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

摘要

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 ～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.