Controlled enzymatic synthesis of oligonucleotides

Abstract

Oligonucleotides are advancing as essential materials for the development of new therapeutics, artificial genes, or in storage of information applications. Hitherto, our capacity to write (i.e., synthesize) oligonucleotides is not as efficient as that to read (i.e., sequencing) DNA/RNA. Alternative, biocatalytic methods for the de novo synthesis of natural or modified oligonucleotides are in dire need to circumvent the limitations of traditional synthetic approaches. This Perspective article summarizes recent progress made in controlled enzymatic synthesis, where temporary blocked nucleotides are incorporated into immobilized primers by polymerases. While robust protocols have been established for DNA, RNA or XNA synthesis is more challenging. Nevertheless, using a suitable combination of protected nucleotides and polymerase has shown promises to produce RNA oligonucleotides even though the production of long DNA/RNA/XNA sequences (>1000 nt) remains challenging. We surmise that merging ligase- and polymerase-based synthesis would help to circumvent the current shortcomings of controlled enzymatic synthesis.

Fig. 1. Pros and cons of chemical automated synthesis and enzymatic synthesis of oligonucleotides.

Controlled enzymatic synthesis really resides at the interface of chemical and enzymatic approaches. Allowing theoretically to reach high quantity, high length and the use of modified nucleotides at the same time.

Fig. 2. Schematic representation of de novo enzymatic synthesis of nucleic acids with template-independent polymerases.

Nucleoside triphosphates blocked at the level of the nucleobase or the sugar (mainly 3’-OH) are incorporated into single-stranded primers by polymerases. The transient blocking group prevents multiple incorporation events from occurring. Following polymerase-mediated incorporation, the masking group is removed and the system is ready for subsequent synthetic cycles.

Fig. 3. Chemical structures of blocked nucleotides used in DNA sequencing by synthesis (SBS) and de novo DNA synthesis.

A Structures of common 3’-O-blocked nucleotides and (B) base-modified analogs. The colors represent different fluorophores or dyes that can be incorporated into DNA during SBS.

Fig. 4. Chemical structures of blocked RNA nucleotides.

The protection pattern can involve only 3’-O-protection (structures 8 and 9) or cis-diol protection (nucleotides 10 and 11). Bs represents any nucleobase.

Fig. 5. Chemical structures of XNA nucleotides.

A Structures of 3’-O-blocked locked nucleic acids (LNAs) and B structures of threose nucleic acids (TNAs) and hexitol nucleic acids (HNAs) nucleotides. Bs represents any nucleobase.

Fig. 6. Alternative biocatalytic method for the production of modified nucleic acids.

This isothermal biocatalytic approach based on the polymerase-mediated incorporation of modified nucleotides into a self-priming hairpin template (gray–blue). (i) Template-dependent synthesis using sugar and phosphate-modified nucleotides and the KOD polymerase; (ii) endonuclease V cleaves the second phosphodiester linkage 3’-downstream of a deoxyinosine moiety present in the template and generates the modified sequence (in red); (iii) after endonuclease-cleavage, the template is available for subsequent rounds of catalytic synthesis.

Fig. 7. Enzymatic synthesis of DNA three-by-three rather than one-by-one.

Chemical structure of a trinucleotide triphosphate stabilized by phosphorothioate linkages. Bs represents any nucleobase.