RNA duplexes with biphenyl substituents as base replacements are less stable than DNA duplexes

Article abstract of DOI:10.1039/b419278h

Introduction of a hydrophobic biphenyl-C-nucleotide pair into a 11-mer RNA duplex is associated with a net penalty in the free energy of duplex formation of 2.0 kcal mol^-1 or 10 °C in T_m, relative to DNA. These differential stabiliti

Full text of DOI:10.1039/b419278h

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RNA duplexes with biphenyl substituents as base replacements are less
stable than DNA duplexes{
Christine Brotschi and Christian J. Leumann*
Received (in Cambridge, UK) 23rd December 2004, Accepted 15th February 2005
First published as an Advance Article on the web 28th February 2005
DOI: 10.1039/b419278h
Introduction of a hydrophobic biphenyl-C-nucleotide pair into
a 11-mer RNA duplex is associated with a net penalty in the
free energy of duplex formation of 2.0 kcal mol21 or 10 uC in
Tm, relative to DNA. These differential stabilities are of
relevance with respect to the transcriptional and translational
aspects of hydrophobic base-pairs.
Unnatural pairs of heteroaromatic base surrogates in DNA,
devoid of the capability to form hydrogen bonds, have recently
been investigated as universal bases,1–3 as tools to probe base-
recognition and insertion fidelity of DNA polymerases,4–8 as
reporter units,9,10 and as novel, orthogonal base-pairs for
expanding the genetic alphabet.11–15 Within the latter context it
was shown that shape mimics of natural base-pairs, as e.g. the
difluorotoluene–methylindole pair, are structurally accommodated
in a DNA double helix in a side-by-side fashion without distortion
of the helix.16 Such pairs were shown to significantly destabilise
Fig. 1 Sequence information (top), chemical structures of the deoxyribo-
a DNA duplex. On the other hand, non shape mimics with
extended surface areas, as e.g. the propynyl isocarbostyril (PICS)
pair,11 can lead to duplex stabilities that match or even excel
those of natural base-pairs, due to partial interstrand stacking
interactions between the units.17–19
and ribonucleoside residues containing the biphenyl base substitutes
(right), and schematic representation of the interstrand stacking motif
(left).
intercalative base arrangements as a function of the nature of the
backbone (RNA or DNA). Here we set out to study the thermal
and thermodynamic properties of this motif in RNA and RNA–
DNA mixed duplexes and to compare the data with that of DNA
duplexes.
In the context of expanding the genetic code, such unnatural
base-pairs should not only recognise each other and be efficiently
replicated on the DNA level. They also have to be efficiently
transcribed and translated. In the latter two processes, DNA–
RNA and RNA–RNA duplexes are central structures along the
information transfer. Thus the critical question arises, how stability
(and enzymatic processing) varies with respect to the nature of
the backbone (RNA vs. DNA). So far there is only little data
available on RNA containing non-hydrogen bonding base
analogues. It was shown that oligoribonucleotides containing
fluorophenyl–fluorobenzimidazol pairs (isosteric to natural base-
pairs) destabilise RNA duplexes by ca. 5–7 uC in Tm.20,21 However,
no data exist so far on RNA duplexes with partially intercalating
aromatic pairs.
Synthetic details and analytical data for the C-nucleoside rBph,
its building block for RNA synthesis as well as the corresponding
oligoribonucleotides (Fig. 1, n 5 0–3), are contained in the
electronic supplementary information.{ Results for the DNA series
were, in part, already reported previously.18,22 The thermodynamic
data for duplex formation in both the DNA and RNA series
were obtained from 1/Tm vs. ln(c) plots (van’t Hoff plots, see
supplementary information{). Thermal melting (Tm) and thermo-
dynamic data are summarized in Table 1.
Introduction of one rBph-pair into the RNA duplex is
associated with a drop in Tm by 10 uC and a loss in the free
energy of duplex formation (DDG25 uC) of 2.5 kcal mol21, relative
to the unmodified duplex. On the other hand, introduction of one
dBph-pair into the DNA duplex leads to a decrease in Tm of only
2.5 uC and a DDG of 0.5 kcal mol21. Additional Bph-pairs were
then introduced into the duplex to investigate whether a stability
enhancing interaction between the aromatic residues, also occurs
in the RNA context. Indeed, introducing a second and a third
consecutive Bph-pair leads to partial recovery of the thermal and
thermodynamic stabilities in RNA. While in the case of DNA,
three Bph-pairs lead to a Tm that is higher by almost 5 uC relative
We recently found that biphenyl C-deoxynucleosides (dBph,
Fig. 1) form stable self-pairs in DNA duplexes due to interstrand
stacking interactions of the phenyl rings.17,18 In the sequence
context given in Fig. 1 we found that this motif is extendable to at
least 7 pairs with increasing thermal stability of the duplex.22 Thus,
this motif can serve as a model to study the energetics of
{ Electronic supplementary information (ESI) available: synthesis and
analytical data of monomer rBph and oligoribonucleotides, and van’t
Hoff plots for the determination of thermodynamic data. See http://
www.rsc.org/suppdata/cc/b4/b419278h/
*leumann@ioc.unibe.ch
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Table 1 Thermal melting data (Tm) from UV–melting curves (260 nm) and free enthalpy of duplex formation from van’t Hoff plots of DNA or
RNA duplexes with the sequence indicated in Fig. 1
na
DNA Tm
RNA Tm
DG25uc (DNA)
DG25uc (RNA)
DDGd (DNA)
DDGd (RNA)
b
b
0
1
2
3
a
45.0
42.5
46.9
49.9
57.1
47.1
51.9
53.2
213.2
212.7
213.9
214.9
218.1
215.6
216.9
217.7
—
—
+0.5
21.2
21.0
b
+2.5
21.3
20.8
c
conditions: 10 mM NaH2PO4, 0.15 M NaCl, pH 7.0; Tm [uC], estimated error: ¡ 0.5 uC. Tm values at 1.2 mM. [ kcal mol21].
DDG 5 DGn+1 2 DGn.
d
to the unmodified duplex, the same situation in the RNA series is
still associated with a reduction in Tm by ca. 4 uC. It emerges,
however, that introduction of any Bph-pair after the first one
leads to a similar increase in thermal stability in both backbone
series, indicating a similar mode of interaction of the additional
hydrophobic pairs.
Table 2 Thermal melting data (Tm) from UV–melting curves (260 nm)
of DNA or RNA duplexes with one Bph-residue in a bulge position, or
of a DNA–RNA hybrid containing one Bph-pair
a
Tm
Entry Structure
Duplex
1
2
3
a
DNA bulge 59-d(GATGAC Bph GCTAG)-39 45.9 (45.0)
39-d(CTACTG’ 2 ‘CGATC)-59
RNA bulge 59-r(GAUGAC Bph GCUAG)-39 49.1 (57.1)
39-r(CUACUG’ 2 ‘CGAUC)-59
We measured CD spectra of the RNA duplexes in order to
screen for major changes in duplex structure upon introduction of
rBph-pairs (Fig. 2). As expected, the unmodified RNA duplex
shows the typical fingerprint of an A-conformation. Introduction
of one or three rBph-pairs does not significantly change this
pattern. The duplex with n 5 1 leads to a slight red shift of the
ellipticity maximum, which is not the case for the duplex with three
rBph-pairs and for the corresponding DNA duplexes.22 Thus
introduction of only one Bph-pair seems to be associated with a
more pronounced structural effect in the RNA series compared to
the DNA series.
DNA–RNA 59-d(GATGACBphGCTAG)-39
hybrid 39-r(CUACUGBphCGAUC)-59
conditions: c 5 1.2 mM in 10 mM NaH2PO4, 0.15 M NaCl,
36.1 (43.5)
pH 7.0. estimated error in Tm: ¡ 0.5 uC; in parenthesis: Tm data of
parent, non-modified duplex.
corresponds to L of the loss in Tm relative to a pure RNA duplex.
Thus we conclude that any accommodation of a Bph-residue in a
duplex that contains at least one RNA strand leads to reduced
thermal stability relative to a pure DNA duplex.
In order to determine the effect on duplex stability of a
hydrophobic residue in an internal bulge position, in DNA relative
to RNA and to determine the effect of one Bph-pair insertion into
a DNA–RNA hybrid, we measured the thermal stabilities of the
duplexes listed in Table 2. Introduction of a Bph-unit in a DNA
bulge stabilises the duplex relative to the unmodified duplex by
0.9 uC, while the same sequence arrangement in the RNA leads to
a destabilisation by 8.0 uC (Table 2, entries 1 and 2). The increase
in stability in the former case is an indication for intercalation
of the Bph-residue into the base-stack. In the case of RNA,
intercalation is either associated with a significant energetic penalty
or does not occur. The introduction of a complete Bph-pair into
a DNA–RNA hybrid (Table 2, entry 3) again leads to a decrease
in Tm, relative to the unmodified duplex, by 7.4 uC, which
It appears that interstrand stacking recognition of a single Bph-
pair is associated with a net penalty in free energy of duplex
formation of 2.0 kcal mol21 in RNA relative to DNA. A possible
explanation for this could lie in the different conformational
families (A vs. B) in which the geometry and the extent of overlap
of the aromatic units of both strands lead to less efficient stacking
in the case of RNA. Another reason could be the reduced
flexibility and dynamics of the RNA backbone relative to DNA.
Indeed, it has been shown before that classical aromatic inter-
calators tend to stabilise double stranded DNA more than RNA
duplexes.23 Along the same lines it was shown recently that an
oligodeoxynucleotide containing a covalently attached pyrenyl
intercalator, pairs preferentially to a DNA relative to an RNA
complement.24 In the context of more complex nucleic acid
structures it was also shown that the i-motif, consisting of pairwise
intercalated C–C+ nucleotide-pairs, is significantly more stable in
DNA as compared to RNA.25
In conclusion, the data presented here show that interstrand
intercalation of hydrophobic base-pairs that can adopt stabilities
of natural base-pairs in DNA, can be significantly less stable in
RNA duplexes or DNA–RNA hybrids.
These results are of interest in the following context. They
indicate that a hydrophobic base-pair that is not a shape mimic
of a natural base-pair, and that takes its interaction energy mainly
from interstrand stacking, may compromise the process of
transcription and translation due to significant changes in base–
base affinity, by changing from the DNA to the RNA backbone
and consequently from a B to an A-helical structure. Thus, this
finding is of importance for the future design of hydrophobic base-
pairs aiming at expanding the genetic alphabet. Furthermore, the
fact that an additional hydrophobic base in a bulge position within
Fig. 2 CD spectra of RNA-duplexes from Table 1 (n 5 0, 1, 3), c 5
3.6 mM, in 10 mM NaH2PO4, 0.15 M NaCl, pH 7.0, T 5 20 uC.
2024 | Chem. Commun., 2005, 2023–2025
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The Swiss National Science Foundation (grant-No. 200020-
100178) is gratefully acknowledged for financial support.
Christine Brotschi and Christian J. Leumann*
Department of Chemistry and Biochemistry, University of Bern,
Freiestrasse 3, CH-3012 Bern, Switzerland.
E-mail: leumann@ioc.unibe.ch; Fax: +41 31 631 3422; Tel: +41 631 4355
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