Automated chemical synthesis of oligonucleotides is of fundamental importance for the production of primers for the polymerase chain reaction (PCR), for oligonucleotide-based drugs, and for numerous other medical and biotechnological applications. The highly optimised automised chemical oligonucleotide synthesis relies upon phosphoramidites as the phosphate precursors and one of the drawbacks of this technology is the poor bench stability of phosphoramidites. Here, we report on the development of an on-demand flow synthesis of phosphoramidites from their corresponding alcohols, which is accomplished with short reaction times, near-quantitative yields and without the need of purification before being submitted directly to automated oligonucleotide synthesis. Sterically hindered as well as redox unstable phosphoramidites are synthesised using this methodology and the subsequent couplings are near-quantitative for all substrates. The vision for this technology is direct integration into DNA synthesisers thereby omitting manual synthesis and storage of phosphoramidites.

Oligonucleotides are increasingly being used as a programmable connection material to assemble molecules and proteins in well‐defined structures. For the application of such assemblies for in vivo diagnostics or therapeutics it is crucial that the oligonucleotides form highly stable, non‐toxic, and non‐immunogenic structures. Only few oligonucleotide derivatives fulfil all of these requirements. Here we report on the application of acyclic l‐threoninol nucleic acid (aTNA) to form a four‐way junction (4WJ) that is highly stable and enables facile assembly of components for in vivo treatment and imaging. The aTNA 4WJ is serum‐stable, shows no non‐targeted uptake or cytotoxicity, and invokes no innate immune response. As a proof of concept, we modify the 4WJ with a cancer‐targeting and a serum half‐life extension moiety and show the effect of these functionalized 4WJs in vitro and in vivo, respectively.

Triplex forming oligonucleotides are used as a tool for gene regulation and in DNA nanotechnology. By incorporating artificial nucleica cids, target affinity and biological stabilitys uperior to that of naturalD NA mayb e obtained. This work demonstrates how ac himeric clamp consisting of acyclic( L)-threoninol nucleic acid (aTNA) and DNA can bind DNA and RNA by the formationof ah ighly stable triplex structure. The (L)-aTNA clamp is released from the targeta gain by the addition of ar eleasing strand in as trand displacement type of reaction. It is shown that the clamp efficientlyi nhibits Bsu and T7 RNA polymerase activity and that polymerase activity is reactivated by displacing the clamp. The clamp was successfully appliedt o the regulation of luciferase expression by reversible binding to the mRNA. When targeting as equence in the double stranded plasmid, 40 %d ownregulation of protein expression is achieved.[a] Dr.

Acyclic (l)-threoninol nucleic acids ((l)-aTNA) containing poly-cytosines are prepared and investigated at various pH values, revealing the formation of a highly stable structure at lower pH that have the characteristics of an i-motif.

Oligonucleotides are increasingly being used as a programmable connection material to assemble molecules and proteins in well‐defined structures. For the application of such assemblies for in vivo diagnostics or therapeutics it is crucial that the oligonucleotides form highly stable, non‐toxic, and non‐immunogenic structures. Only few oligonucleotide derivatives fulfil all of these requirements. Here we report on the application of acyclic l‐threoninol nucleic acid (aTNA) to form a four‐way junction (4WJ) that is highly stable and enables facile assembly of components for in vivo treatment and imaging. The aTNA 4WJ is serum‐stable, shows no non‐targeted uptake or cytotoxicity, and invokes no innate immune response. As a proof of concept, we modify the 4WJ with a cancer‐targeting and a serum half‐life extension moiety and show the effect of these functionalized 4WJs in vitro and in vivo, respectively.