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. 2024 Sep 13;15(1):8009.
doi: 10.1038/s41467-024-52141-8.

Template-dependent DNA ligation for the synthesis of modified oligonucleotides

Nazarii Sabat  1 , Andreas Stämpfli  2 , Steven Hanlon  3 , Serena Bisagni  3 , Filippo Sladojevich  2 , Kurt Püntener  3 , Marcel Hollenstein  4
Affiliations

Template-dependent DNA ligation for the synthesis of modified oligonucleotides

Nazarii Sabat et al. Nat Commun. .

Abstract

Chemical modification of DNA is a common strategy to improve the properties of oligonucleotides, particularly for therapeutics and nanotechnology. Existing synthetic methods essentially rely on phosphoramidite chemistry or the polymerization of nucleoside triphosphates but are limited in terms of size, scalability, and sustainability. Herein, we report a robust alternative method for the de novo synthesis of modified oligonucleotides using template-dependent DNA ligation of shortmer fragments. Our approach is based on the fast and scaled accessibility of chemically modified shortmer monophosphates as substrates for the T3 DNA ligase. This method has shown high tolerance to chemical modifications, flexibility, and overall efficiency, thereby granting access to a broad range of modified oligonucleotides of different lengths (20 → 120 nucleotides). We have applied this method to the synthesis of clinically relevant antisense drugs and ultramers containing diverse modifications. Furthermore, the designed chemoenzymatic approach has great potential for diverse applications in therapeutics and biotechnology.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Overview of existing synthetic protocols and of the co-polymerization of shortmer fragments.
A Common chemical modifications in oligonucleotides. B Overview of solid phase synthesis. C Schematic representation of enzymatic synthesis. D Alternative chemoenzymatic synthesis based on convergent fragment assembly by ligation. D1 Reported assembled single fragment ligation. D2 Reported overlapping stepwise multiple fragment ligation. D3 Our approach is based on self-assembled one-pot multiple-fragment ligation.
Fig. 2
Fig. 2. DNA ligations of repetitive sequences.
Scope and limitations. Gel electrophoresis images PAGE (20%): A Screening of commercial DNA ligases. B–E DNA ligation reactions of shortmer (5 nt) fragments 1–23 using T3 DNA ligase, 5’-FAM labeled DNA primer P1 (22 nt) on complementary DNA templates of different lengths (1x-22 nt, 2x-27 nt, 4x-47 nt, 14x-87 nt). (-) – negative control in the absence of DNA ligase.
Fig. 3
Fig. 3. DNA ligations of diverse sequences. Scope and limitations.
Gel electrophoresis images PAGE (20%): A Study of stepwise ligation of natural shortmers (5 nt) to DNA primer P1 (22 nt) on DNA template 4x-mix (37 not). B Ligation of diverse natural and modified (LNA, phosphorothioate) shortmers (5 nt) to DNA primer P1 (22 not). C Ligation of diverse natural and modified (LNA, phosphorothioate) shortmers (5 nt) to DNA primer P2 (6 nt) on DNA template 4x-mixS (26 not). D Ligation of diverse natural shortmers of different length (5–10 not). E Ligation of diverse natural and variously modified (2’-OMe, 2’-MOE, phosphorothioate, NB) shortmers (5 not); (-) – negative control in the absence of DNA ligase. F Ligation of diverse natural and variously modified (LNA, RNA, RNA-phosphorothioate) shortmers (5 nt); (-) – negative control in the absence of DNA ligase.
Fig. 4
Fig. 4. Synthesis of long and modified oligonucleotides.
Gel electrophoresis images Agarose (4%). A DNA ligations of multiple diverse shortmer fragments (5 nt) F1–F18 to DNA primer P1 on ultramer DNA template TU (107 nt). B DNA ligation of long fragments VL-IL (79 nt) on shorter DNA template TL (31 nt).
Fig. 5
Fig. 5. Synthesis of therapeutic antisense oligonucleotide and its conjugates.
A Synthesis of Fomivirsen. B Synthesis of Fomivirsen FAM conjugate. C Synthesis of Fomivirsen FG (NH2, N3) conjugates. D Synthesis of Fomivirsen Fatty acid (C16:0) conjugate. E Synthesis of Fomivirsen Cholesterol-TEG conjugate. F Synthesis of Fomivirsen LNA gapmer analog.
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