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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. Nucleic acids are key elements in numerous applications such as therapeutics and nanotechnology. However, the synthesis of long and modified oligonucleotides remains challenging and alternative, biocatalytic approaches are needed. Here, the authors discuss recent progress in the controlled enzymatic synthesis of oligonucleotides.

Recent progresses in organic chemistry and molecular biology have allowed the emergence of numerous new applications of nucleic acids that markedly deviate from their natural functions. Particularly, DNA and RNA molecules—coined aptamers—can be brought to bind to specific targets with high affinity and selectivity. While aptamers are mainly applied as biosensors, diagnostic agents, tools in proteomics and biotechnology, and as targeted therapeutics, these chemical antibodies slowly begin to be used in other fields. Herein, we review recent progress on the use of aptamers in the construction of smart DNA origami objects and MRI and PET imaging agents. We also describe advances in the use of aptamers in the field of neurosciences (with a particular emphasis on the treatment of neurodegenerative diseases) and as drug delivery systems. Lastly, the use of chemical modifications, modified nucleoside triphosphate particularly, to enhance the binding and stability of aptamers is highlighted.

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. Chemical modification of DNA is a common strategy to improve the properties of oligonucleotides. Herein, the authors report a robust method based on the template-dependent DNA ligation of shortmer fragments by DNA ligases, which is compatible with numerous chemical modifications and applicable to short (~20 nt) and longer (<100 nt) sequences.

Research on enzymatic RNA synthesis has long been eclipsed by work on DNAbut a new method provides a leap forward for RNA. [AU: OK?].The growing demand for nucleic-acid drugs and vaccines has underscored the dire need for improved technologies to produce long DNA and RNA sequences equipped with chemical modifications. Unlike its DNA cousin, surprisingly little effort has been devoted to the manufacture of therapeutic RNA oligonucleotides longer than short sequences (20-80 nt) used for siRNAs and miRNAs. Writing in Nature Biotechnology, Wiegand et al. 1 now report an alternative, robust approach for template-independent enzymatic synthesis of RNA oligonucleotides, both natural and with therapeutically relevant modifications. Using a mutant polymerase, they demonstrate controlled enzymatic synthesis of chemically modified RNA oligonucleotides of up to 10 nt. Their strategy has the potential to surpass existing methods

Research on enzymatic RNA synthesis has long been eclipsed by work on DNA—but a new method provides a leap forward for RNA.

Many potent antibiotics fail to treat bacterial infections due to emergence of drug-resistant strains. This surge of antimicrobial resistance (AMR) calls in for the development of alternative strategies and methods for the development of drugs with restored bactericidal activities. In this context, we surmised that identifying aptamers using nucleotides connected to antibiotics will lead to chemically modified aptameric species capable of restoring the original binding activity of the drugs and hence produce active antibiotic species that could be used to combat AMR. Here, we report the synthesis of a modified nucleoside triphosphate equipped with a vancomycin moiety on the nucleobase. We demonstrate that this nucleotide analogue is suitable for polymerase-mediated synthesis of modified DNA and, importantly, highlight its compatibility with the SELEX methodology. These results pave the way for bacterial-SELEX for the identification of vancomycin-modified aptamers.

Oligonucleotides are biopolymers that can be easily modified at various locations. Thereby, the attachment of metal complexes to nucleic acid derivatives has emerged as a common pathway to improve the understanding of biological processes or to steer oligonucleotides towards novel applications such as electron transfer or the construction of nanomaterials. Among the different metal complexes coupled to oligonucleotides, ruthenium complexes, have been extensively studied due to their remarkable properties. The resulting DNA-ruthenium bioconjugates have already demonstrated their potency in numerous applications. Consequently, this review focuses on the recent synthetic methods developed for the preparation of ruthenium complexes covalently linked to oligonucleotides. In addition, the usefulness of such conjugates will be highlighted and their applications from nanotechnologies to therapeutic purposes will be discussed.

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. Controlled enzymatic DNA synthesis represents an alternative synthetic methodology that circumvents the limitations of traditional soild-phase synthesis. Here, the authors explore the use of 3’-phosphate as a transient protecting group for the controlled enzymatic synthesis of DNA and XNA oligonucleotides.

Decoration of aptamers with chemical modifications at the level of nucleobases grants access to alternative binding modes, which often result in improved binding properties. Most functional groups involved in such endeavours mimic the side chains of amino acids or are based on sp 2 -dominated moieties. While this approach has met undeniable success, trends in modern drug discovery seem to favor sp 3 -rich compounds over aromatic derivatives. Here, we report the use of a nucleotide modified with the three-dimensional, highly flexible cyclooctatetraene carboxylate (COTc). This nucleotide was engaged in an SELEX experiment against the biomarker PvLDH. Tightly binding aptamers were identified, which displayed dissociation constants in the low nM range, representing a significant improvement compared to previously identified cubamers. These modified aptamers clearly underscore the usefulness of COTc as a bioisostere replacement of aromatic moieties not only in small compounds but also in functional nucleic acids. Chemical modifications of aptamers can enhance binding properties, however, the design of suitable functional groups remains challenging. Here, the authors introduce cyclooctatetraene (COT)-modified nucleotides in SELEX experiments, identifying COT-aptamers with low nM dissociation constants, highlighting the potential of three-dimensional ligands in aptamer discovery.

BsmI, a thermophilic Type IIS restriction endonuclease from Bacillus stearothermophilus , presents a unique structural composition, housing two distinct active sites within a single monomer. Recognition of the non-symmetrical 5’-GAATGC-3’ sequence enables precise cleavage of the top and bottom DNA strands. Synthetic biology interventions have led to the transformation of BsmI into Nb.BsmI, a nicking endonuclease. Here we introduce Nt*.BsmI, tailored for top-strand cleavage, which is inactive on standard double-stranded DNA, but active on bottom-strand nicked DNA, suggesting a sequential cleavage mechanism. Crystallographic structures of pre- and post-reactive complexes with cognate DNA show one major conformational change, a retractable loop possibly governing sequential active site accessibility. The x-ray structures reveal the position of the divalent metal ions in the active sites and the DNA:protein interactions, while the models predicted by Alphafold3 are incorrect. This comprehensive structural and functional study lays a foundation for rational enzyme redesign and potential applications in biotechnology. Functional and structural studies of restriction endonuclease BsmI provide a rationale for its substrate specificity and uncover its peculiar enzymatic mechanism in two sequential steps, opening possible applications through rational engineering