Phosphoramidite solid-phase chemical DNA synthesis (CDS) (Beaucage and Caruthers, 1981) is the standard method for producing de novo single-stranded DNA (ssDNA) and is used on a large scale by commercial vendors to produce oligonucleotides (oligos) for PCR, cloning, and increasingly for de novo construction of genes and plasmids (Kosuri and Church, 2014; Song et al., 2021). CDS methods have evolved to maximize product length while minimizing overall cost and errors, but they are limited to production of oligos of 200 or fewer nucleotides (nt) and require the use of hazardous chemicals such as acetonitrile, tetrahydrofuran, and dichloromethane (Hughes et al., 2011; Kosuri and Church, 2014; Song et al., 2021).

Controlled enzymatic DNA synthesis (EDS) was first developed in the 1950s–1970s (Grunberg-Manago et al., 1955; Bollum, 1962; Chang and Bollum, 1971) with the discovery of the terminal deoxynucleotidyl transferase (TdT) enzyme that adds dNTPs to the 3′-hydroxyl group of DNA. Recent breakthroughs have addressed historical limitations of TdT (Palluk et al., 2018; Song et al., 2021) by evolving more thermally stable TdT enzymes (Chua et al., 2020; Lu et al., 2022), developing reversible 3′-dNTP protecting groups that can be accommodated by TdT (Mathews et al., 2016; Mathews et al., 2017; Hoff et al., 2020), and improving enzymatic coupling efficiency to enable a cyclic, solid-phase-templated extension of DNA (Palluk et al., 2018; Barthel et al., 2020; Hoff et al., 2020). EDS has the added benefit of eliminating the use of hazardous organic chemicals by utilizing a primarily aqueous workflow (Song et al., 2021).

Several EDS-focused companies were launched within the last decade, including Molecular Assemblies and Nuclera (2013), DNA Script (2014), Camena Bioscience (2016), Ansa Biotechnologies (2018), and Kern Systems (2019) (Eisenstein, 2020). Camena Bioscience and DNA Script report greater than 99% coupling efficiency while producing 300- and 280-mer oligos, respectively (Eisenstein, 2020), elevating this technology to a competitive level with chemical synthesis (Masaki et al., 2022). More recently, Ansa Biotechnologies, Inc., reported the enzymatic synthesis of the longest de novo oligonucleotide ever produced in a single synthesis at 1,005 nucleotides long (Ansa Biotechnologies, 2023), and Camena Bioscience reported the production of a whole 2.7-kb plasmid using multi-enzymatic de novo DNA synthesis and gene assembly (Camenabio, 2020). However, EDS products are not readily available yet for purchase from commercial sources, and interested users are limited to early access programs (Ansa and Molecular Assemblies) or purchasing a benchtop EDS instrument (DNA Script).

The Syntax-100™ from DNA Script is the first commercially available benchtop EDS instrument. It is marketed to have a user-friendly platform and essentially aqueous waste stream and can synthesize 96 × 60 mers in 13 hours, including cleanup and quantitation. Here, we evaluate the Syntax-100™ to produce oligos using EDS and compare them to commercial CDS oligos to assemble a synthetic gene encoding green fluorescent protein (GFP). In this brief study, we consider the time of oligo synthesis and gene assembly, cost per oligo/base, sequence accuracy, and waste production.

In this study, we sought to compare CDS- and EDS-sourced oligonucleotides for use in the assembly of the gene encoding GFP. We used the application Primerize (Tian and Das, 2017) to divide a GFP coding sequence (Snapgene, 2023) into overlapping oligos of 60 nt or 90 nt maximum length, a 20 nt overlap, and a minimum annealing temperature of 60°C that were amenable to assembly using previously established polymerase cycling assembly (PCA) methods (Supplementary Table S1) (Stemmer et al., 1995; Dormitzer et al., 2013). The 60 nt oligos provided the maximum length available from our EDS benchtop instrument, and the 90 nt oligos provided a longer yet cost-effective option for CDS oligos that could be used to compare the effects of oligo length on gene assembly. We synthesized replicates of twenty 60 nt oligos via EDS in two independent runs on the Syntax-100™. The total run time for each plate was thirteen hours, including sample cleanup and quantitation. A set of twenty (60 nt max, 25 nmol each) and twelve (90 nt max, 100 nmol each) commercial CDS oligos was ordered and delivered in 2 days.

Our adapted gene assembly approach (Dormitzer et al., 2013) included an initial isothermal assembly of pooled oligos, followed by two rounds of PCR: PCR1 for PCA and PCR2 to enrich the final product (Figure 1A). The CDS 90 and 60 nt oligo assemblies showed a faint band at the expected size (717 bp) after PCR1 and a clear band after PCR2, whereas the two EDS 60 nt oligo sets did not produce a visible full-length product until PCR2 (Figure 1B). Full gene products assembled from each oligo set were TOPO-cloned into pCR4 vectors (Thermo Fisher Scientific, 2023) and sequenced after transformation and colony screening. A minimum of ten transformants were screened and sequenced for each independent assembly (Table 1). We observed that the designed gfp sequence was successfully assembled using each of the four oligo sets (Table 1). Notably, the longer 90 nt CDS oligos did produce a higher percentage of correct sequences (40%) compared to the 60 nt oligo sets (27%, CDS and 24%, EDS). Sequence-validated gfp gene products from each oligo set were sub-cloned into an expression vector, transformed into competent E. coli cells, and analyzed for GFP protein expression and fluorescence. EDS- and CDS-sourced DNA sequences successfully produced functional gene products with confirmed expression and fluorescence of GFP (Figure 1C).

While this work was being completed, the vendor for the Syntax-100™ published an application note on their website detailing a similar set of experiments (DNA Script, 2022). We have summarized those findings in Table 1 along with our independent validation of EDS oligos compared to commercially prepared CDS oligos. Using an analogous gene assembly method, we obtained similar percentages of correctly assembled sequences using EDS oligos (23%) compared to the vendor (24%). However, our results for CDS 60 nt oligos differed, with 27% of our sequenced transformants having the designed sequence compared to only 18% reported by the vendor. The vendor does not state the source of their CDS oligos.

Currently, the most cost-effective way to utilize the Syntax-100™ is to produce 96 × 60-mers (or 80-mers and 120-mers that have recently become available), which is comparable to ordering the equivalent number, length, and quality of oligos from IDT (Table 2) at approximately $0.20–$0.40 per base, if labor and the initial cost of the instrument are not included. The yield for EDS oligos is much lower (0.3 nmol per oligo per well), but in our experiments, EDS oligos were of sufficient quality and quantity to assemble a functional gfp gene. It is worth noting that purchasing a CDS synthetic gfp gene from commercial sources currently costs approximately $280, which is still substantially cheaper than purchasing or producing the assembly oligos by EDS or CDS methods. Finally, we were interested in analyzing the waste generated in our laboratory on a benchtop CDS instrument (MerMade 192E™, LGC Limited) compared to the Syntax-100™ after production of 96 × 60-mers. Our analysis indicated a clear reduction in hazardous chemical waste production in both volume and individual waste components when using the benchtop EDS method (Figure 1D).

This brief study provides an independent validation that EDS oligonucleotides are suitable for use in gene synthesis workflows, with 23% of assembled samples containing the designed gfp sequence (Table 1). Our findings are similar to those described by the vendor in their recently published application note (DNA Script, 2022). Notably, we obtained similar results without adding a correction step in our PCA protocol. Our results for CDS gene assembly indicate a higher rate of correct sequences assembled (40% and 27% compared to 23% for EDS oligos) that may be due to longer starting oligo length (in the case of CDS 90 nt oligos) or the higher reported error rate for EDS (0.4%) versus CDS (0.1%) (Masaki et al., 2022). Further experiments are required to differentiate between errors introduced during DNA synthesis or gene assembly. Both EDS- and CDS-produced oligos used in gene assembly resulted in functional GFP-coding sequences.

Although commercial CDS sources remain the most cost-effective solution for obtaining oligos, it is worth noting that benchtop DNA synthesis instruments (CDS and EDS) provide users with improved access to on-demand DNA synthesis in mobile or austere laboratory settings. In our studies, EDS produced substantially less hazardous waste than a CDS instrument, suggesting this technology could provide a safer and more environmentally friendly alternative for large-scale DNA synthesis. Finally, the recent report of kilobase-length DNA by EDS methods (Ansa Biotechnologies, 2023) suggests that this technology may overcome the inherent length limitations faced by CDS methods. Future studies may include testing additional EDS kits, offering oligo modifications such as the addition of biotin, fluorophores, and quenchers for a variety of downstream applications.