title: Steric inhibition of human immunodeficiency virus type-1 Tat-dependent trans-activation in vitro and in cells by oligonucleotides containing 2'-O-methyl G-clamp ribonucleoside analogues.
abstract: We report the synthesis of a novel 2'-O-methyl (OMe) riboside phosphoramidite derivative of the G-clamp tricyclic base and incorporation into a series of small steric blocking OMe oligonucleotides targeting the apical stem-loop region of human immunodeficiency virus type 1 (HIV-1) trans- activation-responsive (TAR) RNA. Binding to TAR RNA is substantially enhanced for certain single site substitutions in the centre of the oligonucleotide, and doubly substituted anti-TAR OMe 9mers or 12mers exhibit remarkably low binding constants of <0.1 nM. G-clamp-containing oligomers achieved 50% inhibition of Tat-dependent in vitro transcription at approximately 25 nM, 4-fold lower than for a TAR 12mer OMe oligonucleotide and better than found for any other oligonucleotide tested to date. Addition of one or two OMe G-clamps did not impart cellular trans-activation inhibition activity to cellularly inactive OMe oligonucleotides. Addition of an OMe G-clamp to a 12mer OMe-locked nucleic acid chimera maintained, but did not enhance, inhibition of Tat-dependent in vitro transcription and cellular trans-activation in HeLa cells. The results demonstrate clearly that an OMe G-clamp has remarkable RNA-binding enhancement ability, but that oligonucleotide effectiveness in steric block inhibition of Tat-dependent trans-activation both in vitro and in cells is governed by factors more complex than RNA-binding strength alone.
authors.affiliation: Medical Research Council, Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Holmes
authors.forename: Andrey A
authors.initials: AA
authors.lastname: Arzumanov
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Gait
mesh_terms: D019380:Anti-HIV Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D015696:Gene Products, tat / Q000037:antagonists & inhibitors* / Q000235:genetics; D015497:HIV-1 / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008745:Methylation; D009843:Oligoribonucleotides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D009943:Organophosphorus Compounds / Q000737:chemistry; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism; D012263:Ribonucleosides / Q000138:chemical synthesis / Q000737:chemistry; D015533:Transcriptional Activation / Q000187:drug effects*; D054322:tat Gene Products, Human Immunodeficiency Virus
chemical_list: D019380:Anti-HIV Agents; D015696:Gene Products, tat; D009843:Oligoribonucleotides; D009943:Organophosphorus Compounds; D012367:RNA, Viral; D012263:Ribonucleosides; C434331:phosphoramidite; D054322:tat Gene Products, Human Immunodeficiency Virus; D004247:DNA