Evaluation of 3′-phosphate as a transient protecting group for controlled enzymatic synthesis of DNA and XNA oligonucleotides

Abstract

Chemically modified oligonucleotides have advanced as important therapeutic tools as reflected by the recent advent of mRNA vaccines and the FDA-approval of various siRNA and antisense oligonucleotides. These sequences are typically accessed by solid-phase synthesis which despite numerous advantages is restricted to short sequences and displays a limited tolerance to functional groups. Controlled enzymatic synthesis is an emerging alternative synthetic methodology that circumvents the limitations of traditional solid-phase synthesis. So far, most approaches strived to improve controlled enzymatic synthesis of canonical DNA and no potential routes to access xenonucleic acids (XNAs) have been reported. In this context, we have investigated the possibility of using phosphate as a transient protecting group for controlled enzymatic synthesis of DNA and locked nucleic acid (LNA) oligonucleotides. Phosphate is ubiquitously employed in natural systems and we demonstrate that this group displays most characteristics required for controlled enzymatic synthesis. We have devised robust synthetic pathways leading to these challenging compounds and we have discovered a hitherto unknown phosphatase activity of various DNA polymerases. These findings open up directions for the design of protected DNA and XNA nucleoside triphosphates for controlled enzymatic synthesis of chemically modified nucleic acids.

Benzoyl and Pivaloyl as Efficient Protecting Groups for Controlled Enzymatic Synthesis of DNA and XNA Oligonucleotides

“…Lastly, when lower concentrations of triphosphates were used, the occurrence of faster running bands was observed. This probably is the result of hydrolytic degradation of the primer which has been reported for other modified nucleotides, [14,25] Similarly, some weak bands with a gel mobility comprised between that of the n and n+1 products can be observed under specific conditions (Figure 3). These products might occur due to 3’‐phosphorylation of primer P1 by the TdT polymerase [17b,26] .…”

“…Hence, the protecting group needs to be stable under higher pH (i. e. 8.0-9.0) conditions which are required for optimal polymerase activity and exposure to elevated temperatures (ranging from 37 °C to 65 °C depending on the nature of the polymerases). In addition, the blocking group needs to be resistant against the intrinsic esterase [16] and phosphatase [14,17] activity displayed by a number of DNA polymerases. Third, removal of the blocking group should proceed in high yields and involve mild conditions that do not damage the nascent DNA/LNA chain.…”

“…However, controlled enzymatic synthesis has been barely explored for alternate sugar chemistries, particularly of XNAs. [14] Here, we have evaluated the possibility of using different reversible protecting groups for controlled enzymatic LNA synthesis. Among the various terminators, benzoyl and pivaloyl protecting groups appear to display interesting properties since the corresponding nucleotides are well-tolerated by polymerases and single incorporation events are achieved in high yields.…”

DNA synthesis technologies to close the gene writing gap

“…Crucially, this bias extends to protected NTPs used in DNA synthesis 27 , 28 , 80 , prompting researchers to develop methods to mitigate the bias 87 . Additional features of TdT, which impact on the choice of PGs and synthesis efficiency, include the DNA phosphorylation capacity and phosphatase activity of the enzyme 88 – 90 . Thus when a growing oligonucleotide chain is exposed to a mixture of NTPs, TdT would preferentially incorporate certain nucleotides resulting in the synthesis of homopolymeric chains of varying lengths.…”

“…97 – 100 ) and Camena Bioscience appears to favour 2-nitrobenzyl 26 as a 3ʹ-PG 101 . Other PGs are attempted for the protection of 3ʹ-OH and the bases of NTPs 102 – 106 , with parallel efforts focusing on PGs for XNA synthesis 90 , 107 . However, TdT must be able to accommodate the protected nucleotides in its active site, which limits the choice of PGs or requires the re-engineering of the enzyme for compatibility with 3ʹ-PG.…”