A cytosine analog that confers enhanced potency to antisense oligonucleotides

Antisense technology is based on the ability to design potent, sequence-spe cific inhibitors. The G-clamp heterocycle modification, a cytosine analog t hat clamps on to guanine by forming an additional hydrogen bond, was ration ally designed to enhance oligonucleotide/RNA hybrid affinity. A single, con test-dependent substitution of a G-clamp heterocycle into a 15-mer phosphor othioate oligodeoxynucleotide (S-ON) targeting the cyclin-dependent kinase inhibitor, p27(kip1), enhanced antisense activity as compared with a previo usly optimized C5-propynyl-modified p2(kip1) S-ON and functionally replaced 11 C5-propynyl modifications. Dose-dependent, sequence-specific antisense inhibition was observed at nanomolar concentrations of the G-damp S-ONs, A single nucleotide mismatch between the G-clamp S-ON and the p27(kip1) mRNA reduced the potency of the antisense ON by five-fold. ii 2-base-mismatch S- ON eliminated antisense activity, confirming the sequence specificity of G- clamp-modified S-ONs, The G-clamp-substituted p27(kip1) S-ON activated RNas e H-mediated cleavage and demonstrated increased in vitro binding affinity for its RNA target compared with conventional 15 mer S-ONs, Furthermore, in corporation of a single G-clamp modification into a previously optimized 20 -mer phosphorothioate antisense S-ON targeting c-raf increased the potency of the S-ON 25-fold. The G-clamp heterocycle is a potent, mismatch-sensitiv e, automated synthesizer-compatible antisense S-ON modification that will h ave important applications in the elucidation of gene function, the validat ion of gene targets, and the development of more potent antisense-based pha rmaceuticals.