4326-4334 Nucleic Acids Research, 1994, Vol. 22, No. 20
Q-D) 1994 Oxford University Press
Evidence from CD spectra that d(purine) r(pyrimidine) and
r(purine) d(pyrimidine) hybrids are in different structural
classes
Su-Hwi Hung+, Qin Yu, Donald M.Gray* and Robert L.Ratliffl
Program in Molecular and Cell Biology, Mail Stop FO 31, The University of Texas at Dallas,
Box 830688, Richardson, TX 75083-0688 and 1Genetics Group, Life Sciences Division, Los Alamos
National Laboratory, Los Alamos, NM 87545, USA
Received April 19, 1994; Revised and Accepted July 31, 1994
ABSTRACT
CD spectra and difference CD spectra of four d(oligopurine). r(oligopyrimidine) and four r(oligopurine)
d(oligopyrimidine) hybrid duplexes containing mixed
A* T(U) and G C base pairs were compared with the
spectra of four DNA* DNA and four RNA RNA oligomer
duplexes of similar repeating sequences. The 16
duplexes were formed by mixing oligomers that were
24 nucleotides long. The buffer was 0.05 M Na+
(phosphate), pH 7.0. DNA* DNA and RNA RNA oligomer duplexes were used as reference B-form and Aform structures. We found that the CD spectra of
d(purine)* r(pyrimidine) and r(purine) * d(pyrimidine)
hybrid duplexes were different from the CD spectra of
either DNA DNA or RNA RNA duplexes. The data
suggested that these hybrids have intermediate
structures between A-form RNA and B-form DNA
structures. The CD spectra of d(purine) r(pyrimidine)
and r(purine) * d(pyrimidine) hybrid duplexes were
different from each other, but the hybrids in each class
had consistent CD spectra as indicated by nearestneighbor comparisons. Thus, it appeared that the two
types of hybrids belonged to different structural
classes. The negative 210 nm band found in difference
CD spectra was correlated with the presence of an
r(purine) strand in the hybrid duplexes. The melting
temperatures (Tm values) of these hybrids were
compared with the Tm values of the DNA" DNA and
RNA* RNA duplexes. The order of the thermal stability
was: RNA * RNA duplex > r(purine) * d(pyrimidine)
hybrid > DNA * DNA duplex > d(purine)* r(pyrimidine)
hybrid, when comparing analogous sequences.
INTRODUCTION
DNA -RNA hybrid duplexes are involved in replication,
transcription, and reverse transcription. DNA RNA hybrids
(Okazaki fragments)
are formed during lagging strand DNA
synthesis, where a chimeric DNA-RNA strand is paired with
an all-DNA strand. 1'2 Hybrids are formed during transcription
of DNA into RNA. Hybrids are found as intermediates in reverse
transcription of retroviral RNA.3 A model duplex A-form
DNA * RNA hybrid has been built into the cleft of the RNase H
and RNA polymerase active sites of reverse transcriptase.4
DNA * RNA hybrids are substrates for a number of enzymes. For
example, the Okazaki fragment of the RNA strand is removed
by a 5'-3' exonuclease activity of DNA polymerase in Escherichia
coli and is cleaved by an RNase H.5 The RNase H activity found
in reverse transcriptase of human immunodeficiency virus
(HIV-1) degrades the RNA template during synthesis of viral
DNA and is required for HIV-1 replication.6 Even though
DNA- RNA hybrids are involved in important biological
processes, the structures and thermal stabilities of DNA -RNA
hybrids have been less thoroughly studied than those of duplex
DNAs and duplex RNAs.
Milman et al.7 used X-ray fiber diffraction to study a
DNA-RNA hybrid duplex formed between fl single-stranded
DNA and its complementary RNA. Their results suggested that
the geometry of the helical structure resembled that of A-DNA
and differed from that of retroviral RNA. Amott et al.8 showed,
from the X-ray fiber diffraction data of various homopolymer
DNA * RNA hybrids, poly[d(G) * r(C)], poly[r(G) * d(C],
poly[d(A) r(U)], and poly[r(A) d(T)] at relative humidity <
80%, that the A-conformation is the most common secondary
structural type in hybrids. However, model building based on
X-ray fiber diffraction studies of DNA * RNA hybrids
poly[d(A) r(U)] and poly[r(A) - d(T)], at relative humidity >
80%, demonstrated that the RNA strands of poly[d(A) * r(U)] and
poly[r(A) d(T)] can be in an A conformation with a C3'-endo
sugar pucker, while the DNA strands of these hybrids can
maintain a B-like structure with a C2'-endo sugar pucker.8 9
The intermediate, heteronomous structural features of the
homopolymer hybrid poly[r(A) d(T)] was confirmed by 31p
*To whom correspondence should be addressed
+Present address: Department of Biochemistry,
Brandeis University, Waltham MA
02254--9110,
USA
Nucleic Acids Research, 1994, Vol. 22, No. 20 4327
solid state NMR and Raman spectroscopy studies.'0'11 A highresolution NMR study of a synthetic DNA* RNA hybrid
dodecamer containing the consensus Pribnow promoter sequence,
d(CGTTATAATGCG) * r(CGCAUUAUAACG), indicated that
the RNA strand is in an A-conformation (i.e. has C3'-endo
sugars) while the DNA strand is closer to a B-conformation (with
C2'-endo sugars).12 An X-ray crystallography study of
DNA-RNA chimeric duplexes (two self-complementary
duplexes [r(G)d(CGTATACGC)]2 and [d(GCGT)r(A)d(TACGC)]2, as well as the Okazaki fragment d(GGGTATACGC) r(GCG)d(TATACCC)) showed that these duplexes
adopted A-conformations.13 A number of intramolecular
interactions of the ribose 2'-hydroxyl group contribute to the
stabilization of the A-conformation. From these results, one can
conclude that the structures of DNA* RNA hybrids are
polymorphous and their conformation may depend on various
parameters, such as solvent conditions and base compositions.
The thermal stabilities of DNA* RNA hybrid duplexes depend
the type of base pairing and the base sequences. The order
of the thermal stabilities of homopolymer duplexes containing
G C base pairs is poly[r(G) * r(C)] > poly[r(G) * (C)] >
poly[d(G) r(C)] > poly[d(G) d(C)], while the order of the
thermal stabilities of homopolymer duplexes containing A* T/U
base pairs is poly[d(A) d(T)] > poly[r(A) d(T)] >
poly[r(A) r(U)] > poly[d(A) r(U)] .14 In both series, the
r(purine) d(pyrimidine) hybrid is more stable than the
d(purine) * r(pyrimidine) hybrid. The melting temperatures of the
homopolymer duplexes containing A T/U base pairs are unusual
in that the DNA * DNA duplex is more stable than the RNA * RNA
duplex. Since the poly[d(T)]-containing duplexes have higher
Tm values than the poly[r(U)]-containing duplexes, the methyl
group of the thymine residues may exert a stabilizing effect. 14
Recent work by Roberts and Crothers"5 on two
homopurine * homopyrimidine hairpin loop hybrid duplexes
on
-
-
Table 1. Melting temperatures of duplexesa
Duplexes
Tm (°C)
S(pur)
£(260 nm)C
S(pyr) e(duplex)
Hyperchromicity
at 260nm
(%)d
(A) DNA-DNA
d(AG)l2*d(CT)l2
d(AGG)8*d(CCT)8
d(AAG)8-d(CTT)8
d(AAGG)6-d(CCTT)6
59.4± 0.5b
9,300
7,660
7,520
23.0±0.4b
63.4 ± 0.5
9,790
7,590
8,200
15.9 ± 0.5
50.0 ± 0.05
10,300
7,950
8,320
25.6 ± 1.2
57.9 ± 0.0
8,940
7,820
7,640
21.6 ± 2.0
r(AG)12*r(CU)12
69.5 ± 1.0
11,960
8,360
8,750
17.0 ± 0.8
r(AGG)8*r(CCU)8
77.6 ± 0.3
11,540
8,060
9,560
11.5 ± 2.1
()
RNA-RNA
r(AAG)8-r(CUU)8
53.5 ± 1.5
11,960
8,910
9,350
20.4 ± 0.2
r(AAGG)6*r(CCUU)6
70.9 ± 0.0
11,640
8,540
9,300
17.9± 0.5
8,730
13.2±0.05
10.6 ± 0.3
(C) DNA*RNA Hybrid
d(AG)12.r(CU)12
43.2± 0.8
d(AGG)8gr(CCU)8
46.2 ± 0.8
9,890
d(AAG)8-r(CUU)8
d(AAGG)6*r(CCUU)6
33.4± 1.4
9,140
12.0± 1.4
47.4 ± 0.5
8,400
16.9 ± 1.6
r(AG)l2*d(CT)l2
64.0± 0.9
8,510
17.7±0.5
r(AGG)8.d(CCT)8
r(AAG)8*d(CTT)8
r(AAGG)6*d(CCTT)6
69.4 ± 0.5
8,530
11.5 ± 1.6
51.6 ± 1.2
8,570
22.4 ± 3.6
66.4 ± 0.5
8,500
20.4 ± 0.0
(e)
D) RNA-DNA Hybrid
aThe buffer was 0.05 M Na+ (phosphate), pH 7.
bThe error is the range of two or three Tm measurements.
cExtinction coefficients at 20'C. E(pur) and E(pyr) values for the purine- and pyrimidine-containing
oligomer strands were calculated from the extention coefficients (E) of monomers and dimers with the
assumption that molar absorptivity is a nearest-neighbor property and that the oligomers were singlestranded at 20°C, with the exception that the E(260, 20°) values for the d(purine) strands were obtained
from the expression e(260, 20') = [A(260 20o)/A(260 900))] XE260, 90,calc) The e(duplex) values were
based on the concentration of nucleotides in the mixed single strands on the measured optical densities
at 200C.
= 100 X OD(260, 90') - OD(260, 20°)- The optical density at 90'C was corrected
dHyperchromicity ( I)
OD(260, 20°)
for volume expansion of the solution.
eSee above for e of single strands.
4328 Nucleic Acids Research, 1994, Vol. 22, No. 20
containing 67% G C base pairs in the hairpin stems showed that
they had the same order of thermal stabilities as those of the
homopolymer duplexes containing G * C base pairs and A* U/T
base pairs.14 In work on polymer sequences containing a
repeating purine-pyrimidine sequence in each strand, Gray and
Ratliff'6 found that the hybrids were less stable than either the
corresponding DNA* DNA or RNA * RNA duplex, with the order
of Tm values being: poly[r(AC) *r(GU)] > poly[d(AC) -d(GT)]
> poly[r(AC) d(GT)] > poly[d(AC) r(GU)]. A study of the
thermal stabilities of oligonucleotide hybrid duplexes containing
60% Gs C base pairs in mixed purine-pyrimidine sequences also
showed that two pentamer DNA RNA hybrid duplexes are
destabilized relative to either the DNA* DNA or the RNA * RNA
duplex. 17
Circular dichroism (CD) measurements of the two hairpin loop
hybrids studied by Roberts and Crothers15 showed that the
hybrids had substantially different CD magnitudes of the 275 nm
band. The hybrids constituted an intermediate structure and were
not identical to an A form RNA duplex structure. 15 CD spectra
of the repeating sequence polymer hybrids, poly[d(AC) r(GU)]
and poly[r(AC) d(GT)] showed differing degrees of similarity
to that of the RNA * RNA duplex, poly[r(AC) * r(GU)].16 Steely
et al.'8 and Johnson et al.19 used near UV CD spectra and
vacuum UV CD spectra to characterize the structures of two
hybrid duplexes, poly[r(A) d(T)] and poly[r(A) d(U)], and
concluded that the polypurine strands of the hybrids retained
features of an A-form while the polypyrimidine strands had
spectral features of both the A-form and B-form.
In the present work, we used UV CD spectra, difference CD
spectra, nearest-neighbor calculations, and melting profiles to
explore the secondary structures and thermal stabilities of four
d(purine) * r(pyrimidine) and four r(purine) * d(pyrimidine)
repeating sequence oligomer duplexes containing A T(U) and
G * C base pairs (Table 1). The CD spectra of four DNA * DNA
oligomer duplexes and four RNA * RNA oligomer duplexes of
analogous sequences were used as reference B-form and A-form
CD spectra.
-
MATERIALS AND METHODS
The 16 duplexes were formed by mixing homopurine and
homopyrimidine oligomers that were 24 nucleotides long. Eight
single-stranded DNAs (d(AG)12, d(AGG)8, d(AAG)8, d(AAGG)6, d(CT)12, d(CCT)8, d(CTT)8 and d(CCTT)6) and eight
single-stranded RNAs (r(AG)12, r(AGG)8, r(AAG)8, r(AAGG)6,
r(CU)12, r(CCU)8, r(CUU)8, and r(CCUU)6) were chemically
synthesized. The single-stranded DNAs were purified by
NENSORB'm PREP cartridges (du Pont de Nemours & Co.)
to remove the oligomers of incomplete, short lengths. Although
the RNA oligomers could not be similarly purified, a check
showed that at least 90% of the single-stranded r(CU)12
preparation electrophoresed as a single band in a 20% denaturing
polyacrylamide gel (with 7 M urea). Since all of the RNA
duplexes and hybrids melted with a similar ATm (see below),
we assumed that all of the RNA oligomers were of similar purity.
These synthetic DNA or RNA oligomers were dissolved in
distilled, deionized water with molar nucleotide concentrations
of approximately l0-3 M. The oligomers were then diluted with
0.05 M Na+(phosphate), pH 7, to give a final molar nucleotide
concentration of approximately 5 x 10-5 M.
The extinction coefficients at 260 nm of these oligomers were
calculated from the extinction coefficients of monomers and
dimers at 200C20 with the assumption that molar absorptivity is
a nearest-neighbor property and that the oligomers were singlestranded at 20°C. For oligomers that could form intrastrand
structures at 20°C, we assumed that the calculated extinction
coefficients could be used for single-stranded oligomers at 90°C
and then obtained the E(26o) at 20°C from the expression E(260, 200)
- [A(260, 20o)/A(26(, 90°)] X E(260,
900°, ac). The extinction coefficients of the single-stranded oligomers are given in Table 1.
To determine whether duplexes could be formed by mixing
each pair of complementary oligomers, data were obtained for
classical mixing curves. Nine mixtures of molar strand ratios of
approximately 100:0, 80:20, 67:33, 60:40, 50:50. 40:60, 33:67,
20:80, and 0:100 were made with equal molar nucleotide
concentrations (of about 4.6 i 0.7 x 10-5 M) of each pair of
the oligopurine and oligopyrimidine strands. The procedure is
detailed in Gray et al.21 Duplex formation was confirmed by the
demonstration of break points in the absorption and CD mixing
curves near a 50:50 molar ratio of the mixed strands. The specific
sequences of the four sets of DNA-DNA, RNA-RNA, and
hybrid oligomer duplexes that were formed are listed in Table 1.
Digitized CD spectra were obtained using a Jasco J500A
spectropolarimeter. The CD spectra were recorded with the
oligomer single strands and duplexes in 0.05 M Na+
(phosphate), pH 7, 20 or 90°C. The CD spectra of single strands
and duplexes above 220 nm were obtained with samples in a 1
cm pathlength cell and with nucleotide concentrations of
approximately 5 x 10-5 M; spectra at short wavelengths from
220 to 186 nm were obtained with the samples in a 1 mm
pathlength cell and with nucleotide concentrations of
approximately 5 x 10-4 M. The averaged CD spectra of the
single-stranded constituents were subtracted from the CD spectra
of the corresponding duplexes to obtain the difference CD spectra.
Nearest-neighbor calculations were carried out to analyze the
secondary structures within each of the four sets of oligomer
duplexes. The CD spectra of three of the oligomer duplexes in
each set were used to predict the CD spectrum of the fourth
duplex under the assumption that neighbors other than the first
neighbors do not contribute significantly to the CD spectra and
that a given nearest-neighbor base pair is in the same
conformation in all the oligomers compared. For example, the
CD spectrum of the DNA-DNA oligomer duplex, d(AAGG)6 * d(CCTT)6 was calculated from the measured CD spectra
of the three other DNA DNA oligomer duplexes as follows:
AAGG)6*d(CCTT)6=
(3/4) *
[CD()8*d(CTT)8
+
CDd(G)8*d(CCT)8]
(2/4) * [CD(Xmi2.d(cTns 2] + [unavailable 2nd-neighbor terms equal to
(1/24) (IAGA-TCT + IGAG-CTC - IGGACCT - IGAA-Crr)]Such nearest-neighbor calculations took all of the first-neighbor
and almost all of the second-neighbor contributions into account.
The second-neighbor contributions that were unaccounted for
amounted to less than 17% mole fraction, where only four of
the second-neighbor interactions differed between the tetramer
and the combination of the other three duplexes (see above
equation). An analogous nearest-neighbor approximation was also
applied to calculate spectra for the RNA -RNA duplex, r(AAGG) . r(CCUU)6, the d(purine) . r(pyrimidine) hybrid duplex,
d(AAGG)6 * r(CCUU)6, and the r(purine) * d(pyrimidine) hybrid
duplex, r(AAGG)6 d(CCTT)6.
Melting temperatures (Tm values) of the duplexes (at
nucleotide concentrations of about 5 x 10-5 M) were obtained by
Nucleic Acids Research, 1994, Vol. 22, No. 20 4329
monitoring the absorbance at 260 nm in teflon-capped, cylindrical
1 cm cells with a Cary 118 spectrophotometer while the sample
temperature was raised from 20°C to 90°C at a rate of
0.25°C/min using a Neslab MTP-5 programmer. There was less
than 0.3 % loss due to evaporation, as checked by weighing each
sample before and after heating. The A(260) values were
collected at one degree intervals, corrected for volume expansion
of the solution, and smoothed by a sliding 13-point quadraticcubic function.22 Data from the smoothed melting profiles were
then derivatized by a 13-point quintic-sexic function.22 The Tm
value of each oligomer duplex was obtained from the peak value
of the first-derivative curve of the melting profile. ATm values
(the temperature range over which 80% of the hyperchromicity
took place) were 23 4 2, 36 + 3, 37 2, 33 i 2°C for the
DNA* DNA, RNA RNA, d(purine) *r(pyrimidine) hybrid, and
r(purine) d(pyrimidine) hybrid duplexes, respectively. The Tm
of poly[d(AT) * d(AT)] was measured as a control and was found
to be 52.8 + 0.2°C in 0.05 M Na+ (phosphate) pH 7.0. This
value was close to published values,23'24 with the assumption
that the Tm has a dependence on molar Na+ concentration of
ATm/A (log[Na+]) = 18.50C.25
RESULTS AND DISCUSSION
CD of single strands
Sixteen single-stranded oligomers were mixed to make 16
duplexes. The CD spectra of these single-stranded oligomers from
320 to 186 nm are given in Figure 1. The CD spectra of the
single-stranded d(purine) strands in Figure IA were taken at 90°C
because the spectra of the d(purine) strands at 20°C had large
positive bands at 258 nm which were similar to those observed
by Antao et al.26 for the self complex of d(AG)30 (in 0.01 M
Na+ (phosphate), pH < 7.8, 200C) and by Lee et al.27,28 for
the self-complexes poly[d(AG)] and poly[d(AGG)] (in 10 mM
Tris-HCl pH 8, 0.1 mM EDTA, and 0.25 M NaCl, 200C).
The percentage hyperchromicity at 260 nm of the four d(purine)
oligomers used in the present work ranged from 16 to 30% upon
increasing the temperature from 200C to 90°C.
As seen in Figure 1, the spectra within each panel are similar.
The measured CD spectra of the single strands in Figure 1 were
also similar in the positions and signs of bands to the nearestneighbor calculated CD spectra based on spectra of dinucleotides
and monomers at 200C29'30 (data not shown).
CD of double-stranded oligomers
The CD spectra of the four DNAeDNA oligomer duplexes
(-curves in Figure 2A - D) were similar to those of natural
DNAs, in having positive and negative CD bands of moderate
magnitudes at wavelengths above 220 nm and a crossover point
between 248 and 262 nm, characteristic of the B-DNA
conformation.31 The CD spectra of repeating dimer and trimer
DNA * DNA oligomer duplexes were similar to spectra of their
corresponding polymer duplexes32 at wavelengths above 230
nm, which suggested that the end effects did not significantly
affect the spectral features of the oligomer duplexes. The CD
spectra of the RNA * RNA oligomer duplexes (-curves in Figure
2A-D) were similar to the CD spectra of double-stranded natural
RNA -RNA duplexes.33'34 The appearance of a large positive
CD band above 260 nm and a large negative CD band near 210
nm is a characteristic of the A-RNA conformation.
The CD spectra of the four d(purine) * r(pyrimidine) and the
four r(purine) - d(pyrimidine) hybrids are also shown in Figure
2A - D, (curves with x x X, 0 0 0, respectively). At
wavelengths above 250 nm, the CD spectra of the
d(purine) * r(pyrimidine) hybrids were similar to the CD spectra
of the RNA -RNA duplexes, while the CD spectra of the
r(purine) * d(pyrimidine) hybrids were similar to the CD spectra
of the DNA * DNA duplexes. At wavelengths in the range of 250
to 200 nm, the CD spectra of the d(purine) * r(pyrimidine) hybrids
were close to the CD spectra of the DNA * DNA duplexes, while
the CD spectra of the r(purine) d(pyrimidine) hybrids were close
to the CD spectra of the RNA RNA duplexes. The negative CD
bands of the duplexes at 210 nm had magnitudes that were
correlated with the attendant d(purine) or r(purine) single strands
of the duplexes. A similar correlation has been noted in the case
of polymers containing A T/U base pairs.18'19
The longest wavelength positive bands (above 260 nm) in the
CD spectra of the oligomer duplexes were dominated by
contributions of the constituent single strands (Figures 1 and 2).
At shorter wavelengths, a large positive CD band (190-195 nm)
upon duplex formation appeared as new features that arose from
base pairing and the formation of secondary structures.
-
-
Difference CD of hybrids
To further study the secondary structures of the d(purine) * r(pyrimidine) and r(purine) d(pyrimidine) hybrids, difference CD
spectra were obtained by subtracting the CD spectra of the singlestranded constituents from the CD spectra of the duplexes. The
difference CD spectra of the four B-form DNA DNA duplexes
(Figure 3A) had the smallest magnitudes above 210 nm,
indicating that the single strands were relatively unchanged upon
forming the DNA DNA duplexes. The A-form RNA RNA
duplexes (Figure 3B) had difference CD bands above 210 nm
that were larger than those of the DNA-DNA duplexes. A
significant feature in the difference spectra of most of the A form
RNA RNA duplexes was a negative CD band at 207-209 nm.
The two classes of hybrids in Figures 3C and 3D had
characteristic difference CD spectra. Unlike the difference spectra
of the d(purine) *r(pyrimidine) hybrids in Figure 3C, those of
the r(purine) - d(pyrimidine) hybrids in Figure 3D had large 210
nm bands and were like the difference CD spectra of the
RNA * RNA duplexes (Figure 3B) in generally having a couplet
of positive and negative bands below 220 nm. The small
difference CD magnitudes at wavelengths above 260 nm found
in spectra of the r(purine) * d(pyrimidine) hybrids indicated that
the d(pyrimidine) strands in these hybrids had optical activities
similar to those of the free single-stranded components. It
appeared that the pairing of -the r(purine) strands in the
r(purine) d(pyrimidine) hybrids and the RNA . RNA duplexes
was correlated with CD changes common to the spectra in Figures
3B and 3D.
We concluded from the CD spectra (Figure 2) and the
difference CD spectra (Figure 3) that the d(purine) * r(pyrimidine)
and r(purine) . d(pyrimidine) hybrids belonged to two different
structural classes, both of which were intermediate between the
-
DNADNA B-form and RNARNA A-form structures.
Nearest-neighbor analysis of hybrids
Nearest-neighbor calculations, carried out as described in
Materials and Methods, provided an additional means of
comparing the secondary structures of the duplexes. The
4330 Nucleic Acids Research, 1994, Vol. 22, No. 20
calculated CD spectra for the four repeating tetramers (AAGG)6 (CCTT/UU)6 were very close to the measured spectra,
almost within the measurement error, throughout the 186-310
nm spectral range.
Table 2 shows the RMS values to quantitate the spectral
differences of the nearest-neighbor calculations relative to the
measured spectra of the four tetramers (AAGG)6 (CCTT/UU)6.
These values (0.75-1.16 M- Icm- ) were only slighdy smaller
12
10
8
6
4
QU2
I
-2
-4
-6
14
12
10
8
(L6
1J4
2
0
-2
-4
1 80 200 220 240 260 280 300 320 200 220 240 260 280 300 320
WAVELENGTH (nm)
WAVELENGTH (nm)
), r(AGG)8
), d(AGG)8 (---), d(AAG)8 (X x X), and d(AAGG)6 (O0 0). (B) r(AG)12 (
Figure 1. CD spectra of single-stranded oligomers. (A) d(AG)12 (
), r(CCU)8 (---),
), d(CCT)8 (---), d(CTI)8 (x X), and d(CCTT)6 (O 0 0). (D) r(CU)12 (
(---), r(AAG)8 (X X X), and r(AAGG)6 (ooo). (C) d(CT)12 (
r(CUU)8 (X X X), and r(CCUU)6 (O0 0). The solution conditions were 0.05 M Na+ (phosphate), pH 7, 20°C, except that the CD spectra of the d(purine) strands
were obtained at 90°C. Note that the spectra in this figure are on an expanded CD scale that is one-half the magnitude of the scale in the following figures.
Nucleic Acids Research, 1994, Vol. 22, No. 20 4331
than the average of the RMS values (1.04-1.74 M-'cm-' )
derived for the three duplexes used in each of the nearest-neighbor
calculations, also relative to the spectra of tetramers. Thus, the
CD spectra of the oligomer duplexes in each class were not very
dependent on sequence and reflected primarily the secondary
conformations of the duplexes.
24
20
16
12
Uj8
14
0
-4
-8
-12
24
20
16
12
IY 8
wu~ 4
0
-4
-8
-12
180 200 220 240 260 280 300 320 200 220 240 260 280 300 320
WAVELENGTH (nm)
WAVELENGTH (nm)
Figure 2. Average CD spectra of four sets of DNA -DNA, RNA-RNA, d(purine) r(pyrimidine), and r(purine) -d(pyrimidine) oligomer duplexes. (A) d(AG)12 d(CT)12
), r(AG)12 * r(CU)12 (---), d(AG)12 * r(CU)12 ( x x x), and r(AG)12 * d(CT)12 (O0 0 ). Spectra shown are the averages of at least three independently formed
duplexes. The standard deviation of the three CD measurements of an RNA- RNA duplex, r(AG)12 r(CU)12 at various wavelengths (195, 210, 225, 240, 255, 270,
), r(AGG)8 r(CCU)8 (---), d(AGG)8- r(CCU)8 (x x X), and r(AGG)8 d(CCT)8
and 285 nm) was no more than i 0.2 M- cm- . (B) d(AGG)8- d(CCT)8 (
), r(AAG)8 r(CUU)8 (---), d(AAG)8 * r(CUU)8 (Xx x), and r(AAG)8 * d(CTT)8 (O0 0 ). (D) d(AAGG)6 * d(CCTD6 (
),
(O0 0). (C) d(AAG)8 * d(CTT)8 (
r(AAGG)6 r(CCUU)6 (---), d(AAGG)6 r(CCUU)6 (X X X), and r(AAGG)6 d(CCTT)6 (O0 0). The solution conditions were 0.05 M Na+ (phosphate), pH 7, 20°C.
4332 Nucleic Acids Research, 1994, Vol. 22, No. 20
Quantitation of the differences between the classes of duplexes
Table 3 gives the differences as RMS values between pairs of
CD spectra of the analogous sequences. The average RMS value
between the DNA* DNA and RNA * RNA duplexes was 4.80 +
0.26 M- 'cm- 1, which is the quantitation of the CD differences
between the DNA DNA B-form and the RNA RNA A-form
r
d(pur) d(pyr)
24
20
16 -
I'
12
,
8
(L
4
*
tL)
0
-
4
-8
-12
24
20
16
Q 12
-4
-4
-8
-12
180 200 220 240 260 280 300 320 200 220 240 260 280 300 320
WAVELENGTH (nm)
WAVELENGTH (nm)
Figure 3. Difference CD spectra. The panels contain data for difference spectra of the same duplexes as in Figure 2. The averaged spectra of the component individual
single strands were subtracted from the spectra of the duplexes of Figure 2. Spectra of the individual strands were taken under the same conditions used to obtained
), d(AGG)8 d(CCT)8 (---),
the data for Figure 2, except that the spectra of the d(purine) single-strands were taken at 90°C. (A) d(AG)12 d(CT)12 (
), r(AGG)8 r(CCU)8 (---), r(AAG)8 r(CUU)8 (xxx), and
d(AAG)8-d(CTT)8 (XXX), and d(AAGG)6 d(CCTT)6 (000). (B) r(AG)12 r(CU)12 (
), d(AGG)8 r(CCU)8 (---), d(AAG)8 r(CUU)8 (X XX), and d(AAGG)6 r(CCUU)6 (000).
r(AAGG)6 r(CCUU)6 (000). (C) d(AG)12 r(CU)12 (
), r(AGG)8 * d(CCT)8 (---), r(AAG)8 * d(CTT)8 (x x x), and r(AAGG)6 * d(CCTT)6 (O0 0 ).
(D) r(AG) 12 * d(CT)12 (
Nucleic Acids Research, 1994, Vol. 22, No. 20 4333
Table 2. CD spectral differences (as RMS values)a within each class of duplex, compared with the nearest-neighbor (n-n) calculation
| d(AAGG)6.d(CCTT)6
r(AAGG),.r(CCUU)
|
d(AAGG),.r(CCUU)6
r(AAGG)6.d(CCTT)6
1.58
1.37
(AG)12.(CT/U)12
0.89
(AGG) * (CCT/U)g
1.08
1.93
1.84
1.87
(AAG)g (CTTgUU)^
1.16
0.79
1.32
0.78
Averageb
1.04
1.74
1.58
1.34
-
2.51
1 16d
0.74d
075d
0c88d
Nearest-neighbor calculationC
of (AAGG)6 *(CCTTJ6U)
aNumbers are RMS values = [Fx(CD(X)i - CD(X)2)2/n]/2 (in M-lcm-1), where the differences between the CD values of spectra 1 and 2
(CD(X)1 and CD(X)2 were squared and summed over n wavelengths; then the square root was taken. The number of wavelengths, n, was 125
(310 - 186 nm).
__
bThe average was obtained from individual RMS values as follows:
Average = [RMS((AG)12-(CT/U)12) + RMS((AGG)8-(CCT/U)Q) + RMS((AAG)8 (CTT/UU)s )]/3.
cNumbers are RMS deviations = [EX(CD(X)mew - CD(,) ,,)2/n] (in M-lcm-1). The measured CD spectrum was for the repeating tetramer,
and the calculated CD spectrum was a nearest-neighbor combination of the other three spectra. The number of wavelengths, n, was 125 (310-186
nm).
dThese RMS values are larger than previously published values, because CD data have been included at short wavelengths. Over the range
of 310-220 nm, the RMS values for the calcilated versus the measured spectra for the tetramers (0.19-0.57 M -cm') were slightly higher
than the measurement error (0.15-0.28 M-Icm-1).23
Table 3. CD spectral differences (as RMS values)a between the classes of duplexes
RMS values_____ _________
CD(DNA duplex)
CD(DNA duplex)
CD(RNA duplex)
Sequences
CD(RNA dupkx)
-
(AG)I *(CT/U)I,
1
*
(AGG)R (CCT/U)g
(AAG)R * (CTT/UU)g
AAGG)6- (CCTT/UU)6
AVERAGE
CD(DNA duplex)
-
CD(dqw).qpyr))
CD(RNA duplex)
CD(rq&).d(pyr))
- CD(d(pur).r(pyr))
- CD(rw).d(pyr))
- CD(d(pur).rpyr))
-
CD(rwur).d(pur))
5.00
2.45
2.87
2.24
3.07
3.34
5.00
2.59
2.78
1.86
2.82
3.82
4.75
1.97
2.24
1.38
3.27
3.69
4.44
2.62
2.50
1.46
2.60
3.73
4.80 ± 0.26b
2.41 ± 0.30
2.60 ± 0.28
1.74 ± 0.40
2.94 ± 0.29
3.64±0.21
aNumbers are RMS values = [E (CD(X)1 -CD(X)2)2/n]'2 (in M 1cm-'). The number of wavelengths, n, was 125 (310-186
were obtained using the averaged spectra from two to ten measurements of each type of duplex.
bStandard deviation of RMS values.
duplexes. The CD spectra of the d(purine) r(pyrimidine) and
r(purine) * d(pyrimidine) hybrid duplexes had average RMS values
of 2.41 + 0.30 M-Icm'-, showing that the two types of hybrid
duplexes had structural differences but were nevertheless closer
to each other than were the DNA * DNA B-form and the
RNA * RNA A-form duplexes (See Table 3, columns 1 and 2 of
RMS values).
As shown in the 3rd and 5th columns of Table 3, the
d(purine) * r(pyrimidine) hybrid duplexes had CD spectra equally
different from those of the DNA * DNA B-form and the
RNA * RNA A-form. On the other hand, as seen from the values
in 4th and 6th columns, the r(purine) d(pyrimidine) hybrid
duplexes had spectra and secondary structures closer to the
RNA . RNA A-form than to the DNA- DNA B-form.
The RMS values within each class were substantially smaller
than those values between the classes of duplexes (Tables 2 and
3). Therefore, the four DNAe DNA duplexes listed in Table 1
belong to a similar class of B conformation, and the four
nm).
Values
RNA * RNA duplexes belong to the same class of A conformation.
Quantitation of the spectral differences within and between the
d(purine) * r(pyrimidine) and the r(purine) * d(pyrimidine) hybrid
duplex classes shows that the four d(purine) * r(pyrimidine)
duplexes and the four r(purine) * d(pyrimidine) duplexes can be
distinguished as belonging to different conformational classes of
intermediate structures.
Thermal stability of hybrids
Table 1 shows the melting temperatures (Tm values) and percent
hyperchromicities (%H) of the four classes of oligomer duplexes.
Tm values were determined from the peak values of the
derivatives of the melting profiles. Within a given class of duplex,
a higher (G +C) content in the duplexes was correlated with
higher Tm values. The r(purine)-containing oligomer duplexes
always melted at higher temperatures than the corresponding
d(purine)-containing oligomer duplexes. The RNA * RNA
oligomer duplexes had the highest Tm values, which were
4334 Nucleic Acids Research, 1994, Vol. 22, No. 20
2 -8°C higher than those of the r(purine) *d(pyrimidine) oligomer
duplexes of corresponding sequences. The Tm values of
r(purine) d(pyrimidine) oligomer hybrid duplexes were 2-8°C
higher than those of the DNA DNA oligomer duplexes. The
d(purine) * r(pyrimidine) oligomer hybrid duplexes had the lowest
Tm values, which were 10-17°C lower than those of the
DNA* DNA oligomer duplexes. The order of the thermal
stabilities of the duplexes was always: RNA RNA >
r(purine) d(pyrimidine) > DNA* DNA > d(purine) r(pyrimidine). These results were consistent with the thermal stability
data obtained by Roberts and Crothers15 on hairpin loop hybrids
with all purines in one strand and all pyrimidines in the other.
Ratmeyer et al.35 also recently reported the same order of
stability for a series of duplexes having the repeating tetramer
sequence (GAAG)3' (CTTC/CUUC)3.
CONCLUSIONS
The d(purine) * r(pyrimidine) and r(purine) * d(pyrimidine)
oligomer hybrid duplexes exhibit structures that are intermediate
between the A and B conformations. The d(purine) * r(pyrimidine)
hybrids are in a conformational class different from that of the
r(purine) * d(pyrimidine) hybrids, but the hybrids within each class
have similar structures as indicated by nearest-neighbor
calculations and a comparison of RMS values between and within
the classes of duplexes. Since the CD spectra of three hybrids
within each class can be used to predict the spectrum of a fourth
hybrid, and since CD is approximately a nearest-neighbor
property,23 it should be possible to predict the CD spectrum of
any other hybrid sequence having all purines on one strand and
all pyrimidines on the other. The Tm values of the
r(purine) * d(pyrimidine) hybrids are higher than the Tm values
of the d(purine) * r(pyrimidine) hybrids, suggesting that
d(pyrimidine) oligomers will be better candidates than d(purine)
oligomers for antisense drugs.
ACKNOWLEDGEMENTS
We are grateful for support from NIH Research Grant GM-19060
from the National Institute of General Medical Sciences, Grant
AT-503 from the Robert A.Welch Foundation, and a grant from
Cytoclonal Pharmaceuticals, Inc., of Dallas, TX. This material
is also based in part upon work supported by the Texas Advanced
Technology Program under Grant No. 9741-036.
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