Triplex-DNA stabilization by hydralazine and the presence of anti-(triplex DNA) antibodies in patients treated with hydralazine

Abstract

Hydralazine is an antihypertensive drug that elicits andti-nuclear antibodies in patients as an adverse effect. We investigated the ability of hydralazine to promote/stabilize the triplex DNA form of poly(dA).2poly(dT). Under conditions of low ionic strength, the polynucleotide melted as a double helix with a melting temperature (Tm) of 55.3 degrees C. Hydralazine destabilized this duplex form by reducing its Tm to 52.5 degrees C. Spermidine (2.5 microM), a natural polyamine, provoked the triplex form of poly(dA)-.2poly(dT) with two melting transitions, Tm1 of 42.8 degrees C corresponding to triplex-->duplex+single-stranded DNA and Tm2 of 65.4 degrees C, corresponding to duplex melting. Triplex DNA thus formed in the presence of spermidine was further stabilized by hydralazine (250 microM) with a Tm1 of 53.6 degrees C. A similar stabilization effect of hydralazine was found on triplex DNA formed in the presence of 5 mM Mg2+. CD spectra revealed conformational perturbations of DNA in the presence of spermidine and hydralazine. These results support the hypothesis that hydralazine is capable of stabilizing unusual forms of DNA. In contrast with the weak immunogenicity of DNA in its right-handed B-DNA conformation, these unusual forms are immunogenic and have the potential to elicit anti-DNA antibodies. To test this possibility, we analysed sera from a panel of 25 hydralazine-treated patients for anti-(triplex DNA) antibodies using an ELISA. Our results showed that 72% of sera from hydralazine-treated patients contained antibodies reacting toward the triplex DNA. In contrast, there was no significant binding of normal human sera to triplex DNA. Taken together our data indicate that hydralazine and related drugs might exert their action by interacting with DNA and stabilizing higher-order structures such as the triplex DNA.

Structural specificity effects of trivalent polyamine analogues on the stabilization and conformational plasticity of triplex DNA

“…In the absence of spermidine, the CD spectrum of poly(dA).2poly(dT) had a negative band at 250 nm and three positive bands at 220, 260 and 280 nm. These spectral features are similar to that previously reported for the duplex form of this polynucleotide [15,16,27]. In the presence of low concentrations of spermidine, triplex formation was indicated by an inversion of the positive peak centred at 220 nm with a new negative peak at 210 nm (inset).…”

“…These data demonstrate that the triplex form of poly(dA).-2poly(dT) is conformationally ultrapolymorphic and assumes a variety of conformations in the presence of spermidine. There was no major conformational alteration in the CD spectrum of poly(dA).poly(dT) duplex in the presence of spermidine under the same buffer conditions as used in the present study [27].…”

Unraveling the structure and biological functions of RNA triple helices

“…Antibodies have the ability to recognize a specific molecule with high affinity. Although nucleic acids are often times weakly immunogenic, several antibodies against triple helices have been reported in the literature, including one that can discriminate between poly(U•A‐U) versus poly(U•dA‐dT) triple helices (Rainen & Stollar, 1977; Stollar & Raso, 1974; Thomas, Seibold, Adams, & Hess, 1995). Therefore, a straightforward approach to map RNA triple helices would be to perform an immunopurification using an anti‐RNA triple helix antibody followed by deep sequencing to identify bound nucleic acids.…”

Biotransformation of Hydrazine Dervatives in the Mechanism of Toxicity

“…It is suggested that these modified bases are antigenic and are responsible for lupus [34]. Hydralazine has been shown to intercalate into DNA [35, 36] and stabilize triplex-DNA in the presence of Mg 2+ and sperimidine. Interestingly, this triplex-DNA elicited an immunological response, and patients contained antibodies that reacted with this unusual form of DNA [36].…”

“…Hydralazine has been shown to intercalate into DNA [35, 36] and stabilize triplex-DNA in the presence of Mg 2+ and sperimidine. Interestingly, this triplex-DNA elicited an immunological response, and patients contained antibodies that reacted with this unusual form of DNA [36]. Therefore, it is suggested that formation of antibodies to such triplex DNA may, in part, be responsible for lupus formation.…”