Comparison of triple helix formation by polypurine versus polypyrimidine oligodeoxynucleotides when conjugated to a DNA intercalator.
Biological applications of triplex forming oligonucleotides will require the development of oligomers with high avidity and specificity. We examined the binding enhancement resulting from intercalator conjugation to both parallel design (polythymidine T15) and antiparallel design (polypurine AG15, for binding a 15 base pair polypurine-polypyrimidine sequence in the IL-2R alpha gene enhancer) oligomers under various ionic strength and temperature conditions. Oligonucleotides were conjugated through a urea link to 6,9 diamino-3-methoxy acridine (to give T15C and AG15C). Intercalator conjugation dramatically enhanced the specific triplex binding avidity (Kd = 5 nM for AG15C and 275 nM for T15C at 25 degrees C, compared to 2 microM for AG15 and > 50 microM for T15 at 25 degrees C), without detectable binding to an inappropriate target sequence. Surprisingly, triplex formation with AG15C occurred at lower Mg2+ concentrations than with T15C. AG15 and AG15C showed rapid Mg2+ dependent self association, but not T15C or T15. T15C triplex formation occurred rapidly (completion in less than 4 min), while AG15C bound to its target sequence more slowly over 20-24 h. Thus, binding constants in the low nanomolar range are now achievable with intercalator conjugated polypurine antiparallel binding oligonucleotides, a prerequisite for biological applications of such agents.
Orson
F M
FM
Veterans Affairs Medical Center Research Center on AIDS and HIV Infections, Houston, TX, USA.
Klysik
J
J
Glass
G A
GA
Kinsey
B M
BM
D001483:Base Sequence; D002021:Buffers; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007364:Intercalating Agents / Q000737:chemistry*; D007376:Interleukin-2 / Q000235:genetics; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry*; D011687:Purines / Q000737:chemistry*; D011743:Pyrimidines / Q000737:chemistry*; D013696:Temperature
D002021:Buffers; D007364:Intercalating Agents; D007376:Interleukin-2; D009841:Oligonucleotides; D011687:Purines; D011743:Pyrimidines; D008274:Magnesium