A 15-base acridine-conjugated oligodeoxynucleotide forms triplex DNA with its IL-2R alpha promoter target with greatly improved avidity.
Attachment of 6,9-diamino-2-methoxyacridine to the 5' end of a purine-rich oligodeoxynucleotide targeting a 15 bp oligopurine oligopyrimidine stretch in the promoter region of the interleukin-2 receptor alpha chain (IL-2R alpha) gene results in an approximately 500-fold increase in its triplex forming avidity as determined by both band shift assay and DMS footprinting (Kd lowered from 2.5 microM to 5 nM). This oligonucleotide participates in Mg(2+)-dependent three-stranded DNA formation in which it is oriented antiparallel relative to the purine strand of the target duplex as determined by acridine moiety sensitized photoreactivity with the target duplex DNA. The oligonucleotides used in these studies were synthesized with a 3-amino-2-hydroxypropyl group at the 3' end to protect against exonucleolytic degradation for future in vivo applications. The 3'-amino group underwent partial removal, probably during the NaOH deprotection step. Both the 3'-amino and the 3'-free forms of the oligo have the same binding avidity and specificity. The interaction of the third strand with its target is sequence specific and can be essentially abolished by a point G-->T transversion 4 bases away from the 3' end of the target oligopurine block or severely reduced by other mutations within the target duplex. Thus, the attachment of the acridine moiety to the 5' end of the oligonucleotide does not seem to substantially compromise the sequence specificity of binding. Additionally, the oligonucleotide composed of G and A nucleotides was found to be superior to the oligonucleotide containing G and T residues since the difference in avidity of binding to the same target site was 17-fold.
Klysik
J
J
Veterans Affairs Medical Center Research Center on AIDS and HIV Infections, Houston, Texas, USA.
Kinsey
B M
BM
Hua
P
P
Glass
G A
GA
Orson
F M
FM
D000166:Acridines / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D007364:Intercalating Agents / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D015375:Receptors, Interleukin-2 / Q000235:genetics*
D000166:Acridines; D007364:Intercalating Agents; D009838:Oligodeoxyribonucleotides; D015375:Receptors, Interleukin-2; D004247:DNA; D011188:Potassium