DNA synthesis technologies to close the gene writing gap

Abstract

Synthetic DNA is of increasing demand across many sectors of research and commercial activities. Engineering biology, therapy, data storage and nanotechnology are set for rapid developments if DNA can be provided at scale and low cost. Stimulated by successes in next generation sequencing and gene editing technologies, DNA synthesis is already a burgeoning industry. However, the synthesis of >200 bp sequences remains unaffordable. To overcome these limitations and start writing DNA as effectively as it is read, alternative technologies have been developed including molecular assembly and cloning methods, template-independent enzymatic synthesis, microarray and rolling circle amplification techniques. Here, we review the progress in developing and commercializing these technologies, which are exemplified by innovations from leading companies. We discuss pros and cons of each technology, the need for oversight and regulatory policies for DNA synthesis as a whole and give an overview of DNA synthesis business models.

Towards the controlled enzymatic synthesis of LNA containing oligonucleotides

“…While most efforts to improve the efficiency of de novo DNA synthesis are centered around TdT-mediated, template independent oligonucleotide production, template-dependent approaches are also emerging ( Hoff et al, 2020 ; Hoose et al, 2023 ; Van Giesen et al, 2023 ). A main advantage of template-dependent synthesis is the plethora of polymerases that have been engineered to display very lax substrate requirements and which might be capable of incorporating blocked nucleotides.…”

“…Hence, various enzymatic methods are currently developed to alleviate the shortcomings of solid-phase synthesis of nucleic acids. In this context, controlled enzymatic synthesis represents a promising approach where temporarily blocked nucleoside triphosphates are incorporated sequentially into DNA mainly by template-independent polymerases such as the terminal deoxynucleotidyl transferase (TdT) ( Jensen and Davis, 2018 ; Lee et al, 2019 ; Sarac and Hollenstein, 2019 ; Doricchi et al, 2022 ; Lu et al, 2022 ; Wang et al, 2022 ; Ashley et al, 2023 ; Hoose et al, 2023 ; Van Giesen et al, 2023 ). The blocking groups can be affixed either at the 3′-hydroxyl moiety to prevent further nucleophilic attack on the α-phosphorous of incoming nucleoside triphosphates ( Bollum, 1962 ; Mackey and Gilham, 1971 ; Chen et al, 2010 ; Hutter et al, 2010 ; Gardner et al, 2012 ; Chen et al, 2013 ; Mathews et al, 2016 ; Jang et al, 2019 ) or on the nucleobase which then act as inhibitors of polymerases ( Bowers et al, 2009 ; Palluk et al, 2018 ).…”

Construction and Applications of Mammalian Cell-Based DNA-Encoded Peptide/Protein Libraries

“… Chip-Synthesized Primer Libraries The recently emerged chip-based oligonucleotide synthesis can help produce hundreds of thousands of customized primers simultaneously. This enables researchers to perform the rational design of variant libraries, instead of traditional tiled saturation mutagenesis libraries . For instance, Ivarsson et al designed a peptide library containing over 50,000 C-terminal heptapeptides, printed the oligonucleotides on microarray slides, and generated a peptide-phage (ProP-PD) display library with further PCR-amplification and cloning steps .…”

Towards the controlled enzymatic synthesis of LNA containing oligonucleotides

“…While most efforts to improve the efficiency of de novo DNA synthesis are centered around TdTmediated, template independent oligonucleotide production, template-dependent approaches are also emerging. (Hoff et al, 2020;Van Giesen et al, 2022;Hoose et al, 2023) A main advantage of templatedependent synthesis is the plethora of polymerases that have been engineered to display very lax substrate requirements and which might be capable of incorporating blocked nucleotides. On the other hand, template-dependent synthesis leads to the formation of dsDNA rather than ssDNA products but this can be circumvented by immobilizing products on solid-support or to an extent by using universal templates.…”

“…In this context, controlled enzymatic synthesis represents a promising approach where temporarily blocked nucleoside triphosphates are incorporated sequentially into DNA mainly by templateindependent polymerases such as the terminal deoxynucleotidyl transferase (TdT). (Jensen and Davis, 2018;Lee et al, 2019b;Sarac and Hollenstein, 2019;Doricchi et al, 2022a;Lu et al, 2022;Van Giesen et al, 2022;Wang et al, 2022;Ashley et al, 2023;Hoose et al, 2023) The blocking groups can be affixed either at the 3'-hydroxyl moiety to prevent further nucleophilic attack on the -phosphorous of incoming nucleoside triphosphates (Bollum, 1962;Mackey and Gilham, 1971;Chen et al, 2010;Hutter et al, 2010;Gardner et al, 2012;Chen et al, 2013;Mathews et al, 2016;Jang et al, 2019) or on the nucleobase which then act as inhibitors of polymerases. (Bowers et al, 2009;Palluk et al, 2018) While robust protocols have been established for DNA, (Palluk et al, 2018;Lee et al, 2019b;Jung et al, 2022;Venter et al, 2022;Wang et al, 2022) changing the sugar chemistry to ribose (RNA) or to more complex modification patterns deviating from natural systems (xenonucleic acids, XNAs (Chaput and Herdewijn, 2019;Chaput et al, 2020)) raises yet unmet challenges.…”