title: A cytosine analog that confers enhanced potency to antisense oligonucleotides.
abstract: Antisense technology is based on the ability to design potent, sequence-specific inhibitors. The G-clamp heterocycle modification, a cytosine analog that clamps on to guanine by forming an additional hydrogen bond, was rationally designed to enhance oligonucleotide/RNA hybrid affinity. A single, context-dependent substitution of a G-clamp heterocycle into a 15-mer phosphorothioate oligodeoxynucleotide (S-ON) targeting the cyclin-dependent kinase inhibitor, p27(kip1), enhanced antisense activity as compared with a previously optimized C5-propynyl-modified p27(kip1) S-ON and functionally replaced 11 C5-propynyl modifications. Dose-dependent, sequence-specific antisense inhibition was observed at nanomolar concentrations of the G-clamp 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. A 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 RNase H-mediated cleavage and demonstrated increased in vitro binding affinity for its RNA target compared with conventional 15-mer S-ONs. Furthermore, incorporation 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-sensitive, automated synthesizer-compatible antisense S-ON modification that will have important applications in the elucidation of gene function, the validation of gene targets, and the development of more potent antisense-based pharmaceuticals.
authors: Gilead Sciences, 333 Lakeside Drive, Foster City, CA 94404, USA. mflanagan@sunesis-pharma.com
authors: W M
authors: WM
authors: Flanagan
authors: J J
authors: JJ
authors: Wolf
authors: P
authors: P
authors: Olson
authors: D
authors: D
authors: Grant
authors: K Y
authors: KY
authors: Lin
authors: R W
authors: RW
authors: Wagner
authors: M D
authors: MD
authors: Matteucci
mesh_terms: D000818:Animals; D001483:Base Sequence; D018797:Cell Cycle Proteins; D002460:Cell Line; D002522:Chlorocebus aethiops; D050760:Cyclin-Dependent Kinase Inhibitor p27; D003596:Cytosine / Q000031:analogs & derivatives*; D015195:Drug Design; D004791:Enzyme Inhibitors; D007668:Kidney; D008869:Microtubule-Associated Proteins / Q000037:antagonists & inhibitors / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D020319:Oligodeoxyribonucleotides, Antisense / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology*; D010078:Oxazines / Q000494:pharmacology; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics; D013329:Structure-Activity Relationship; D013873:Thionucleotides; D014162:Transfection; D025521:Tumor Suppressor Proteins
chemical_list: D018797:Cell Cycle Proteins; D004791:Enzyme Inhibitors; D008869:Microtubule-Associated Proteins; D020319:Oligodeoxyribonucleotides, Antisense; D010078:Oxazines; D012333:RNA, Messenger; D013873:Thionucleotides; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D003596:Cytosine; C039203:phenoxazine