Watson-Crick base-pairing properties of bicyclo-DNA
Watson-Crick base-pairing properties of bicyclo-DNA
Martin
Bolli
+
,
Huldreich U.
Trafelet
and
Christian
Leumann*
Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012
Bern
,
Switzerland
Received August 28, 1996;
Revised and Accepted October 14, 1996
ABSTRACT
A series of sequences of the DNA analog bicyclo-DNA, 6-12 nucleotides in length and containing all four natural
nucleobases, were prepared and their Watson-Crick pairing properties with complementary RNA and DNA, as well as in
its own series, were analyzed by UV-melting curves and CD-spectroscopy. The results can be summarized as follows: bicyclo-DNA forms stable Watson-Crick duplexes with complementary RNA and DNA, the
duplexes with RNA generally being more stable than those with DNA. Pyrimidine-rich bicyclo-DNA sequences form duplexes of equal or slightly increased stability
with DNA or RNA, whereas purine-rich sequences show decreased affinity to complementary DNA and RNA when
compared with wild-type (DNA-DNA, DNA-RNA) duplexes. In its own system, bicyclo-DNA prefers antiparallel strand alignment and strongly
discriminates for base mismatches. Duplexes are always inferior in stability
compared with the natural ones. A detailed analysis of the thermodynamic
properties was performed with the sequence 5
'
-GGATGGGAG-3
'
[middot]5
'
-CTCCCATCC-3
'
in both backbone systems. Comparison of the pairing enthalpy and entropy terms
shows an enthalpic advantage for DNA association (
[Delta][Delta]
H = -18 kcal[middot]mol
-1
) and an entropic advantage for bicyclo-DNA association (
[Delta][Delta]
S = 49 cal[middot]K
-1
[middot]mol
-1
), leading to a
[Delta][Delta]
G25
o
C of -3.4 kcal[middot]mol-1 in favor of the natural duplex. The salt dependence of
T
m for this sequence is more pronounced in the case of bicyclo-DNA due to increased counter ion screening from the solvent. Furthermore
bicyclo-DNA sequences are more stable towards snake venom phosphodiesterase by a factor of 10-20, and show increased stability in fetal calf serum by a factor of
8 compared with DNA.
INTRODUCTION
Oligonucleotide analogs, displaying strong and sequence specific binding to single-stranded RNA or double-stranded DNA and exhibiting resistance to enzymic degradation are
potential candidates for therapeutic applications as inhibitors of protein expression (
1
-
4
). Among the whole family of DNA analogs, those containing defined structural modifications in the sugar-phosphate part gain special interest since the study of their supramolecular interactions can also contribute to the understanding of the structural and energetic
factors that define order mode and specificity in DNA association. Within this
context we recently introduced the DNA-analog `bicyclo-DNA' (Fig.
1
). This analog was initially designed to stabilize complex formation with
complementary natural nucleic acids entropically by exhibiting a higher degree
of preorganisation of its single strands for duplex formation due to the
conformationally locked sugar structure of the underlying bicyclo-deoxynucleosides (Fig.
1
; ref.
5
).
We have demonstrated that decamers of bicyclo-deoxyadenosine [bcd(A
10
)] and bicyclothymidine [bcd(T
10
)] bind to their natural RNA and DNA complements as well as with each other,
forming double and triple helical structures. Compared with natural DNA, duplex formation is associated with (numerically) reduced pairing enthalpy and entropy terms, having compensatory effects on the free energy of
duplex formation (
6
,
7
). Complexes of bcd(A
10
) with complementary DNA or RNA are thermodynamically more stable than those of bcd(T
10
).
MATERIALS AND METHODS (ref. 10)
Synthesis and characterization of oligo-bicyclodeoxy- nucleotides
The synthesis of the bicyclodeoxynucleosides, the corresponding cyanoethyl
phosphoramidites (and allyl phosphoramidite in the case of bicyclothymidine) for oligonucleotide assembly as well as the starter units bound to the solid support is described elsewhere (
5
,
7
). Automated bicyclo-DNA synthesis was performed on a Pharmacia LKB Gene Assembler Special DNA synthesizer on a 1.0-1.5 [mu]mol scale using the modified protocol for bicyclo-DNA assembly, allowing for a prolonged coupling (6 min) and detritylation (60 s) time relative to the synthesis
of natural DNA oligomers (
7
). Coupling yields were in the range of 98% per step.
End-detritylated oligonucleotides were detached from solid support and frayed from protecting groups by standard deprotection (25% aq. NH
3
, 55oC, 10-20 h). In the cases where allyl phosphoramidites were used, Pd(0)
catalyzed allyl deprotection according to the method of Hayakawa
et al
. (
11
) preceded the ammonia treatment. Purification was performed by HPLC (Pharmacia
LKB 2249 gradient pump, UV-detection 260 nm) on reversed phase stationary phase (Aquapore RP 300, 220 * 4.6 mm, Brownlee, linear gradient of max. 80% CH
3
CN in 0.1 M aq. triethylammonium acetate, pH 7.0) and anion exchange stationary phase (Nucleogen DEAE 60-7, Macherey&Nagel, linear gradient of max. 1 M KCl in 20 mM NaH
2
PO
4
, pH 6.0, H
2
O:CH
3
CN 4:1; or Mono Q HR 5/5, Pharmacia, linear gradient of max. 1 M NaCl in 10 mM
aq. NaOH).
The isolated oligonucleotides were desalted over SEP-PAK cartridges (Waters). The homogeneity of the collected fractions was additionally secured in the case of the sequences bcd(CGCGAATTCGCG) and bcd(GCGAATTGCG) by capillary electrophoresis (Waters Quanta 4000, capillary: J&W Scientific (Fisons), 75cm * 75[mu]m, polyacrylamide gel filled (5%T, 5%C), buffer: 100 mM Tris-borate, 7 M urea, pH 8.3). All bicyclo-deoxyoligonucleotides, with the exception of bcd(G
6
), were analyzed by MALDI-TOF mass spectrometry as described (
12
) and were within 2% of the expected mass (monoanionic form). Analytical data as well as yields for the bicyclo-oligomers used in this study are in the supplementary material.
Natural oligodeoxynucleotides containing bicyclo-deoxynucleosides were synthesized according to standard phosphoramidite chemistry using
the modified cycle, described above, for the introduction of the modified nucleosides only. Oligoribonucleotides were prepared as described (
13
) (coupling time, 17 min), purified by HPLC (DEAE anion exchange) and desalted
over Sephadex G-10 (BioRad).
Enzyme digestions
Enzyme digestions of oligonucleotides were performed by treating 1.25 ml of a solution containing 1.5 OD
260
oligonucleotide in 180 mM NaCl, 12 mM Tris-HCl, pH 7.0 at 37oC with 6 mU of snake venom phosphodiesterase and 125 U of alkaline
phosphatase (Boehringer, Mannheim) and followed by recording the increase in UV-absorption (260 nm) as a function of time. Half life times (Table
4
) were directly determined from these curves.
Extinction coefficients of oligo-bicyclodeoxynucleotides
Extinction coefficients of oligo-bicyclodeoxynucleotides were obtained from the same enzyme hydrolysis
curves according to the general formula
{roman epsilon} ( o l i g o ) = {{{{{roman {a b s}}} sub {2 6 0}} ( {roman {s t
a r t}} )} over {{{{roman {a b s}}} sub {2 6 0}} ( {roman {e n d}} )}} cdot
{sum from {i = 1} to n} {{roman epsilon} sub i} ( m o n o )
where abs
260
(start) means the initial absorption, abs
260
(end) the absorption after complete digestion of the oligomer and [epsilon](
mono
) the experimentally determined extinction coefficient of the bicyclodeoxynucleosides at 260 nm (bcd(A) = 13700, bcd(C) = 6200, bcd(G) = 10700,
bcd(T) = 8700). Completeness of digestion of the oligomers to the free
nucleosides was reassured by reversed phase HPLC analysis (vide supra) and identification of the products by coinjection with authentic material. Extinction coefficients for natural DNA
oligomers were calculated as described (
14
).
UV-melting curves
UV-melting curves were measured on a Varian Cary 3E UV/VIS spectrophotometer
equipped with a temperature controller and a multi cell peltier block,
interfaced to a Compaq ProLinea 3/25 ZS computer. A temperature gradient of 0.5oC/min was applied and a heating-cooling-heating cycle was used. At temperatures below 15oC, the cell compartment was flushed with nitrogen to
prevent condensation of water on the cuvettes. Sample solutions were covered with a thin layer of dimethylpolysiloxane (Sigma) in order to prevent evaporation of water. In all cases, heating and cooling curves were
superimposable indicating reversible equilibrium conditions.
T
m
data were defined as the maxima of the first order derivative of the melting
curves and were shown to correspond within +-1oC to those determined at half of the maximal hyperchromicity after
baseline correction. Thermodynamic data for duplex formation were obtained as described (
15
).
CD-spectra
CD spectra were recorded on a Jasco J-500A spectropolarimeter connected to a PC via a IF-500 II (Jasco) interface. Temperature was controlled by a Julabo F20
circulating bath and measured directly in the cell (path length 10 mm).
RESULTS
Complementary base-pairing of bicyclo-DNA with natural DNA and RNA
We synthesized a series of bicyclo-DNA sequences and analyzed their binding affinities to complementary DNA
and RNA by UV-melting curves. All duplex melting curves reflect highly cooperative
melting transitions and are completely reversible. Dominant self aggregation
phenomena of the single strands could be excluded in all cases.
T
m
data and sequences of the hybrid duplexes investigated are reproduced in Table
1
. Inspection of the data leads to a picture in which replacement of purine-rich sequences by bicyclo-DNA decrease duplex stability whereas replacement of pyrimidine-rich DNA sequences by bicyclo-DNA does not. As often observed, complementary base-pairing with RNA is more efficient as with DNA.
Structures of hybrid duplexes with complementary RNA were followed by CD-spectroscopy in the case of the sequence bcd(GGATGGGAG) and bcd(CTCCCATCC). These
spectra are similar to that of the corresponding all RNA duplex (Fig.
2
). Small but clear differences arise in the relative ellipticities in the region near to the maximum positive cotton effect at 270
nm.
Left-handed bicyclo-DNA?
On the basis of the self-complementary hexamer sequence (CG)
3
we investigated the influence of bicyclodeoxynucleotides on its possibility to undergo a salt induced B -> Z conformational transition by CD-spectroscopy. While under neutral conditions (10 mM Tris-HCl, pH 7.0), the natural duplex d(CG)
3
(10 [mu]M,
T
m
= 46.2oC, 0.15 M NaCl) clearly switches to a Z conformation upon raising the NaCl
concentration from 0.15 to 4 M, substitution of bicyclo- deoxycytidine for deoxycytidine [(bcdC-dG)
3
, 10 [mu]M,
T
m
= 51.0oC, 0.15 M NaCl], or bicyclo-deoxyguanosine for deoxyguanosine [(dC-bcdG)
3
, 10 [mu]M,
T
m
= 51.4oC, 0.15 M NaCl] completely abolishes this conformational transition (Fig.
4
). No doubt that also the completely bicyclic duplex bcd(CG)
3
(10 [mu]M,
T
m
= 38.5oC, 0.15 M NaCl) showed no tendency to adopt a left handed Z-conformation either.
The fact that any substitution of a bicyclo-deoxynucleoside for a deoxynucleoside in the left handed DNA forming
sequence (CG)
3
abolishes Z-DNA formation is not unexpected and can be explained with the inability of
the bicyclo-G nucleoside to adopt a 3'-endo furanose conformation and the bicyclo-C nucleoside to adopt a synclinal conformation around
the C(4')-C(5') bond (torsion angle [gamma]) as required for Z-DNA formation (
26
).
Enzymic stability of bicyclo-DNA
We specified the degree of resistance of bicyclo-DNA towards the stability of 3' exonucleases, the latter being mostly responsible for nucleolytic
degradation of oligonucleotides in plasma (
27
). The sequences shown in Table
4
were subjected to hydrolysis catalyzed by the 3'-exonuclease snake venom phosphodiesterase and the corresponding
half-life times determined by UV-spectroscopy.
DISCUSSION
Effects of the bicyclo-DNA backbone on Watson-Crick duplex stability
From the sequences investigated it appears that Watson-Crick duplexes made entirely of bicyclonucleotides are distinctly less
stable compared with those in the natural DNA series. The stability of bicyclo-DNA-DNA or bicyclo-DNA-RNA duplexes follow the same trend especially if
purine-rich bicyclo-DNA sequences are involved. This compares inversely proportional to
the relatively high stability of Hoogsteen base-paired bicyclo-DNA duplexes (
9
) and supports the view that bicyclo-DNA purine nucleotides, in contrast to natural DNA, preferentially accept
complementary strands at the Hoogsteen face of the nucleobases and not at the
Watson-Crick face (Fig.
5
)-this as a direct consequence of the structural alteration of the backbone
within torsion angle [gamma].
Thermodynamic properties
We have reported earlier that duplex formation in the bcd(A
10
)[middot]bcd(T
10
) series is entropically favored and enthalpically disfavored with respect to the corresponding natural duplex and thus seems to be a general property of bicyclo-DNA (
6
,
7
). However, since the bicyclic duplex is Hoogsteen or reversed Hoogsteen base paired (
9
) and the natural one Watson-Crick base paired, this comparison needed a further confirmation on the basis of a sequence, that
adopts the same (Watson-Crick) duplex constitution in both systems. These prerequisites were given with the sequence A (Table
2
). Assuming validity of the two state dissociation model, the thermodynamic data
obtained from UV-melting curves (Table
3
) show the same trend: loss of pairing enthalpy and gain of pairing entropy.
Again, we attribute the enthalpic loss to strain in the duplex caused by the
structural alterations around torsion angle [gamma] in bicyclo-DNA, and the entropic gain, at least in part, to the reduced
flexibility of the sugar-phosphate backbone. To what extent differential solvation of the
backbones in DNA and bicyclo-DNA affects the entropy term is unknown so far.
The higher dependence of duplex formation from monovalent cation concentration
relative to natural DNA seems to be independent of the base sequence and
association mode and seems to be a general property of the altered backbone
structure in bicyclo-DNA. This has already been observed in bicyclo-DNA Hoogsteen duplexes (
7
) as well as in duplexes formed in the [alpha]-bicyclo-DNA series (
30
,
31
). In all cases the number of counter ions screened from the solvent is higher
with respect to the corresponding natural duplexes. This higher demand may have
its origin in the additional ethylene bridge that directly perturbs solvation
of the phosphate groups, or more likely, in an extended conformation of the
bicyclo-DNA single strands compared with the natural ones (assuming that
intrastrand phosphate distances in the stacked duplexes are about equal in both
systems).
Enzymic stability
The linking phosphodiester groups in bicyclo-DNA are higher substituted than in natural DNA. One would therefore expect
this unit to be less of a substrate for enzymic degradation than that of
natural DNA. Much to our surprise, however, bicyclo-DNA is only moderately more stable against 3' exonucleases (Table
4
). The stability against snake venom phosphodiesterase (SVP) is slightly dependent from the base sequence and, as in the case of natural DNA,
seems to be moderately higher in the case of duplexed sequences with respect to single strands as deduced from the self-complementary dodecamer 5'-CGCGAATTCGCG-3' in both backbone systems. In general terms,
modifications on the [alpha]-side of the natural nucleosides [as in [alpha]-DNA (
32
,
33
), [alpha]-bicyclo-DNA (
30
,
31
) or 2'-O-alkyl-RNA (
34
)] seem to increase the enzymic stability more efficiently than the modifications on the [beta]-side in bicyclo-DNA. The moderately enhanced 3'-exonuclease stability of bicyclo-DNA is also reflected in the serum experiment with the DNA sequence
containing a 3'-bicyclo-deoxynucleotide cap.
CONCLUSIONS
The results presented here, together with earlier findings on Hoogsteen and
reversed Hoogsteen pairing of oligopurine/oligopyrimidine strands in bicyclo-DNA (
9
), now allow for a generalized description of the differences in the association properties within the two
pairing systems (bicyclo-DNA and natural DNA).
(i) Bicyclo-DNA strongly prefers the Hoogsteen association mode and discriminates the
Watson-Crick association mode and thus behaves opposite to what is known from
natural DNA. Furthermore, bicyclo-DNA Hoogsteen and reversed Hoogsteen duplexes are of higher thermodynamic
stability relative to the Watson-Crick duplex of natural DNA for a given purine sequence motif (
9
). (ii) Within the Watson-Crick pairing regime both, natural and bicyclo-DNA strongly prefer antiparallel over parallel strand alignment and discriminate base-base mismatches. (iii) Bicyclo-DNA, however, does not discriminate between the parallel (Hoogsteen) and the antiparallel (reversed Hoogsteen)
arrangement upon duplex formation and thus behaves differently from natural DNA for which a
parallel Hoogsteen duplex was reported (
35
), but for which the antiparallel reversed Hoogsteen pairing-mode is only described in the context of DNA triple helix formation by
oligopurine strands (
36
) or oligomers containing deoxyguanosine and thymidine (
37
).
Besides serving as a model for the study of structure/association mode relations in DNA, bicyclo-DNA also shows interesting antisense properties. Due to their strong pairing and enhanced nuclease stability,
pyrimidine rich bicyclo-DNA sequences can advantageously be used in the recognition of single-stranded RNA. Furthermore bicyclothymidine is an efficient
substitute for natural thymidine in DNA duplex recognition by oligonucleotides. We have shown earlier that substitution of bicyclothymidine for thymidine in a pyrimidine DNA sequence exerts a stabilizing effect on
triple helix formation in the parallel binding motif (
29
).
ACKNOWLEDGEMENTS
We thank ISIS Pharmaceuticals (Carlsbad, CA) for performing the serum resistance
test. Financial support from the Swiss National Science Foundation (grant No.
20-42107.94), the Wander-Stiftung Bern and Ciba-Geigy AG is gratefully acknowledged.
7 Tarköy,M., Bolli,M. and Leumann,C. (1994) Helv. Chim. Acta,77, 716-744.
8 Egli,M., Lubini,P., Bolli,M., Dobler,M. and Leumann,C. (1993) J. Am. Chem. Soc., 115, 5855-5856.
9 Bolli,M., Litten,C., Schütz,R. and Leumann,C. (1996) Chem. Biol.,3, 197-206.
10 Bolli,M. (1994) Nukleinsäure-Analoga mit Konformationell Eingeschränktem Zucker-Phosphat-Rückgrat (`Bicyclo-DNA'): Synthese und Eigenschaften. Dissertation, University of Bern.
11 Hayakawa,Y., Wakabayashi,S., Kato,H. and Noyori,R. (1990) J. Am. Chem. Soc., 112, 1691-1696.
30 Bolli,M., Lubini,P., Tarköy,M. and Leumann,C. (1994) In Sanghvi,Y.S. and Cook,P.D. (eds), Carbohydrate Modifications in Antisense Research. ACS Symposium Series, 580, Washington, DC, pp. 100-117.
31 Bolli,M., Lubini,P. and Leumann,C. (1995) Helv. Chim. Acta,78, 2077-2096.
