Modulation of Cm/T, G/A, and G/T Triplex Stability by Conjugate Groups in the Presence and Absence of KCl

• Published in: Biochemistry (Easton) 1997-12, Vol.36 (48), p.14816-14826
• Main Authors: Gamper, Howard B, Kutyavin, Igor V, Rhinehart, Rebecca L, Lokhov, Sergei G, Reed, Michael W, Meyer, Rich B
• Format: Article
• Language: English
• Subjects: Base Composition; DNA - chemistry; DNA - drug effects; HLA-DQ Antigens - genetics; HLA-DQ beta-Chains; Humans; Nucleic Acid Conformation - drug effects; Nucleic Acid Denaturation - drug effects; Nucleic Acid Hybridization - drug effects; Oligodeoxyribonucleotides; Potassium Chloride - pharmacology
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi971339+

Apparent equilibrium association constants were determined by gel mobility shift analysis for triple strand formation between a duplex target containing a 21 base long A-rich homopurine run and several end-modified Cm/T (pyrimidine motif; Cm = 5-methylcytosine), G/A (purine motif), and G/T (purine-pyrimidine motif) triplex-forming oligonucleotides (TFOs). Incubations were carried out for 24 h at 37 °C in 20 mM HEPES, pH 7.2, 10 mM MgCl2, and 1 mM spermine. The purine motif triplex was the most stable (K a = 6.2 × 108 M-1) even though the TFO self-associated as a linear duplex. Conjugation of a terminal hexanol or cholesterol group to the G/A-containing TFO reduced triplex stability by 1.6- or 13-fold, whereas an aminohexyl group or intercalating agent (acridine or psoralen) increased triplex stability by 1.3- or 13-fold. These end groups produced similar effects in Cm/T and G/T triplexes, although the magnitude of the effect sometimes differed. Addition of 140 mM KCl to mimic physiological conditions decreased stability of the G/A triplex by 1900-fold, making it less stable than the Cm/T triplex. The inhibitory effect of KCl on G/A triplex formation could be partially compensated for by conjugating the TFO to an intercalating agent (30−350-fold stabilization) or by adding the triplex selective intercalator coralyne (1000-fold stabilization). Although the G/T triplex responded similarly to these agents, the stability of the Cm/T triplex was unaffected by the presence of coralyne and was only enhanced 1.4−2.8-fold when the TFO was linked to an intercalating agent. In physiological buffer supplemented with 40 μM coralyne, the G/A triplex (K a = 3.0 × 108 M-1) was more stable than the Cm/T and G/T triplexes by factors of 300 and 12, respectively.