for the development of enzymatic synthesis has been to balance the choice of a 3’-reversible terminator that is both an efficient substrate for TdT and is rapidly removable under mild conditions. By merging the creativity, experience and expertise of chemists and protein engineers, novel combinations of modified dNTPs and tailored TdTs are being developed to fine-tune this DNA synthesis “engine” and to deliver on the promise of writing DNA efficiently and economically.

Future outlooks

Enzymatic synthesis of DNA is at the beginning of it’s technology lifecycle arc. There is tremendous progress to be made in the commercial implementation of this technology. Novel hardware and high performance enzymes with long shelf-life, to name two, are all part of the future development of enzymatic DNA synthesis. The versatility of the technology is yet to be fully demonstrated or explored. One notable example is large-scale synthesis of nucleic acids for therapeutics, agricultural and nucleic acid-based materials. The potential of recycling reactants for multiple cycles would have a dramatic impact on the costs of such synthesis; currently, this opportunity does not exist for chemical synthesis. Another example is the emerging application of DNA as information storage system. The DNA “hard drives” of the future represent an infinitely larger demand for synthetic DNA compared to Life Sciences applications, requiring alternative implementations of enzymatic DNA synthesis, with a focus on lowered cost and higher throughput while maintaining fidelity. Like how silicon has accelerated the computing innovation, enzymatic DNA synthesis could be the linchpin for the next era of technology advancement. It promises to finally bring “writing” on par with reading and editing DNA.

Written by J. William Efcavitch, Ph.D., Co-Founder and Chief Scientific Officer, Molecular Assemblies and Stefan Lutz, Ph.D., Senior Vice President, Research, Codexis, Inc.