Triple helix formation: binding avidity of acridine-conjugated AG motif third strands containing natural, modified and surrogate bases opposed to pyrimidine interruptions in a polypurine target.
A critical issue for the general application of triple-helix-forming oligonucleotides (TFOs) as modulators of gene expression is the dramatically reduced binding of short TFOs to targets that contain one or two pyrimidines within an otherwise homopurine sequence. Such targets are often found in gene regulatory regions, which represent desirable sites for triple helix formation. Using intercalator-conjugated AG motif TFOs, we compared the efficacy and base selectivity of 13 different bases or base surrogates in opposition to pyrimidines and purines substituted into selected positions within a paradigm 15-base polypurine target sequence. We found that substitutions closer to the intercalator end of the TFO (positions 4-6) had a more deleterious effect on the dissociation constant (K d) than those farther away (position 11). Opposite T residues at position 11, 3-nitropyrrole or cytosine in the TFO provided adequate binding avidity for useful triplex formation (K ds of 55 and 110 nM, respectively). However, 3-nitropyrrole was more base selective than cytosine, binding to T >/=4 times better than to A, G or C. None of the TFOs tested showed avid binding when C residues were in position 11, although the 3-nitropyrrole-containing TFO bound with a K d of 200 nM, significantly better than the other designs. Molecular modeling showed that the 3-nitropyrrole.T:A triad is isomorphous with the A.A:T triad, and suggests novel parameters for evaluating new base triad designs.
Orson
F M
FM
The Veterans Affairs Medical Center, Department of Internal Medicine, Baylor College of Medicine, Building 109, Room 226, VAMC, 2002 Holcombe, Houston, TX 77030, USA. forson@bcm.tmc.edu
Klysik
J
J
Bergstrom
D E
DE
Ward
B
B
Glass
G A
GA
Hua
P
P
Kinsey
B M
BM
D000166:Acridines / Q000378:metabolism*; D000225:Adenine / Q000378:metabolism*; D005786:Gene Expression Regulation; D005821:Genetic Techniques; D006147:Guanine / Q000378:metabolism*; D007364:Intercalating Agents / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000138:chemical synthesis*; D011687:Purines / Q000378:metabolism*; D011743:Pyrimidines / Q000378:metabolism*; D011758:Pyrroles / Q000494:pharmacology
D000166:Acridines; D007364:Intercalating Agents; D009841:Oligonucleotides; D011687:Purines; D011743:Pyrimidines; D011758:Pyrroles; C094911:3-nitropyrrole; D006147:Guanine; D000225:Adenine