Biosensor detection of triplex formation by modified oligonucleotides.
Due to the instability of DNA oligonucleotides in biological solutions, antisense or antigene therapies aimed at modulation of specific gene expression will most likely require the use of oligonucleotides with modified backbones. Here, we examine the use of a surface plasmon resonance biosensor (BIAcore) to compare triplex-directed binding of modified oligonucleotides targeted to a region of the murine c-myc promoter. We describe optimization of experimental conditions to minimize nonspecific interactions between the oligonucleotides and the sensor chip surface, and the limitations imposed by certain backbones and sequence types. The abilities of pyrimidine oligonucleotides with various modified backbones to form specific triple helices with an immobilized hairpin duplex were readily determined using the biosensor. Modification of the third-strand oligonucleotide with RNA or 2(')-O-methyl RNA was found to enhance triplex formation, whereas phosphorothioate or phosphotriester substitutions abrogated it. A comparison of these results to DNase I footprinting experiments using the same oligonucleotides showed complete agreement between the two sets of data.
Department of Medicine and James Graham Brown Cancer Center, University of Louisville, Louisville, KY, USA.
Paula J
PJ
Bates
James F
JF
Reddoch
Pintusorn
P
Hansakul
Amy
A
Arrow
Roderic
R
Dale
Donald M
DM
Miller
D004247:DNA / Q000032:analysis* / Q000138:chemical synthesis* / Q000737:chemistry; D018983:DNA Footprinting; D003850:Deoxyribonuclease I / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009841:Oligonucleotides / Q000737:chemistry*; D011743:Pyrimidines / Q000378:metabolism; D020349:Surface Plasmon Resonance / Q000379:methods*
D009841:Oligonucleotides; D011743:Pyrimidines; C051802:triplex DNA; D004247:DNA; D003850:Deoxyribonuclease I; C030986:pyrimidine