Tricyclo-DNA containing oligonucleotides as steric block inhibitors of human immunodeficiency virus type 1 tat-dependent trans-activation and HIV-1 infectivity.
Replication of human immunodeficiency virus type 1 (HIV-1) is controlled by a variety of viral and host proteins. The viral protein Tat acts in concert with host cellular factors to stimulate transcriptional elongation from the viral long terminal repeat (LTR) through a specific interaction with a 59-residue stem-loop RNA known as the trans-activation responsive element (TAR). Inhibitors of Tat-TAR recognition are expected to block transcription and suppress HIV-1 replication. In previous studies, we showed that 2'-O-methyl (OMe) oligonucleotide mixmers containing locked nucleic acid (LNA) residues are powerful steric block inhibitors of Tat-dependent trans-activation in a HeLa cell reporter system. Here we compare OMe/LNA mixmer oligonucleotides with oligonucleotides containing tricyclo-DNAs and their mixmers with OMe residues in four different assays: (1) binding to the target TAR RNA, (2) Tat-dependent in vitro transcription from an HIV-1 DNA template directed by HeLa cell nuclear extract, (3) trans-activation inhibition in HeLa cells containing a stably integrated firefly luciferase reporter gene under HIV-1 LTR control, and (4) an anti-HIV beta-galactosidase reporter assay of viral infection. Although tricyclo-DNA oligonucleotides bound TAR RNA more weakly, they were as good as OMe/LNA oligonucleotides in suppressing in vitro transcription and trans-activation in HeLa cells when delivered by cationic lipid. No inhibition of in vitro transcription and trans-activation in HeLa cells was observed for tricyclo-DNA/OMe mixmers, even though their affinities to TAR RNA were strong and their cell distributions did not differ from oligonucleotides containing all or predominantly tricyclo-DNA residues. Tricyclo-DNA 16-mer showed sequence-specific inhibition of beta-galactosidase expression in an anti-HIV HeLa cell reporter assay.
Ivanova
Gabriela
G
Medical Research Council, Laboratory of Molecular Biology, Cambridge CB2 2QH, UK.
Reigadas
Sandrine
S
Ittig
Damian
D
Arzumanov
Andrey
A
Andreola
Marie-Line
ML
Leumann
Christian
C
Toulmé
Jean-Jacques
JJ
Gait
Michael J
MJ
D019380:Anti-HIV Agents / Q000378:metabolism / Q000494:pharmacology*; D001483:Base Sequence; D015967:Gene Expression Regulation, Viral / Q000187:drug effects*; D015696:Gene Products, tat / Q000037:antagonists & inhibitors* / Q000235:genetics; D017930:Genes, Reporter; D016325:HIV Long Terminal Repeat / Q000187:drug effects; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D009838:Oligodeoxyribonucleotides / Q000378:metabolism / Q000494:pharmacology*; D015533:Transcriptional Activation / Q000187:drug effects; D054322:tat Gene Products, Human Immunodeficiency Virus
D019380:Anti-HIV Agents; D015696:Gene Products, tat; D009838:Oligodeoxyribonucleotides; D054322:tat Gene Products, Human Immunodeficiency Virus; D008156:Luciferases