Template-Independent Enzymatic Oligonucleotide Synthesis (TiEOS): Its History, Prospects, and Challenges

Abstract

There is a growing demand for sustainable methods in research and development, where instead of hazardous chemicals, an aqueous medium is chosen to perform biological reactions. In this Perspective, we examine the history and current methodology of using enzymes to generate artificial single-stranded DNA. By using traditional solid-phase phosphoramidite chemistry as a metric, we also explore criteria for the method of template-independent enzymatic oligonucleotide synthesis (TiEOS). As its key component, we delve into the biology of one of the most enigmatic enzymes, terminal deoxynucleotidyl transferase (TdT). As TdT is found to exponentially increase antigen receptor diversity in the vertebrate immune system by adding nucleotides in a template-free manner, researchers have exploited this function as an alternative to the phosphoramidite synthesis method. Though TdT is currently the preferred enzyme for TiEOS, its random nucleotide incorporation presents a barrier in synthesis automation. Taking a closer look at the TiEOS cycle, particularly the coupling step, we find it is comprised of additions > n+1 and deletions. By tapping into the physical and biochemical properties of TdT, we strive to further elucidate its mercurial behavior and offer ways to better optimize TiEOS for production-grade oligonucleotide synthesis.

Terminal Deoxynucleotidyl Transferase in the Synthesis and Modification of Nucleic Acids

“…This can be achieved by using nucleoside triphosphate analogues equipped with a temporary 3′‐ O ‐blocking group that terminates the elongation after single nucleotide incorporation (structures 11 – 14 in Scheme ). After removal of the excess amount of nucleotide and the TdT, the terminator group can be deprotected to recover the 3′‐hydroxy functionality, allowing for further elongation cycles (Scheme ) …”

“…This blocking group is of interest because of its small size (comparable to the size of a OCH 3 group), ease of removal (with a simple sodium nitrite treatment), and good recognition by DNA polymerases . Lastly, the 3′‐ O ‐azidomethyl and 3′‐ O ‐allyl ( 12 and 13 in Scheme , respectively) moieties have also been proposed as transient protecting groups for the development of potent reversible terminators …”

“…After removal of the excess amount of nucleotide and the TdT,t he terminator group can be deprotected to recover the 3'-hydroxy functionality, allowing for further elongation cycles (Scheme2). [104] These nucleoside triphosphates, the so-called "reversible terminators", are used in the context of next-generation sequencing, although template-dependentp olymerases are necessary. [105] In this context, Mathews et al reported photocleavable 3'-O-(2-nitrobenzyl)-2'-deoxyribonucleoside triphosphates (such as 14 in Scheme 1) and 3'-O-(4,5-dimethoxy-2-nitrobenzyl)-2'-deoxyribonucleoside triphosphates, in which the 3'-O-nitrobenzyl and 4,5-dimethoxy-2-nitrobenzyl groups can be removedw ith a l = 365 nm UV-light source.…”

“…[110] Lastly, the 3'-O-azidomethyl [111] and 3'-O-allyl [112] (12 and 13 in Scheme 1, respectively)m oietiesh ave also been proposed as transientp rotecting groups for the development of potent reversible terminators. [104] In template-free enzymatic DNA synthesis, it is still ab ig challenge to achieveq uantitative coupling efficiencies by using 3'-O-blockedd NTPs.Recently,anew way for the de novo DNA synthesis has been described, and it makes use of aT dT-dNTP conjugate. In this approach, no 3'-O-blocking groups are required, and the nucleobase of ad NTP is covalently linked to the active site of the TdTt hrough either ad isulfidem oiety or ap hotocleavable linker (e.g.,s ee TdT-TTP 10 in Scheme 1).…”

Photon-directed Multiplexed Enzymatic DNA Synthesis for Molecular Digital Data Storage

“…Our system utilizes a combination of commercial photolabile materials and maskless lithography technology with the added benefit of circumventing the use of expensive physical photomasks, special phosphoramidites, harsh organic solvents and the accumulation of toxic waste from chemical synthesis. Additionally, since metal cation cofactors are essential for polymerase catalysis, the presented method will be potentially suitable for controlled synthesis using mutated enzymes with enhanced attributes such as higher processivity or the ability to incorporate modified nucleotides 21 . While enzyme-based oligonucleotide synthesis technology is still in its infancy, we envision that bridging overall cleaner and faster DNA oligonucleotide synthesis with cutting-edge, high-density array photolithographic techniques will pave the way for its widespread adoption and further promote practical molecular data storage technology.…”