ABSTRACT
The 24mer deoxyoligonucleotide 3
'
-d(T)10-
5'
-5'
-d(C)4
- d(A)10-
3'
(psC4) with an uncommon 5'
-p-5'
phosphodiester linkage was designed to enable the formation of a hairpin
structure with unusual parallel-stranded stem. As reference hairpin structure with an antiparallel-stranded stem, the 24mer 5'
-d(T)10
-d(C)4
-d(A)10
-3'
(apsC4) was chosen. The behaviour of these oligonucleotides at different
temperatures, DNA and salt concentrations was characterised by a combination of
UV melting, CD, CD melting, infrared and Raman spectroscopy, infrared melting
and analytical ultracentrifugation. The parallel-stranded hairpin structure was found to be formed by psC4 only under
conditions of low DNA concentration and low salt concentration. Increase of the
NaCl concentration beyond the physiological level or high DNA concentration
supports the formation of intermolecular multi-stranded structures. The experimental data are in agreement with a four-stranded complex formed by two molecules of psC4. The base pairing
model of this asymmetric four-stranded complex is based on the pyrimidine motif of a triple helix with
two bifurcated hydrogen bonds at the O4 of the thymine each directed towards
one of the amino protons of both adenines. In contrast, the reference oligonucleotide apsC4 forms only an antiparallel-stranded hairpin under all experimental conditions.
The antiparallel orientation of complementary strands in right-handed A- and B-form and left-handed Z-form helices is a fundamental structural principle
of DNA structure that shapes its physical and biological capacity. However, 10
years ago, it was predicted that d(A)6
[middot]d(T)6
might form a parallel-stranded, right-handed double helical structure with reverse Watson-Crick base pairing (
1
). Recently several authors have published their experimental results (
2
-
9
) which demonstrate the existence of a parallel double-stranded helix (ps DNA) in solution. The sequence constraints for the formation of ps DNA in these systems were obtained,
either by using oligonucleotides containing tracts of A and T residues (
2
,
3
) or by the introduction of alternating AT or GA segments (
4
,
5
). Beside parallel-stranded duplexes, a special 24mer DNA oligonucleotide 3'-d(T)10
-5'-5'-d(C)4
-d(A)10
-3' (psC4) with an uncommon 5'-p-5' phosphodiester linkage in the loop
between the T and C residues was designed to facilitate the formation of an
intramolecular hairpin structure (Fig.
1
A) with a parallel-stranded stem (ps hairpin) (
6
,
8
). Low DNA concentration and temperature >20oC were found to favour the parallel-stranded hairpin (ps hairpin) formation. Considering investigations of ps
hairpin formation by the very similar DNA oligonucleotide 3'-d(T)8
-5'-5'-d(C)4
-d(A)8
-3', the formation of multimeric antiparallel-stranded structures was proposed at DNA concentration higher
than 2.5 mM (
8
,
9
).
Oligodeoxynucleotides psC4 and apsC4 were synthesized by using automated phosphoramidite chemistry on a DNA synthesizer (Applied Biosystems Model 394). 5'-
O
-dimethoxytrityl-2'-deoxynucleoside-3'-
O
-(2-cyanoethyl-
N
,
N
-diisopropyl) aminophosphanes were purchased from Perseptive Biosystems GmbH and 5'-CE-phosphoramidite from GLEN Research. The standard
solutions for activation, oxidation and detritylation were also from these companies. Supports consisted of a thin layer (3-5%) of polystyrene grafted onto a polytetrafluoroethylene core (
16
). Polymer-supported oligonucleotides were cleaved from supports and deprotected by treatment
with 28% aqueous ammonia solution for 6-12 h at 55oC. Purification was carried out on a BioRad Model 2700 system using
a mono Q HR5/5 anion-exchange column from Pharmacia. The salt content was adjusted to ~1 Li per phosphate by LiClO4
precipitation and elution on a Sephadex G10 column. The sample purity was
checked by 15% polyacrylamide gel electrophoresis under denaturing conditions
(8 M urea, 90 mM Tris-borate, 5 mM EDTA, pH 8.3, stained by 0.4 [mu]g/ml ethidium bromide and visualized by fluorescence). Experiments were carried out with samples dissolved in 10 mM Na-cacodylate, pH 7.2 (unless stated otherwise) to which various
amounts of NaCl or MgCl2
were added, as indicated. In all experiments, samples were heated at 75oC for 5 min and slowly cooled to room temperature after each addition of
salt. D2
O was purchased from Aldrich.
The UV absorption measurements were performed on a Cary 1E spectrophotometer
(Varian) equipped with a thermostated cell holder. The molar extinction
coefficients of the oligonucleotides at 260 nm and 80oC were apsC4, 8900 M-1
cm-1
; psC4, 9000 M-1
cm-1
and pd(T)10
, 8800 M-1
cm-1
(
6
). All concentrations are designated on a per oligomer basis unless otherwise
mentioned.
The thermal denaturation was followed at four different wavelengths: 257, 260,
266 and 280 nm. The heating rate was 0.5oC/min. Changes in the heating or cooling rates (0.2-1.0 oC/min) did not affect the thermodynamic parameters. Absorbance
readings were collected at 0.25oC intervals in the temperature range 8-85oC.
CD spectra were recorded on a Jasco Model 720 CD dichrograph using 1 cm path
length cell. The sample temperature was controlled by circulating water bath.
Spectra reported are averages of two scans. The CD melting curves were
collected at 0.5oC intervals between 2 and 85oC.
The data from the thermal transition experiment were analyzed according to a
concerted two-state model for the helix to coil transition as described by Ramsing
et al
. (
17
). The initial parameters for
T
m
and [Delta]HvH
at each wavelength were determined from a derivative plot of the thermal
transition curve computed with Savitzky-Golay filters. The linear parameters [epsilon]0h
, [beta]h
, [epsilon]0c
and [beta]c
were estimated from for the two linear segments of the thermal transition curves (
17
). The initial parameters were improved by the non-linear least-squares minimization routine based on the Levenberg-Marquardt algorithm using Tablecurve 2D from Jandel
Scientific.
Molecular mass of the oligonucleotides was determined by sedimentation
equilibrium experiments using an analytical ultracentrifuge XL-A from Beckman. About 70 [mu]l of the samples dissolved in 10 mM Na-cacodylate buffer, pH 7.2, containing different amounts of NaCl were filled in the chamber of six-channel cells and centrifuged 2 h at 36 000 r.p.m. (overspeed)
followed by 20 h at 30 000 r.p.m. (equilibrium speed) at different temperatures
between 5 and 40oC. The radial concentration distribution at sedimentation equilibrium was
recorded with 10 repeats at three different wavelengths, mostly using 255, 260 and 265 nm. Molecular mass (M) of the oligonucleotides was obtained from the radial
concentration distribution (cr
) by a non-linear fitting procedure according equation
1 using our program Polymol (
18
).
cr
= cro
@exp[MK(r2
- {roman r} sub {roman o} sup 2 )
1
with
{roman K} = {{( 1 - {roman {rho {v bar}}} ) {{roman omega} sup 2}} over {2 {roman {R T}}}}
2
or from the linear fit d ln c/d(r2
) by equation
3
{roman M} = {{2 {roman {R T}}} over {( 1 - {roman {rho {v bar}}} ) {{roman omega} sup 2}}} cdot {{{roman d} ln
{roman c}} over {{{{roman d} ( {roman r}} sup 2} )}}
3
R is the gas constant, T the absolute temperature, [omega] the angular velocity and cro
the concentration at the radial reference position. The data for the buoyancy
term ( 1 - {roman {rho {v bar}}} ) were taken as Equation
values from Cohen and Eisenberg (
19
).
FT-IR measurements were performed using a Bruker IFS-66 FT-IR spectrometer equipped with MCT and DGTS detectors. The DNA
solutions were placed in a demountable temperature-controlled liquid cell (Harrick) with CaF2
windows. Path lengths were 6 [mu]m for samples in H2
O buffer and 6 or 56 [mu]m for samples in D2
O buffer. The resolution was set to 2 cm-1
, 32 interferograms were accumulated and coadded, and Fourier-transformed using the Happ/Genzel apodization function. The DNA spectrum
was obtained by subtraction of the buffer spectrum from the spectrum of the DNA solution at the respective temperature. The unsmoothed DNA spectra were used for further analysis. Standard procedure of Fourier
deconvolution of the spectra was carried out (
20
) and performed by using Lorentzian bandwidth of 13 cm-1
and a resolution enhancement factor 2. Thermal studies were carried out at a
linear heating rate of 0.75oC/min while recording 32 interferograms in 1 min. Plots of intensity at
1625 cm-1
versus temperature and band position of the C=O stretching vibration around
1695 cm-1
versus temperature were evaluated from the original spectra at 0.75oC temperature intervals. These plots show the thermal transition of the
helix to the coil state. The transition temperature was determined using the
first derivative of the transition curves. The DNA triple helix was prepared by
direct mixing of the oligonucleotide apsC4 with decadeoxythymidylate, pd(T)10
, in aqueous solution. About 1.5 [mu]l droplets of these samples were deposited in cells equipped with ZnSe
windows.
Glass capillary tubes with the DNA oligomer solutions were mounted directly in
the sample illuminator of the Raman spectrometer (Jobin Yvon T 64000) equipped with a CCD detector. Raman spectra were excited with the 488 nm line of an argon laser (Innova 90,
Coherent Inc.) using 100 mW of radiant power at the sample.
The melting curves and hyperchromicity profiles of the oligonucleotide psC4
(Fig.
2
, curves 1 and 2, and Fig.
3
) in buffer with NaCl concentration of <= 100 mM determined by UV absorption spectroscopy were characteristic of ps
DNA, in according to previous studies (
6
,
17
). UV melting analysis of psC4 in the presence of 100 mM NaCl reveals a
cooperative monophasic melting transition with
T
m
= 38.8oC and [Delta]HvH
= 190 kJ/mol. The corresponding values for the reference hairpin apsC4 are
T
m
= 49.5oC and [Delta]HvH
= 209 kJ/mol. Figure
3
A shows the hyperchromicity profile of psC4 and apsC4 at the same NaCl
concentration (100 mM). The relative absorption increase due to helix-coil transition displays a strong wavelength dependence that is in the
case of psC4 characteristic for ps DNA (
6
,
17
).
PsC4 exhibits slight differences in the CD spectral range 230-290 nm in comparison with the reference hairpin apsC4 in a buffer with 100 mM
NaCl. In particular the CD difference spectra (psC4-apsC4) show positive peaks at 247 nm and negative peaks at 260 and 284 nm
(Fig.
4
A) in accordance with previous studies (
6
,
8
). Smaller difference peaks were observed upon increase of the NaCl
concentration between 230 and 290 nm (Fig.
4
A), while a stronger negative peak at 217 nm occurs. Duplex structures are
characterized by a positive peak whereas triplexes exhibit mainly a negative
peak between 200 and 230 nm (
21
). Thus, the strong decrease of the intensity of the peak at 217 nm could
reflect the formation of triple helical structure by psC4 at higher NaCl
concentrations. Therefore, we measured the CD melting curves at 217 nm to
follow the melting of the supposed triple helix. Figure
4
B shows the CD melting curve of psC4 at 217 nm in a buffer with 1 M NaCl. The CD-derived
T
m
(21.4oC) for the observed transition between 2 and 40oC is identical with the
T
m
(22.4oC) of transition I in the UV melting curve. Between 40 and 90oC the [Delta][epsilon] value of the CD melting curves decreases both for psC4 and apsC4. The results suggest formation of an
intermolecular triple helix by two psC4 molecules as the intermolecular structure of psC4 at
high NaCl concentration.
Sedimentation equilibrium runs were carried out at different NaCl concentrations
for psC4 as well as apsC4. The radial concentration distribution for psC4 in
100 mM NaCl at 5oC is presented as linear slope d ln c/d(r2
) in Figure
5
A. From these values a molecular mass of 8350 Da was calculated using equation
3. This result exceeds the monomeric molecular mass of 7860 somewhat indicating a
weak association. In comparison to psC4 the slope obtained for apsC4 is
something lower resulting in a molecular mass of 7800 Da. When increasing the
salt concentration we get higher molecular masses for psC4 as derived from Figure
6
. In the presence of 1 M NaCl values of 15 400 " 200 were obtained, suggesting nearly all psC4 molecules are involved in
intermolecular dimer formation. In contrast to this behaviour apsC4 remains in
the monomeric state even at the high salt concentration (Fig.
5
B).
Striking differences between the spectra of psC4 and apsC4 were found in the region of the C=O stretching vibration at 1650-1700 cm-1
at 1 mM oligomer concentration. The infrared spectra of the D2
O solutions of psC4 and apsC4 show a frequency shift of the vibration bands at
1663 cm-1
and 1697 cm-1
of the antiparallel-stranded reference hairpin apsC4 to new band positions of the psC4 at 1665, 1688 and 1696 cm-1
, respectively (Fig.
7
, curves 1 and 2). These values of psC4 are in very good agreement with the band
position of the C=O stretching vibrations of the parallel-stranded duplex ps-D1D2 (Fig.
7
, curve 3) indicating the formation of a ps hairpin by psC4 at 1 mM oligomer concentration (
22
). No further differences in the infrared spectra of the ps hairpin and the aps hairpin were found in D2
O or H2
O solution (data not shown).
The Raman spectrum of psC4 in H2
O displays a less intensive band at 1346 cm-1
relative to the band at 1377 cm-1
as compared with the spectra of apsC4 (data not shown). The band at 1346 cm-1
is mainly due to the NC8H/NC2H bending mode of adenine (
26
). For triple helices of poly(U*A-U) type, interaction at the N7 position was identified by a decrease in the intensity of the 1344 cm-1
band (
26
). This finding supports the intermolecular multi-stranded helix formation by psC4 like derived from the infrared spectra.
The data reported in this paper give a new insight to the hairpin- dimer/multimer equilibrium of parallel-stranded hairpins. In previous studies, it was already suggested that parallel-stranded hairpins can form higher order antiparallel-stranded structures at increased salt or oligonucleotide concentration (
4
,
7
-
9
). Especially for the DNA oligonucleotide p3'-d(T)8
-5'-5'-d(C)4
-d(A)8
-3', the formation of concatameric structures in the conventional
antiparallel manner (Fig.
1
C) was proposed at DNA concentration higher than 2.5 mM (
8
,
9
). Our experimental results are more consistent with a transition from parallel-stranded hairpin to dimeric multi- stranded structures by psC4 upon increase of ionic strength or DNA
concentration.
The biphasic melting profile of psC4 at high salt concentrations, combined with
the results of the sedimentation experiments, offer strong experimental
evidence that this oligonucleotide can adopt both the parallel-stranded hairpin and intermolecular dimer structure. According to the
melting experiments in buffers with 1 M NaCl, the oligonucleotide psC4 exists
at room and lower temperatures in an equilibrium between intermolecular
structure and ps hairpin since the melting profile of this molecule is biphasic and the transition I is concentration dependent. Several alternative
intermolecular structures of the oligonucleotide (Fig.
1
B-F) are compatible with these results. Therefore, we investigated the
intermolecular structure-ps hairpin equilibrium of psC4 by sedimentation experiments to obtain
more definite information about the intermolecular structure. Ross
et al
. reported the utility of the sedimentation equilibrium experiments to study the
duplex-hairpin equilibrium of conventional antiparallel-stranded hairpins (
27
). Our study is based on the assumption that the partial specific volumes and
the number of bound cations do not differ for the intermolecular and hairpin
structure of psC4. Even when this assumption is not strictly fulfilled our data
clearly show that the molecular weight of the intermolecular structure is
equivalent to two molecules of psC4, representing a dimeric structure.
The CD melting experiments gave us more information about the strand orientation
of the dimeric structure of psC4. Several authors used the increase of CD
signal at 217 nm to detect the triplex to duplex transition in CD melting
curves (
21
,
28
,
29
). Perfectly formed triple helix structures like dT*dA-dT show a negative peak at 217 nm. The dimeric structure formed by psC4 at
high salt (1 M NaCl) and low temperature (8oC) shows a positive CD signal at 217 nm, but the disruption of the dimeric structure is accompanied by a strong increase of the CD amplitude at 217 nm in the CD melting curve (Fig.
4
B). Moreover the CD signal at 217 nm goes to higher values as one traverses the temperature range in which the first UV melting event occurs. Thus, we concluded that the dimeric structure of psC4 could probably represent a multi-stranded structure based on a triple helix according to the pyrimidine motif (Fig.
1
D or E).
It has been shown that most of the synthetic hairpin sequences studied, which
include fully (
10
,
11
) and partly (
14
) matched palindromic sequences, can exist in solution as a mixture of two
ordered forms identified as the monomeric hairpin and the dimeric duplex
structures. In principle, the oligonucleotide psC4 could also form dimeric duplex structures with parallel orientation of the strands (Fig.
1
B), but the hyperchromicity profile and the CD spectra of the dimeric structure of psC4 are more characteristic for an antiparallel-stranded helix (
6
,
7
). The formation of partially base-paired antiparallel dimeric structure by psC4 (Fig.
1
C) can be excluded by the remarkable increase of the hyperchromicity in the UV melting curve by formation of the dimeric structure (Fig.
2
). Furthermore a dimer with partially base-paired antiparallel structure should be very unstable and is expected to
form concatamers.
We used the FT-IR and Raman spectroscopy to probe in more detail the structure of the
multi-stranded complex formed by psC4 at high salt concentration. These experiments were done at 10-3
M DNA concentration. Only at 1.5 mM oligomer concentration or lower and without
extra addition of NaCl it was possible to observe IR spectra of psC4
characteristic for parallel-stranded structures. Upon increase of the oligomer concentration or
addition of NaCl the infrared spectra of psC4 show remarkable changes,
especially in the base pairing region between 1740 and 1500 cm-1
, indicating the formation of a new structure. The same behaviour was also
observed by Germann and co-workers (
8
,
9
) for a slightly different parallel-stranded hairpin structure (like mentioned above) using NMR spectroscopy.
Our interpretation of the infrared spectra of psC4 leads to the conclusion that
intermolecular multi-stranded structures based on a triple helix are formed by psC4 at high NaCl and/or high DNA concentrations. The triple helix is formed according to the pyrimidine motif with the
third strand oriented parallel to the dA strand. Recently, Taillandier and co-workers presented IR spectra of triple helices formed by dT*dA-dT base triplets with the same strand orientation (
24
,
25
). They also observed an almost complete disappearance of the adenine band at about 1622 cm-1
, an appearance of a new band at 1633 cm-1
due to the thymines involved in Hoogsteen base pairing and a relatively low
position of the C=O stretching vibration (mainly from C4=O4) of thymine at 1659
cm-1
. Furthermore the Raman spectra of psC4 compared with the apsC4 suggest triple
helix formation of psC4. There is one possibility to form a triple helix
according to the pyrimidine motif by two psC4 molecules (Fig.
1
D), but in this structure the second dA strand is still not involved in base
pairing. A non base-paired dA is expected to show a strong band around 1622 cm-1
in the infrared spectra (
30
). In contrast, our infrared spectra of psC4 display only a very small intensity
at 1622 cm-1
, probably due to the residual ND2
and ring vibration of the non base-paired cytosines in the loop. Thus, we have to assume that nearly all
adenine amino protons are involved in hydrogen bonds. Considering this
assumption we propose the formation of a four-stranded complex by two psC4 molecules as shown in Figure
1
E. At very high DNA concentrations, the tetraplex structure may be formed
preferably by multimeric chains (Fig.
1
F). The base pairing model of this asymmetric four-stranded complex is based on the pyrimidine motif of a triple helix with
two bifurcated hydrogen bonds at the O4 of the thymine each directed towards one of the amino protons of both adenines (Fig.
9
).
We described here the equilibrium between parallel-stranded hairpin and intermolecular dimer (multimer) of the
oligonucleotide psC4 in terms of temperature, DNA and salt concentration. In contrast to the duplex formation of conventional antiparallel-stranded hairpins with short stems, the oligonucleotide psC4 can form intermolecular multi-stranded dimers (multimers) at conditions of high DNA concentration
(above 1.5 mM strand) and/or higher NaCl concentrations than physiological
ones. Based on our FT-IR and Raman spectroscopic data we propose the formation of an asymmetric
four-stranded complex by two psC4 molecules. Interestingly, the conventional
antiparallel-stranded reference hairpin apsC4 remains in the hairpin structure under all experimental conditions used. Structures similar to the described asymmetric four-stranded complex may be involved in recombination and repair
processes (
37
).
We would like to thank C. Kraft (Max-Delbrück-Zentrum, Berlin) and J. Liquier (Laboratoire de Spectroscopie
Biomoleculaire, Université Paris XIII) for the help in Raman Spectroscopy. This work was supported
by the Deutsche Forschungs-gemeinschaft (DFG), Fonds der Chemischen Industrie (FCI), and the Thuringian Ministry of Science, Research and Culture.
*To whom correspondence should be addressed. Tel: +49 3641 657560; Fax: +49 3641
657520; Email: hfri@hodler.molebio.uni-jena.de
REFERENCES
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