Researchers use synthetic oligonucleotides as building blocks to assemble complete genes, fueling diverse therapeutic innovations. While outsourcing oligonucleotide production to external cores often leads to significant research delays, emerging gene synthesis technologies that enable in-house gene assembly offer researchers unprecedented autonomy and efficiency.
Download this Explainer Article from DNA Script to learn about a cutting-edge gene synthesis approach that transforms gene assembly processes and accelerates pharmaceutical research.
A cutting-edge gene synthesis approach enables researchers to construct genes with speed, adaptability, and cost effectiveness.
Advancements in synthetic biology have revolutionized the field of gene synthesis. Now, researchers are targeting longer and more complex nucleic acid sequences, using synthetic oligonucleotides to assemble complete genes and even entire genomes. As the need for synthetic genes continues to grow, emerging technologies that transform gene assembly processes offer researchers unprecedented autonomy and efficiency.
HOW CAN SCIENTISTS SPEED UP GENE ASSEMBLY?
Like building a molecular LEGO set, researchers assemble custom genes using short single-stranded DNA oligonucleotides as building blocks. By employing computational tools, researchers determine the optimal nucleic acid sequence for each gene segment and design them with overlapping ends to facilitate their assembly.
The next step is physically generating these gene pieces. Traditionally, DNA production relies on an organic chemistry technique known as phosphoramidite synthesis, which involves the stepwise addition of chemically modified nucleotides to a growing DNA chain (1). This method requires harsh organic chemicals and specialized infrastructure, limiting its usability in conventional laboratory settings. As a result, researchers typically turn to external vendors for oligonucleotide synthesis. After receiving synthesized oligonucleotides, they use molecular assembly methods like polymerase cycling assembly (PCA) to build complete gene constructs (2,3). Alternatively, researchers may acquire short double-stranded DNA fragments from vendors, which they then assemble into longer genes, or outsource the synthesis of entire gene constructs.
However, outsourcing DNA synthesis comes with a price and causes significant research delays due to the time required for ordering, production queues, and shipping. Researchers may have to wait for several weeks to obtain the synthesized oligonucleotides they need. To circumvent these setbacks, in-house gene assembly has emerged. With this approach, researchers can generate their own oligonucleotides and assemble genes directly within their own research laboratories, completing the entire gene assembly process within three days.
HOW DOES IN-HOUSE GENE ASSEMBLY WORK?
Enzymatic DNA synthesis (EDS) offers an alternative approach to oligonucletoide synthesis by utilizing gentle aqueous conditions and enzymes, rather than relying on organic chemical reactions. EDS exploits the polymerase enzyme, terminal deoxynucleotidyl transferase (TdT), which can incorporate nucleotides into DNA sequences without the need for a template (4).
Building upon EDS technology, DNA Script’s team of scientists successfully developed a two-step oligonucleotide synthesis process using the fully automated SYNTAX platform. The SYNTAX platform operates in a multiwell plate format, where each well contains a resin-based solid support coated with a customized cleavable DNA molecule called initiator DNA. In the first step, TdT enzymes elongate the initiator DNA using nucleotides with a 3’-hydroxyl group, known as a reversible terminator, ensuring precise incorporation of one nucleotide per cycle. Subsequently, the platform adds an acidic reagent to remove the reversible terminator, preparing the strand for another nucleotide addition. This cycle repeats until the oligonucleotide sequence reaches the desired length. Upon completing the last synthesis cycle, the printer enzymatically cleaves the resulting oligonucleotides from the initiator DNA cut site.
The SYNTAX platform comes with reagents, including enzymes, nucleotides, and initiator DNA. It can synthesize up to 96 oligonucleotides simultaneously in less than a day. Once the synthesis is complete, the printer purifies, quantifies, and normalizes the products, providing ready-touse oligonucleotides. The built-in software offers efficient management of DNA synthesis runs, reagents, and results, streamlining oligonucleotide synthesis and facilitating rapid experiment iteration. With same-day oligonucleotide synthesis, researchers can swiftly assemble desired gene constructs and verify sequences, enabling sustainable success of their in-house gene assembly projects.
HOW DOES IN-HOUSE GENE ASSEMBLY IMPACT PHARMACEUTICAL RESEARCH AND OTHER SCIENTIFIC FIELDS?
In-house gene assembly eliminates potential bottlenecks during synthetic gene construction, speeding up innovation in various areas of pharmaceutical research and development. During cell and gene therapy development, researchers can swiftly modify and refine gene sequences and test their therapeutic effects in target cells. Similarly, in vaccine development, in-house gene assembly offers researchers the flexibility to design and produce DNA constructs for antigens, adjuvants, and delivery systems. Furthermore, in-house gene assembly enables researchers to generate synthetic genomes or edit existing genomes to create novel biological systems. This control over gene synthesis shortens typical cell and gene therapy project timelines from eight weeks to one week, vaccine projects from ten to three weeks, and genome engineering projects from nine weeks to one week.
In addition to speed and flexibility, in-house gene assembly helps avoid costly delays. The traditional approach of relying on service providers for gene constructs may sometimes cause days to weeks of unnecessary delays, potentially resulting in daily financial losses from $500K to $1M and leading to missed market opportunities (5). By taking control of the gene assembly process, researchers can minimize the risk of costly setbacks, optimize resource utilization, and achieve cost-effective progress in drug development pipelines.
The benefits of adopting in-house gene assembly also extend beyond healthcare. In agriculture, it allows genetic modification of crops to enhance their disease resistance, yield, and nutritional value (6). In waste management, it expedites micro-organism engineering for effective plastic waste degradation (7). The chemical industry can also benefit by using in-house gene assembly for the biological production of novel chemicals and metabolic engineering, offering eco-friendlier alternatives to traditional chemical synthesis methods (8).
Proven Success
The oligonucleotides produced by the SYNTAX DNA printer exhibit exceptional success rates in assembling large kilobase (kb)-sized genes. In a proof-of-concept study, DNA Script scientists assembled the 1.7 kb Influenza A hemagglutinin (HA) gene from 60 and 120 nucleotide-long oligonucleotides printed on the SYNTAX system (9). The team cloned the assembled gene into vectors and transformed it into bacterial cells. Remarkably, after overnight incubation, 100 percent of the picked clones contained the correctly sized HA gene fragment. In another study, the DNA Script team constructed the genes for green fluorescent protein and monomeric red fluorescent protein using SYNTAX-printed oligonucleotides (10). Within three days, they successfully obtained full-length clones that expressed functional fluorescent proteins, demonstrating the accuracy and robustness of the in-house gene assembly process. These achievements highlight the promising potential of in-house gene assembly for more rapid and precise creation of complex genetic constructs.
REFERENCES
1. Beaucage, S. L. & Caruthers, M. H. Deoxynucleoside phosphoramidites—A new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedron Letters 22, 1859–1862 (1981).
2. Stemmer, W. P. C., Crameri, A., Ha, K. D., Brennan, T. M. & Heyneker, H. L. Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene 164, 49–53 (1995).
3. TerMaat, J. R., Pienaar, E., Whitney, S. E., Mamedov, T. G. & Subramanian, A. Gene synthesis by integrated polymerase chain assembly and PCR amplification using a high-speed thermocycler. Journal of Microbiological Methods 79, 295–300 (2009).
4. Motea, E. A. & Berdis, A. J. Terminal deoxynucleotidyl transferase: The story of a misguided DNA polymerase. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 1804, 1151–1166 (2010).
5. Gouglas, D. et al. Estimating the cost of vaccine development against epidemic infectious diseases: a cost minimisation study. The Lancet Global Health 6, e1386–e1396 (2018).
6. Wang, Y. & Demirer, G. S. Synthetic biology for plant genetic engineering and molecular farming. Trends in Biotechnology 0, (2023).
7. Mohanan, N., Montazer, Z., Sharma, P. K. & Levin, D. B. Microbial and Enzymatic Degradation of Synthetic Plastics. Frontiers in Microbiology 11, (2020).
8. Li, M. & Borodina, I. Application of synthetic biology for production of chemicals in yeast Saccharomyces cerevisiae. FEMS Yeast Research 15, 1–12 (2015).
9. DNA Script. Rapidly assemble genes in your laboratory using automated enzymatic oligo synthesis. https://www.dnascript.com/wpcontent/uploads/2023/06/ACD_GeneAssembly_PCA_Poster_VF.pdf
10. DNA Script. Gene Assembly Application Note. https://www.dnascript.com/wp-content/uploads/2022/09/DNAScript-Gene-Assembly_AppNote_version1.0.pdf