Targeted inhibition of transcription elongation in cells mediated by triplex-forming oligonucleotides.
Triple-helix-forming oligonucleotides (TFOs) bind in the major groove of double-stranded DNA at oligopyrimidine small middle dotoligopurine sequences and therefore are candidate molecules for artificial gene regulation, in vitro and in vivo. We recently have described oligonucleotide analogues containing N3'-P5' phosphoramidate (np) linkages that exhibited efficient inhibition of transcription elongation in vitro. In the present work we provide conclusive evidence that np-modified TFOs targeted to the HIV-1 polypurine tract (PPT) sequence can inhibit transcriptional elongation in cells, either in transient or stable expression systems. The same constructs were used in transient expression assays (target sequence on transfected plasmid) and in the generation of stable cell lines (target sequence integrated into cellular chromosomes). In both cases the only distinguishable feature between the cellular systems is the presence of an insert containing the wild-type PPT/HIV-1 sequence, a mutated version with two mismatches, or the absence of the insert altogether. The inhibitory action induced by np-TFOs was restricted to the cellular systems containing the complementary wild-type PPT/HIV-1 target, and consequently can be attributed only to a triple-helix-mediated mechanism. As a part of this study we also have applied an imaging technique to quantitatively investigate the dynamics of TFO-mediated specific gene silencing in single cells.
Faria
M
M
Laboratoire de Biophysique, Muséum National d'Histoire Naturelle, Institut National de la Santé et de la Recherche Médicale U. 201-Centre National de la Recherche Scientifique UMR 8646, 43 rue Cuvier, 75005 Paris, France.
Wood
C D
CD
Perrouault
L
L
Nelson
J S
JS
Winter
A
A
White
M R
MR
Helene
C
C
Giovannangeli
C
C
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D005786:Gene Expression Regulation; D009841:Oligonucleotides / Q000378:metabolism*; D014158:Transcription, Genetic
D017931:DNA Primers; D009841:Oligonucleotides