Synthesis and structure determination of phosphonate oligonucleotides

Synthetic DNA and RNA-based oligonucleotides have opened new frontiers in therapy owing to their ability to interfere with gene expression (antisense, siRNA) and bind a wide spectrum of targets (aptamers). However, such nucleic acid therapeutics do not have a clinical potential due to their inherent structure made of repeating 3’-5’-phosphodiester bonds, which renders them prone to fast chemical and enzymatic degradation under physiological conditions. Therefore, we propose to investigate artificial oligonucleotides (XNAs) containing in their backbone a P-C bond instead of the natural P-O bond (phosphonate XNAs) as more stable nucleic acid alternatives. In the initial stage of this project, the synthesis of suitably phosphorylated precursors of nucleoside phosphonates, which are necessary for the solid phase synthesis of modified oligomers, will be performed. Two types of chemistries will be explored including 3-hydroxy-2-(phosphonomethoxy)propyl (HPMP) and phosphonomethylthreosyl (PMT) monomers, and selected sequences comprising these building blocks will be prepared in large amounts. The second part of the project will focus on the structural characterization of phosphonate oligonucleotide homo- and heteroduplexes (with DNA) using solution 1D and 2D NMR experiments. Knowledge on high-resolution structures of these phosphonate nucleic acids are essential to guide the in silico design of XNA processing enzymes that are needed for XNA aptamer selection (XNA-SELEX).