(Received for publication, October 11, 1996, and in revised form, January 14, 1997)
From the Department of Chemistry-Biology, University of Québec at Trois-Rivières, C.P. 500 TR, Québec, Canada G9A 5H7
Diethylstilbestrol (DES), a synthetic estrogen, is known to be a carcinogen in human and in animals. This study was designed to examine the interaction of DES with yeast RNA in aqueous solution at physiological pH with drug/RNA-phosphate (P) molar ratios of 1/80, 1/40, 1/20, 1/10, 1/4, and 1/2. Fourier transform infrared (FTIR) difference spectroscopy was used to determine the drug binding mode, the binding constant, the sequence selectivity, and RNA secondary structure in the RNA·DES complexes.
Spectroscopic evidence showed that at low drug concentration (1/80 and
1/40), DES is intercalating through both Gua-Cyt and Ade-Urd base pairs
with minor interaction with the backbone PO2 group
(external binding). The calculated binding constant of K ~ 8.5 × 104 M1 at a drug
concentration of 3.12 × 10
4 M shows
that DES is a weaker intercalator than those of the methylene blue,
acridine orange, and ethidium bromide. At high drug content (r > 1/40, where r represents the
DES/RNA-phosphate molar ratio), a partial helix destabilization occurs
with no alteration of RNA conformation upon drug complexation. However,
a comparison with DNA·DES complexes showed that drug intercalation
causes major reduction of the B-DNA structure in favor of A-DNA with no
participation of the backbone PO2 group in the DES·DNA
complexation.
DES1 (see Structure
1), a synthetic estrogen, is known to be a carcinogen
both in human and in animals (1, 2). It was used world wide from the
1940s until 1970s to prevent miscarriages in women, while at the same
time, it was found to cause cancer in experimental animals. Although
DES application has been banned for pregnancy, it remains in use for
other clinical purposes such as estrogen replacement therapy for
hormone deficiency. The DES-induced carcinogenesis has been considered
to be due to the high hormonal potency of this synthetic estrogen and
possible DES·DNA adduct formation in vivo (3).
Radioactively labeled DES was found to bind DNA in vivo
(3-5) and in vitro (6), but the nature of complexation with
nucleic acids could not be clarified in these investigations. The major
problem in defining the nature of drug-DNA interaction was mainly
related to the instability of DNA·DES and DNA·DESQ complexes
(diethylstilbestrol-4,4"-quinone, DESQ, a metabolic intermediate
product derived from DES oxidation) formed in vivo and
in vitro (6, 7). Similarly, the possibility of DNA
intercalate formation with both DES and DESQ was not included (7, 8).
It has also been suggested that DES-DNA adduct formation may occur
under oxidative stress (9, 10). However, a number of biological and
biochemical effects of DES were noted, which depended on metabolic
activation of stilben and are commonly associated with genotoxic
activity (11). Since the DES-induced carcinogenesis is related to its
complexation with nucleic acids (2), the structural analysis of DES
complexes with DNA and RNA and their constituents has major biological
importance. Our recent structural characterization of DES·DNA
complexes showed that DES is a weak intercalator with affinity toward
Ade-Thy-rich region (12). However, at high drug concentration, a
partial helix opening occurs with major reduction of B-DNA structure in
favor of A-DNA (12). The present study is also designed to investigate
the interaction between RNA and DES in vitro and to
establish correlations between spectral changes and drug binding mode,
binding constant sequence selectivity, RNA stability, and conformation,
using FTIR difference spectroscopy. In recent years, we have used
vibrational spectroscopy (infrared and Raman) for the structural
characterization of several DNA·drug (12-14), DNA·carbohydrate
(15), DNA·cation (16, 17), and protein complexes (18). We believe
FTIR spectroscopy can also be used here to obtain structural
information regarding the nature of DES-RNA interaction and the effect
of drug complexation on the biopolymer secondary structure in aqueous
solution.
Structure 1.
In this work, FTIR difference spectroscopy is applied to study the interaction between yeast RNA and DES in water-ethanol solution at physiological pH, with drug/RNA-phosphate molar ratios of 1/80 to 1/2. Evidence for the drug binding mode, sequence preference, RNA secondary structure, and helix stability is provided. Furthermore, comparisons were made with those of the corresponding DNA·DES complexes and the results are reported here.
Yeast RNA sodium salt was purchased from Sigma and used as supplied. DES was from Sigma and recrystallized from ethanol/water mixture. Other chemicals were reagent grade and used without further purification.
Preparation of Stock SolutionSodium-RNA was dissolved to 2% (w/w, 0.05 M phosphate) in 0.05 M NaCl and 1 mM sodium cacodylate solution (pH 7.30) at 5 °C with occasional stirring. DES solution of 0.3-12 mM was also prepared in ethanol (DES is not soluble in water). Mixtures of drug and RNA were prepared by adding DES solution dropwise to RNA solution with constant stirring to give the desired drug/RNA molar ratios of 1/80, 1/40, 1/20, 1/10, 1/4, and 1/2 at a final RNA concentration of 1% w/w or 0.025 M RNA-phosphate. Solution pH was 7.30-6.80, and the infrared spectra were recorded 4 h after initial mixing of drug and RNA solution. The infrared spectra of DES·RNA complexes with r > 1/2 could not be recorded in solution due to precipitation.
Infrared spectra were recorded on a BOMEM DA3-0.02 Fourier transform
infrared spectrometer equipped with nitrogen-cooled HgCdTe detector and
a KBr beam splitter. The solution spectra were taken on AgBr windows
with spectral resolution of 2-4 cm1 and 100-500 scans.
Water subtraction was carried out as in our previous report (16). A
good water subtraction was achieved by a flat base line around 2200 cm
1, where the H2O combination mode is
located (19). The difference spectra ((RNA solution + DES solution) - (RNA solution)) were produced, using the RNA band at 913 cm
1 as internal reference. This band, due to
ribose-phosphate vibration exhibits no spectral changes (intensity or
shifting) upon drug complexation, and it is cancelled on spectral
subtraction. The difference spectra contain several positive and
negative derivative features in the region of 1800-600
cm
1, whose amplitudes are less than 20% of the original
peaks, with an estimated error of ± 5% (absorbance). Several
positive features in the difference spectra of DES·RNA complexes are
coming from DES vibrations and are not due to RNA vibrational mode
(properly labeled).
The intensity ratios of several RNA in-plane vibrations related to Ade-Urd, Gua-Cyt, and the backbone PO2 groups were measured as a function of drug concentration with error of ± 5%. These intensity ratio variations were used to quantitatively measure the amounts of drug base and drug-PO2 bindings. The detailed intensity ratio measurements and spectral manipulations are described in our previous publication (16).
The calculation of the binding constant was also carried out as reported for other drug complexes with DNA, RNA, and mononucleotides (20-23). By assuming that there is only one type of interaction (intercalation) between drug and RNA molecule, Equations 1 and 2 can be established.
![]() |
(Eq. 1) |
![]() |
(Eq. 2) |
At low drug concentration
(r = 1/80 and 1/40), DES (Structure 1)
intercalates in the Gua-Cyt and Ade-Urd-rich regions. Evidence for this
comes from minor intensity increase (10%) of the RNA in-plane
vibrations at 1698 cm1 (Gua, Urd) and 1654 cm
1 (Urd, Gua, Ade, Cyt) (24-29). The increase in the
intensity was associated also with the shift of the band at 1698 cm
1 toward a lower frequency at 1695 cm-1,
while the band at 1654 cm
1 exhibited no shifting upon DES
complexation (Figs. 1 and 2). Similarly,
the mainly adenine band at 1608 cm
1 (19) gained intensity
(20%) on drug interaction (Fig. 2). The positive derivative features
at 1690-1689 cm
1 (Gua, Urd), 1656-1650
cm
1 (Urd, Gua, Ade, Cyt), 1610-1608 cm
1
(Ade), and 1250-1245 cm
1 (PO2) in the
difference spectra of DES·RNA complexes are arising from the increase
in the intensity of the RNA bands at 1698, 1654, 1608, and 1244 cm
1 (Figs. 1 and 2, r = 1/80 and 1/40).
The observed spectral changes are related to drug intercalation with
both Gua-Cyt and Ade-Urd base pairs. However, at this stage, the drug
intercalation with the Gua-Cyt bases is larger than that of the
Ade-Urd-rich region. The calculated binding constant K ~ 8.5 × 104 M
1 obtained at a
drug concentration of 3.12 × 10
4 M is
indicative of DES being a weak intercalator. The distributions of drug
around DNA bases were about 35% with the Gua-Cyt and 25% with Ade-Urd
base pairs (Fig. 3). The RNA backbone PO2
anti-symmetric band at 1244 cm
1 (24, 26) also gained
intensity (10%) upon drug complexation (Fig. 2). This is indicative of
some degree of DES-PO2 interaction (external binding) in
these DES·RNA complexes. However, the amount of drug-phosphate
binding is less than 20% (Fig. 3). Similar increases in the intensity
of DNA in-plane vibrations were observed in the Raman spectra of
several porphyrins and their metal derivatives, intercalated in the
Ade-Thy- or Gua-Cyt-rich regions of the synthetic and native DNAs (30,
31). DES intercalation with DNA resulted also in major spectral changes
(intensity increase and shifting) of several DNA in-plane vibrations,
related to the Ade-Thy and Gua-Cyt base pairs (12). The possibility of
the external binding with the Ade-Thy, Gua-Cyt, or PO2
donor groups have also been examined for several DNA intercalators
(31-34).
The band at 1488 cm1 related mainly to the cytosine
vibrations (24) exhibited no major intensity variations or shifting
upon DES interaction (Fig. 2). This is indicative of less perturbation of cytosine bases in DES·RNA complex formation.
A comparison with the infrared spectra of other strong DNA and RNA
intercalators, such as ethidium bromide, acridine orange, and methylene
blue (recorded in our laboratory), showed that DES is a weaker
intercalator than these pigments. The calculated binding constants for
DNA·pigment and RNA-pigment complexes are ranging from
105 M1 to 106
M
1 (21-23, 35-38). These pigments are
intercalated in both Gua-Cyt, Ade-Thy, or Ade-Urd-rich regions with no
major sequence preference and with minor drug-phosphate interaction
(external binding), similar to RNA·DES complexes investigated
here.
At r > 1/40, DES interaction causes a partial helix
destabilization. Evidence for this comes from major increase (20%) in the intensity of RNA bands at 1698, 1654, 1608, and 1244 cm1 (Fig. 2, r = 1/20). A similar
increase of intensity was observed for several DNA or RNA in-plane
vibrations upon thermal denaturation and acid fixation (16, 23). The
partial helix opening increases the chance of drug binding (externally)
to different RNA donor sites that are available on local helix melting.
At r > 1/20, some decrease in the intensity (20-30%)
of several RNA bands at 1698, 1654, 1488, and 1244 cm
1
are observed as results of minor perturbations of ribose-phosphate backbone geometry, upon drug complexation (Fig. 2, r = 1/10). Evidence for such structural changes comes from the shift of the infrared marker bands at 1698 (Gua, Urd) to 1689 cm
1,
1244 (PO2 stretch) to 1247 cm
1, and 861 (sugar-phosphate) to 870 cm
1. However, since the infrared
indicators at 810 and 870 cm
1 related to ribose in
C3
-endo/anti-conformation (25, 39) exhibited no major shifting, the
RNA remains in the A-family structure in these DES·RNA complexes
(Fig. 1). No further drug-RNA interaction occurred at r > 1/10, due to the lack of spectral changes (intensity or shifting)
observed for RNA and drug vibrational frequencies (Fig. 2). No major
differences are also observed between the difference spectra obtained
for the DES·RNA complexes with r = 1/10 and those of
the corresponding drug-RNA complexes with r = 1/4 and
1/2 (Figs. 1 and 2).
Additional evidence for DES·RNA interaction comes also from the major
spectral shiftings of several DES vibrational frequencies (12). The
shifts of the bands at 1612 cm1 (DES C
C stretches) to
1610 cm
1; 1462 (DES C
O stretches) to 1460 cm
1; 1045 (DES C
C stretches) to 1040 cm
1, and 836 (DES ring vibrations) to 838 cm
1 are related to the DES·RNA interaction (Fig. 1).
However, it should be noted that at low drug concentration, most of the
DES infrared vibrations were masked by the strong RNA vibrations, while
at high drug content, some of the RNA vibrations were overlapped by
drug vibrational frequencies (Fig. 1). However, these overlappings did
not interfere with conclusive interpretation of our infrared results
regarding the RNA·DES complexes. The positive derivative features
observed at 1610, 1513, 1460, 1372, 1250, 1038, 850, and 838 cm
1 in the difference spectra of the DES·RNA complexes
are coming from DES vibrations, and they are not related to the RNA
molecule (Fig. 1; r = 1/10).
Recent spectroscopic results on the DES·DNA complexes showed that at low drug concentration, DES is a weak intercalator with major affinity toward Ade-Thy-rich region, whereas at high drug content, DES binding extended to Gua-Cyt base pairs with no drug-PO2 interaction (12). At high DES content, a major helix destabilization was also observed with a partial reduction of B-DNA structure in favor of A-DNA (12). However, on the basis of the FTIR spectroscopic results on RNA·DES complexes presented here for the first time, it is clearly evident that at low drug concentration, DES intercalates in both Ade-Urd- and Gua-Cyt-rich regions of RNA with some degree of drug-PO2 interaction (external binding). At high drug content, a partial helix opening occurs, while RNA remains in the A-family structure. The reason is mainly due to less flexibility of RNA structure (mainly A-conformation) (40) with respect to the polymorphic nature of DNA conformations, which readily adapt B, A, C, D, and Z structures (41, 42). The local helix melting provides additional binding sites for the DES-biopolymer interaction (groove binding) via Gua-Cyt and Ade-Urd bases donor groups. A partial helix destabilization occurred for DNA at high DES content (r > 1/10), while RNA local helix opening was observed at lower drug concentration (r > 1/40). This can also be due to a complete DNA double helicity with respect to RNA structure, which contains a partial non-helical structure, and to minor differences in some of the H-bonding arrangements (40, 43).