Applications of Terminal Deoxynucleotidyl Transferase Enzyme in Biotechnology

Abstract

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Towards the controlled enzymatic synthesis of LNA containing oligonucleotides

“…With blocked nucleotides 1–7 at hand, we sought to explore the possibility of constructing modified and natural oligonucleotides using controlled enzymatic synthesis. In this context, template-independent DNA polymerases such as the terminal deoxynucleotidyl transferase (TdT) ( Sarac and Hollenstein, 2019 ; Ashley et al, 2023 ) are often considered as prime candidates for de novo synthesis of single-stranded DNA oligonucleotides ( Lee et al, 2019 ; Jung et al, 2022 ; Lu et al, 2022 ; Wang et al, 2022 ). While the TdT polymerase is rather tolerant to a broad array of structurally modified nucleotides, it catalyzes the incorporation of single LNA nucleotides which then act as chain terminators even in the absence of 3′- O -blocking groups ( Kuwahara et al, 2009 ; Kasahara et al, 2010 ; Flamme et al, 2021 ).…”

“…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 ).…”

Towards the controlled enzymatic synthesis of LNA containing oligonucleotides

“…With blocked nucleotides 1-7 at hand, we sought to explore the possibility of constructing modified and natural oligonucleotides using controlled enzymatic synthesis. In this context, templateindependent DNA polymerases such as the terminal deoxynucleotidyl transferase (TdT) (Sarac and Hollenstein, 2019;Ashley et al, 2023) are often considered as prime candidates for de novo synthesis of single-stranded DNA oligonucleotides. (Lee et al, 2019a;Jung et al, 2022;Lu et al, 2022; While the TdT polymerase is rather tolerant to a broad array of structurally modified nucleotides, it catalyzes the incorporation of single LNA nucleotides which then act as chain terminators even in the absence of 3'-O-blocking groups.…”

“…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.…”

An enzyme cascade enables production of therapeutic oligonucleotides in a single operation

“…Owing to the limitations of existing synthetic methods, currently marketed therapies are produced in <50% yield with modest purities (~90%) and phosphorothioate (PS)-modified sequences are produced as complex mixtures of stereoisomers. Given these challenges, several elegant approaches to oligonucleotide production have been developed, including the use of nucleoside 3′-oxazaphospholidine derivatives (10), phosphorus(V) reagents (11)(12)(13), chiral phosphoric acid catalysts (14), and enzymatic approaches that use terminal deoxyribonucleotidyl transferases (15)(16)(17)(18)(19). Although these strategies offer improvements in stereocontrol, step economy, and/or reduced solvent consumption, they all share the same fundamental approach of stepwise chain extension by sequential coupling and deprotection steps.…”