Anti-gene effect in live cells of AG motif triplex-forming oligonucleotides containing an increasing number of phosphorothioate linkages.
The murine Ki-ras promoter contains a unique polypurine--polypyrimidine [poly(R.Y)] sequence between -290 and -320 from the 3' boundary of exon phi. Previously we demonstrated triplex formation and transcription inhibition promoted by GT and AG oligonucleotides directed against this site [Alunni-Fabbroni et al. (1996) Biochemistry 35, 16361--16369]. In this work, we have investigated triplex formation and anti-gene activity of five 20-mer AG motif triplex-forming oligonucleotides specific for the Ki-ras poly(R.Y) target, derived from 5'-AGGGAGGGAGGAAGGGAGGG (20AG) by replacing an increasing number of phosphodiester linkages with phosphorothioate linkages (S(i)-20AG; i = 2, 3, 4, 5, 19). Electrophoretic mobility-shift experiments (EMSA) showed that four thioate oligonucleotides, S(i)-20AG (i = 2, 3, 4, 5), recognized the Ki-ras target and exhibited dissociation constants similar to that of 20AG: K(d) = 12 +/- 2 nM, while the all-thioate S(19)-20AG exhibited a K(d) of 128 +/- 15 nM. Moreover, the binding between the Ki-ras promoter and oligonucleotides S(i)-20AG (i = 2, 3, 4, 5, 19) was characterized by DMS/piperidine and DNase I footprinting experiments. We observed that the introduction in the phosphodiester oligonucleotide 20AG of sulfur atoms reduced its aggregation significantly and increased its nuclease resistance. Transient transfection experiments using preformed triplexes with a recombinant plasmid containing the reporter chloramphenicol acetyltransferase (CAT) gene under the control of Ki-ras promoter showed that oligonucleotides S(i)-20AG (i = 2, 3, 4, 5, 19) promote a strong inhibition of up to 75% of the CAT expression when compared with control Ki-ras unspecific oligonucleotides. Taken together, these data provide a guideline for designing triplex-forming effector molecules capable of controlling Ki-ras expression in vivo.
Cogoi
S
S
Department of Biomedical Sciences and Technologies, School of Medicine, University of Udine, Piazzale Kolbe 4, 33100 Udine, Italy.
Rapozzi
V
V
Quadrifoglio
F
F
Xodo
L
L
D000225:Adenine; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D018983:DNA Footprinting; D018900:Dinucleotide Repeats; D004591:Electrophoresis, Polyacrylamide Gel; D017930:Genes, Reporter; D011905:Genes, ras; D006147:Guanine; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000737:chemistry; D013237:Stereoisomerism; D013489:Suppression, Genetic; D013873:Thionucleotides / Q000737:chemistry* / Q000378:metabolism; D014162:Transfection
D009841:Oligonucleotides; D013873:Thionucleotides; C051802:triplex DNA; D006147:Guanine; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; D015719:Single-Strand Specific DNA and RNA Endonucleases; D000225:Adenine