Probing the furanose conformation in the 2′-5′strand of isoDNA:RNA duplexes by freezing the nucleoside conformations

Article abstract of DOI:10.1039/c0cc05402j

Sugar conformations in the isoDNA strand of isoDNA:RNA duplexes are preferred S-type locked/frozen in contrast to N-type locked conformations preferred in DNA:RNA duplexes. The Royal Society of Chemistry.

Full text of DOI:10.1039/c0cc05402j

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COMMUNICATION
Probing the furanose conformation in the 20–50strand of isoDNA : RNA
duplexes by freezing the nucleoside conformationsw
Namrata Erande, Anita D. Gunjal, Moneesha Fernandes and Vaijayanti A. Kumar*
Received 6th December 2010, Accepted 4th February 2011
DOI: 10.1039/c0cc05402j
Sugar conformations in the isoDNA strand of isoDNA : RNA
duplexes are preferred S-type locked/frozen in contrast to
N-type locked conformations preferred in DNA : RNA duplexes.
studies on single-stranded 20,50-RNA9a and 20,50-d(G4C4)9b
implied an extended structure in which the sugar conforma-
tions were N-type. The C30-endo or N-type sugar pucker has
also been observed for single-stranded 20,50-RNA in crystal
structures.10 Thus, the structural requirements of the isoRNA/
isoDNA : RNA duplexes (predominant S-type geometry of the
20,50-strand in isoRNA/isoDNA : RNA duplex) do not match
with the sugar conformations at the single stranded 20,50-
oligomer level. In the single-stranded 20,50-oligomer, the
stereoelectronic (gauche and anomeric) effects of the 20-hydroxy
group, would accentuate the preference for 30-endo (N-type)
sugar conformations in the absence of the 30-OH group.
While modelling the isoDNA : RNA duplex, it was therefore
assumed that the RNA strand would impose its structure11 on
the isoDNA strand, and the individual nucleosides in the
isomeric DNA strand would be required to assume S-type
i.e., C20-endo conformation while binding to the complemen-
tary RNA strand to give rise to a stable duplex. Thus, it was
suggested that the iso-linked DNA would be under conforma-
tional and topological constraints in the duplex.8
20-50-Phosphodiester-linked RNA occurs naturally (Fig. 1).1
It could have been a primary substitute to the natural
30,50-linkage as it is the major product of many non-template,
non-enzymatic oligomerizations of nucleotide monomers
depending on conditions,2 but it is not used to encode the
genetic information. In 1991, Damha et al. synthesized the
20,50-linked RNA (isoRNA Fig. 1) that was found to exhibit
self-pairing as well as pairing with RNA,3 but its complexation
with complementary DNA was not observed. The duplexes of
isoRNA with complementary RNA (isoRNA : RNA) showed
very similar CD patterns as the natural 30,50-DNA : RNA
duplexes, from which it was deduced that the overall structures
of these complexes would be comparable i.e., compact A-form
duplex structure.4 NMR studies in solution for self-pairing
isoRNA duplexes suggested a similar structure in which the
ribose sugars assumed C20-endo (S- type) puckering for the
isoRNA strands.5 From the similarity found in the CD spectra
of isoRNA : RNA and the self-pairing isoRNA duplexes,
along with the NMR studies for the latter duplex, it was
deduced that the 20,50-RNA strand in the isoRNA : RNA
duplex might also assume the structural features of a natural
DNA strand and the sugar residues would adopt S-type
i.e., C20-endo conformations. Molecular modelling studies
reported by Yathindra et al.5 also have suggested that to arrive
at the compact A-type overall structure of the isoRNA : RNA
duplex, the S-type or C20-endo geometry of repeating nucleo-
tides is stereochemically favoured in the isoRNA strand of the
duplex. Very similar results were found independently by
Breslow6 and Switzer7 for the 20,50-linked 30-deoxyribonucleic
acids (isoDNA) as well. CD spectroscopy,7a and modeling
studies8 predicted that the sugar conformations in the isoDNA
strand of isoDNA : RNA duplexes would have predominantly
S-type sugar conformations. Contrary to this, the NMR
Considering these predictions, we hereby report the study of
the effects of the incorporation of monomers which bear either
locked or frozen S-type/N-type sugar conformations. Such
studies are not previously reported for the isoDNA : RNA
duplexes, although the literature is abundant with the locked
or frozen DNA analogues in DNA : RNA duplexes that have
Division of Organic Chemistry, National Chemical Laboratory,
Pune 411008, India. E-mail: va.kumar@ncl.res.in;
Fax: +91 20 25902623; Tel: +91 20 25902623
w Electronic supplementary information (ESI) available: 1H, 13C, 31P
NMR, mass spectral data for compounds II–IV, HPLC profiles and
mass spectra for the modified oligomers 2–9, and melting curves for
the complexes with RNA. See DOI: 10.1039/c0cc05402j
Fig. 1 30-50-DNA/RNA and 20-50-isoDNA/isoRNA in compact and
extended conformations.
c
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Chem. Commun., 2011, 47, 4007–4009 4007

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an entropic advantage.12,13 We envisaged that applying such
structural locks on the isoDNA strand should allow us to
understand the structural preferences of the sugars in the
isomerically-linked strand of the chimeric duplexes and thereby
provide experimental proof for the proposed structural pre-
ferences. The specific RNA binding of isoDNA, supplemented
by favoured geometrical features for complex formation and
enzymatic stability, would have potential applications in
antisense therapeutics.
According to the proposed model,11 the induced S-type
conformations in the isoDNA strand obtained by either
chemically locking or freezing the structure conducive for
RNA binding should positively enhance the strength of bind-
ing to RNA and vice-versa for N-type conformations. Such
experimental work is often important and necessary, as the
predicted 20/30-endo conformational preferences of nucleosides
using MD simulations sometimes do not agree with the
experimental observations.14
We compared the sugar conformations of 30-deoxy uridine,
the S-type/N-type locked systems and also of the 30-ribofluoro/
xylofluoro nucleosides (Fig. 2, R1 = R2 = H, and ESIw).
30-Deoxyuridine was shown to be in 97% N-type,15 i.e., C30-endo
sugar pucker which is consistent with the O40-C10-C20-O20
gauche effect as well as the anomeric effect which brings the
nucleobase in pseudoaxial orientation.10 The 30-O-40-
C-methylene-linked uridine (US)15 (C20-endo locked) and the
xylo-30-O-50-C-methylene-linked uridine (UN)16 (C30-endo
locked) would lock the monomer in S-type and N-type
conformations, respectively (Fig. 2, I & II, R1 = R2 = H, and
ESIw). In compound II, the flexible 5-membered ring probably
allows some amount of S-type character. The monomeric
conformations of 30-deoxy-30-ribofluoro uridine (rUF)17 III,
and 30-deoxy-30-xylofluoro uridine (XUF)18 IV, were found to
be almost frozen in S-type and N-type conformations, respec-
tively (ESIw). The %S for each chosen unit was calculated
from the H10–H20 NMR coupling constants as earlier reported
(ESIw).15 Compounds I–Ib were synthesized by using the
reported procedure.15 Transformation of II–IV (R1 = R2 =
H, Fig. 2) to the DMT derivatives IIa–IVa (R1 = DMT, R2 =
H, Fig. 2) and subsequent conversion to 20-O-phosphoramidite
derivatives, yielded the desired monomeric building blocks
(Fig. 2, IIb–IVb) for incorporation into isoDNA oligomers.
We chose to synthesize a biologically relevant sequence used
for miRNA downregulation.19 The 20-50-linked isoDNA
sequence DNA1 was synthesized using commercially available
monomeric building blocks and standard phosphoramidite
chemistry on solid supports using an automated Bioautomation
DNA Synthesizer. Incorporation of the modified units
(Ib–IVb) at the center and towards the 20-end could be
effectively achieved to get the modified oligomers. The oligomers
(DNA1–DNA9) listed in Table 1 were cleaved from the solid
support, purified by HPLC and characterized by MALDI-TOF
mass spectrometry (ESIw).
The thermal stabilities (melting temperatures, Tms) of
isoDNA : DNA and isoDNA : RNA duplexes containing
N-type locked, S-type locked, ribo-fluoro and xylo-fluoro
modifications were determined and compared with the
unmodified duplex to study their hybridization properties.
The results of UV-Tm studies of complexes with complementary
RNA are summarized in Table 1. The unmodified DNA1
and modified 20,50-linked oligomers (DNA2–DNA9) were
found to bind selectively only to complementary RNA,
exhibiting sharp monophasic melting transitions, while no
transitions were observed for the complexes with complementary
DNA. The complexes formed with oligomers containing an
increasing number of N-type locked monomer (DNA4, DNA5)
Table 1 UV-Tm (1C) values of isoDNA : RNA duplexesa
No
Sequences (50 - 20)
Tm 1C DTm 1C
50.5
51.0 +0.5
52.8 +2.3
DNA 1 DNA 1 CACCATTGTCACACTCCA
DNA 2 DNA 2 CACCATTGTCACACUSCCA
DNA 3 DNA 3 CACCATTGUSCACACUSCCA
DNA 4 DNA 4 CACCATTGTCACACUNCCA
—
49.4
ꢀ1.1
ꢀ7.7
ꢀ4.8
ꢀ6.0
0.0
DNA 5 DNA 5 CACCATTGUNCACACUNCCA 42.8
DNA 6 DNA 6 CACCATTGTCACACXUFCCA
DNA 7 DNA 7 CACCATTGXUFCACACXUFCCA 44.5
45.7
DNA 8 DNA 8 CACCATTGTCACACrUFCCA
50.5
DNA 9 DNA 9 CACCATTGrUFCACACrUFCCA 51.7 +1.2
a
Melting temperatures (Tms) were obtained from the maxima of the
first derivatives of the melting curves (A260nm versus temperature),
measured in buffer containing 10 mM sodium phosphate, 150 mM
sodium chloride, pH 7.4, using 1 mM concentration of each of the two
complementary strands. Each experiment was repeated at least thrice
and the values are accurate to ꢁ0.5 1C. Complementary RNA
sequence = 50-UGGAGUGUGACAAUGGUG-30. Tm of DNA
50-CACCATTGTCACACTCCA-30 with complementary RNA =
59.0 1C. S-locked uridine (US), N-locked uridine (UN), 3’-ribo-
3’-fluoro uridine (rUF), 3’-xylo-3’-fluoro uridine (XUF).
Fig. 2 30-Deoxyribo nucleosides and proposed locked/frozen 30-endo
and 20-endo nucleoside analogues (I–IV).
c
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with complementary RNA were considerably destabilized
compared to the control 20,50-sequence (DTm E ꢀ1.1 to
ꢀ7.7 1C). In contrast, the oligomers containing the S-type
locked monomers (DNA2, DNA3) effected modest stabilization
of the complex with RNA (DTm E +0.5 to +2.3 1C).
Oligomers bearing locked S-type units thus formed more
stable complexes with RNA compared to the unmodified
complex. Apparently, the S-type locked conformation at the
modified site would be in compliance with the predicted S-type
conformations of the isoDNA strand in the isoDNA : RNA
duplex and therefore could stabilize the duplex. The imparted
stability due to pre-organization in this geometry was not
found to be as large as in the case of 30,50-LNA : RNA
duplexes (DTm E + 4 1C/mod).13a This could be because
the S-type conformations would bring the nucleobases in
pseudoequatorial position in which, the stacking and hydro-
gen bonding interactions are not as strong as in the N-type
sugar geometry,10 when the nucleobase assumes pseudoaxial
orientation, as in LNA : RNA duplexes. The significant
destabilization of the isoDNA : RNA complex by locking the
sugar conformation in N-type geometry was also in compli-
ance with the predicted geometry of the isoDNA : RNA
duplex. The 30-deoxy-30-xylofluoro uridine, found to be
in E 100% N-type geometry, caused destabilization of the
duplex (Table 1, DTm E ꢀ4.8 to ꢀ6.0 1C) but the oligomer
with the 30-deoxy-30-ribofluoro uridine modifications in which
the sugar geometry is S-type, showed similar melting behaviour
as unmodified duplex (DTm E + 1.0 1C). These results
indicate that N-type to S-type conformational change that
the 30-deoxyuridine presumably undergoes while in duplex
state is to some extent resisted by 30-deoxy-30-xylofluoro
riboside due to the favourable O40-C40-C30-F30 gauche effect
in N-type sugar geometry, considering comparable steric
interactions between fluorine and hydrogen atoms15 (Fig. 3).
For the 30-deoxy-30-ribofluoro derivative the preferred
sugar pucker would be S-type again due to dominating
O40-C40-C30-F30 gauche effect. The frozen conformation could
not increase the stability of the duplexes when present in the
oligomer probably due to its inability to further strengthen
the stacking and hydrogen bonding interactions in S-type
geometry of the sugar when the base orientation becomes
pseudoequatorial.
Stabilization of isoDNA : RNA duplexes by S-locked/frozen
monomer units and destabilization of the same by N-locked/
frozen monomers provides a proof, for the first time, for the
prediction that in stable isoDNA : RNA duplexes, the DNA
strand would prefer to assume S-type geometry. As the oligomers
bind only to the RNA targets and chemical modifications are
known to make them compatible with biological environments,15
this work opens up an entirely new paradigm of oligonucleotides
for development as antisense therapeutics.
This work was supported by CSIR, New Delhi (NWP0036)
and in part by Wellcome Trust, UK. NE got her fellowship
from UGC, New Delhi.
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Fig. 3 O40-C40-C30-F30 gauche effect in 30-deoxy-30-xylofluoro and
30-deoxy-30-ribofluoro sugars.
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Chem. Commun., 2011, 47, 4007–4009 4009