Antisense oligonucleotides with increased binding affinity for RNA targets will be developed using carbocyclic nucleoside modifications. The natural modified nucleoside pseudouridine (psi) will be invested in the context of a deoxyribonucleotide and a 2'- methyl ribonucleotide in gapmer phosphorothioate antisense oligonucleotides. The chemical preparation of phosphoramidites will follow literature precedent. Gapmer oligonucleotides targeted against C-raf kinase message with psi as a deoxy residue will be prepared, along with oligomers having 2'-O- methyl psi in the wings. The affinity of oligonucleotides for RNA targeted will be measured, along with their ability to support RNAse H- mediated cleavage. Modified and control oligonucleotides will be incorporated into cell systems, and the biological advantage gained from incorporation of psi will be determined.
Commercialization of antisense therapeutics against many potential target diseases is limited by undesirable properties of the current generation of compounds. Identification of modifications which enhance affinity, or improve biophysical properties would provide cost reduction via shorter sequences of higher specificity in vivo.
