Systematic Investigation of Tether Length and Phosphorus Configuration in Backbone Constrained Macrocyclic Nucleic Acids to Modulate Binding Kinetics for RNA.

We recently described a chemical strategy to pre-organize a trinucleotide subunit in a conformation suitable for Watson-Crick base pairing for modulating the binding kinetics of single-stranded oligonucleotides (ONs) using bis-phosphonate esters bridging hydrocarbon tethers to provide 11- and 15-membered macrocyclic analogues. In this manuscript, we describe the synthesis of all eight P-stereoisomers of macrocyclic 12-, 13-, 14-, and 16-membered hydrocarbon-bridged nucleotide trimers, their incorporation into ONs, and biophysical characterization of the modified ONs. The size of the macrocyclic tether and configuration at phosphorus had profound effects on hybridization kinetics.

The Combination of Mesyl-Phosphoramidate Inter-Nucleotide Linkages and 2'--Methyl in Selected Positions in the Antisense Oligonucleotide Enhances the Performance of RNaseH1 Active PS-ASOs.

Antisense oligonucleotides (ASOs) that mediate RNA target degradation by RNase H1 are used as drugs to treat various diseases. Previously we found that introduction of a single 2'--methyl (2'-OMe) modification in position 2 of the central deoxynucleotide region of a gapmer phosphorothioate (PS) ASO, in which several residues at the termini are 2'-methoxyethyl, 2' constrained ethyl, or locked nucleic acid, dramatically reduced cytotoxicity with only modest effects on potency. More recently, we demonstrated that replacement of the PS linkage at position 2 or 3 in the gap with a mesyl-phosphoramidate (MsPA) linkage also significantly reduced toxicity without meaningful loss of potency and increased the elimination half-life of the ASOs.

Backbone Hydrocarbon-Constrained Nucleic Acids Modulate Hybridization Kinetics for RNA.

The binding affinity of therapeutic oligonucleotides (ONs) for their cognate RNA is determined by the rates of association () and dissociation (). Single-stranded ONs are highly flexible and can adopt multiple conformations in solution, some of which may not be conducive for hybridization. We investigated if restricting rotation around the sugar-phosphate backbone, by tethering two adjacent backbone phosphonate esters using hydrocarbon bridges, can modulate hybridization kinetics of the modified ONs for complementary RNA.