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

Autor: Howard Gamper, Rich B. Meyer, Michael W. Reed, Igor V. Kutyavin, Rebecca L. Rhinehart, Sergei G. Lokhov
Rok vydání: 1997
Předmět:
Zdroj: Biochemistry. 36:14816-14826
ISSN: 1520-4995
0006-2960
DOI: 10.1021/bi971339+
Popis: 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 C(m)/T (pyrimidine motif; C(m) = 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 degrees C in 20 mM HEPES, pH 7.2, 10 mM MgCl2, and 1 mM spermine. The purine motif triplex was the most stable (Ka = 6.2 x 10(8) 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 C(m)/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 C(m)/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 C(m)/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 microM coralyne, the G/A triplex (Ka = 3.0 x 10(8) M-1) was more stable than the C(m)/T and G/T triplexes by factors of 300 and 12, respectively.
Databáze: OpenAIRE
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