Watson-Crick and Hoogsteen tri-base pairing: A co-crystal structure of a 2: Mplex of 6-isopropyluracil and adenine nucleobases

Article abstract of DOI:10.1039/c3ra40766g

The formation of a tri-base pairing from free nucleobases is rare. A co-crystal structure obtained from an equimolar mixture of 6-isopropyluracil and adenine shows the formation of a tri-base pairing, where one molecule of free adenine is adhered to two molecules of the free pyrimidine nucleobase, involving both Watson-Crick and Hoogsteen base-pairing interactions. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3ra40766g

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Watson–Crick and Hoogsteen tri-base pairing: a co-
crystal structure of a 2 : 1 complex of 6-isopropyluracil
and adenine nucleobases3
Cite this: RSC Advances, 2013, 3, 7282
Received 13th February 2013,
Accepted 20th March 2013
K. Radhakrishnan, Laxmi Narayana Burgula and Lal Mohan Kundu*
DOI: 10.1039/c3ra40766g
www.rsc.org/advances
1
9
The formation of a tri-base pairing from free nucleobases is rare. A
co-crystal structure obtained from an equimolar mixture of
(A:T:A), at low temperature. The complex was reported to be
formed due to quasi-Watson–Crick and Hoogsteen base pairing.
Here, we present a Watson–Crick and Hoogsteen tri-base
6
-isopropyluracil and adenine shows the formation of a tri-base
ip
ip
pairing, where one molecule of free adenine is adhered to two
molecules of the free pyrimidine nucleobase, involving both
Watson–Crick and Hoogsteen base-pairing interactions.
pairing co-crystal structure ( U:A: U) formed by adenine and
ip 20a–d
6-isopropyluracil ( U)
in a 1 : 2 ratio. 6-Isopropyluracil was
used to replace thymine, since the 6-isopropyl substituent can
render a higher hydrophobic property compared to the methyl
group on thymine. The co-crystal was obtained when equimolar
quantities of 6-isopropyluracil were mixed with adenine in water–
methanol (2 : 1 v/v), followed by slow evaporation of the solvents
at ambient temperature. One molecule of the adenine nucleobase
was found to be flanked by two molecules of the uracil derivative,
forming a U:A: U tri-base pair (Fig. 1A). Careful crystal structure
analysis showed that part of the adenine is hybridized to a
6-isopropyluracil, through Watson–Crick base-pairing. The other
part of the adenine is bound to a second pyrimidine base via
Hoogsteen interactions.
To the best of our knowledge, such a co-crystal structure,
involving both Watson–Crick and Hoogsteen interactions from the
free nucleobases has not been reported. Interestingly, although
the N1 position of the pyrimidine and the N9 position of the
adenine (where a sugar moiety is attached in DNA or RNA) were
not protected, they did not participate in forming the ‘tri-base
pairing’ structure. The structure of the complex is in good
agreement with the reported X-ray diffraction studies obtained
from short oligonucleotides. Hecke et al. demonstrated the crystal
structure of an undecamer oligonucleotide sequence, which forms
a T:A:T triple helix via Watson–Crick–Hoogsteen base pairing.
The observation of such a co-crystal could be important in
developing modified oligonucleotides and molecular architec-
tures.
Fig. 1B shows the ORTEP diagram of the U:A: U complex at
ambient temperature. The structure demonstrates the formation
of strong Watson–Crick base pairing where N10–H10 and C20–O4
of a U are strongly H-bonded to N7 and N8–H8A of the adenine,
respectively. Hoogsteen base paring could be observed, involving
N4 of the imidazole ring and the amine group (N8–H8B) of the
adenine forming strong H-bonds with a second U molecule (N1–
H1A and C10LO1, respectively). Thus, the amine group of the
The double helical structure of naturally occurring DNA is largely
attributed to Watson–Crick base pairing interactions, along with
1,2
pi-stacking forces. Usually, adenine forms a H-bonded complex
with thymine, whereas guanine forms a base-pair with cytosine.
Hoogsteen base-pairs were also found in nature, where the
3–5
ip
ip
formation of a triple helix was more probable. The formation of
such triple helices could be important to selectively modulate gene
expression, by controlling gene transcription. Hoogsteen first
reported
a crystal structure of a 1 : 1 complex between
1
-methylthymine and 9-methyladenine in which N7 and C6-NH
2
6
of adenine form an unusual complex with the thymine moiety.
Later on, a number of crystal structures, NMR and melting
temperature studies of the short oligonucleotides were reported to
elucidate various Hoogsteen interactions,
protein complexes and G-quadruplex.
3
,5,7–9
including RNA–
Chemically modified
10
11,12
oligonucleotides, such as peptide nucleic acid, efficiently form a
triple helix involving both Watson–Crick and Hoogsteen interac-
13–16
tions.
Hoogsteen interactions were also demonstrated with
complementary nucleobases stabilized by metal-ion coordina-
17,18
tions.
However, there is rare evidence from the co-crystal
5
structure of the free nucleobases to illustrate such complex
formation involving both Watson–Crick and Hoogsteen interac-
tions. The co-crystal structure from the free nucleobases is indeed
very difficult to achieve, primarily due to the poor solubility of the
nucleobases in organic solvents. Recently, Chandrasekhar and co-
workers reported the crystal structure of a complex, in which one
molecule of thymine is flanked by two molecules of adenine
22,23
ip
ip
24
ip
Department of Chemistry, Indian Institute of Technology Guwahati, North Guwahati,
7
81039, Assam, India. E-mail: lmkundu@iitg.ernet.in; Fax: +91-3612582349;
Tel: +91-3612582326
Electronic supplementary information (ESI) available. CCDC 917227. For ESI and
crystallographic data in CIF or other electronic format see DOI: 10.1039/c3ra40766g
ip
3
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ip
ip
Fig. 2 Supramolecular arrangement of the U:A: U co-crystal showing layered
structures. Water molecules act as a bridge between the layers.
interactions were also observed in the crystal lattice. Relatively
strong pi-stacking (3.636 Å) occurred between the pyrimidine base
and the five-membered imidazole ring of the adenine. Another pi-
ip
stacking (3.953 Å) was observed involving the U and the six-
3
membered pyrimidine ring of the adenine (see the ESI ).
ip
ip
In addition to the X-ray diffraction study, we also analysed the
Fig. 1 (A) shows schematic presentation of the U:A: U complex. (B) represents the
1
ORTEP diagram of the co-crystal.
solution-phase HNMR spectrum, after dissolving the co-crystal in
1
DMSO-d
6
(Fig. 3). The HNMR clearly proves the presence of
ip
1
adenine, as well as U (see ESI
3
for the HNMR spectra of adenine
ip
25
adenine is involved in two kinds of hydrogen bonding, which has
also been observed in DNA triple helix structures. Four water
molecules were found to be present in the unit cell, participating
in H-bond formation with N6 and N5–H5 of adenine, along with
O4 and O1 of the two pyrimidine bases (Table 1). Water molecules
were also found to act as a ‘bridge’ between two layers in the
supramolecular arrangement of the complex. The supramolecular
architecture, as depicted in Fig. 2, shows the formation of planar
layers interconnected by the water molecules. As is evident, the
water molecules create a pentagonal void space between the layers
in the supramolecular assembly. Two types of pi-stacking
and U, under identical conditions). Interestingly, the deshield-
ing effects by hydrogen-bond formation were not observed on the
chemical shift of those protons involved in Watson–Crick and
Hoogsteen interactions in the co-crystal structure, possibly due to
26,27
6
disruption of the H-bonds in DMSO-d solution.
Conclusions
This article demonstrates a rare co-crystal structure derived from
the complexation of a modified pyrimidine nucleobase, 6-iso-
propyluracil, and adenine in their free forms. The crystal structure
ip
ip
Table 1 H-bond distances and interactions in the U:A: U co-crystal as depicted in the ORTEP diagram (Fig. 1B)
…
…
…
…
D–H
A
D–H/Å
H
A /Å
D–H A /Å
D–H A/u
Symmetry
…
N8–H8A O4
0.86
0.86
0.86
0.86
0.86
0.86
0.86
0.96
0.96
2.11
2.12
1.97
1.95
2.02
2.02
1.94
2.60
2.59
2.962(4)
2.956(5)
2.827(4)
2.801(4)
2.874(5)
2.867(5)
2.782(4)
2.974(6)
3.410(6)
173
165
176
171
173
166
165
104
144
1 2 x, 1 2 y, 1 2 z
21 + x, y, z
1 2 x, 1 2 y, 1 2 z
1 2 x, 1 2 y, 1 2 z
1 + x, y, z
1 2 x, 2y, 1 2 z
x, 21 + y, z
…
N8–H8B O1
…
N10–H10 N7
…
N50–H23 O3
…
N1–H1A N4
…
N51–H9 O2
…
N5–H5 O5
…
C2–H2C N51
…
C2–H2C O2
1 2 x, y, 1 2 z
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4
1
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Fig. 3 HNMR spectrum (400 MHz) of the U:A: U co-crystalline material dissolved
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6
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ip
2
21
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ip
ip
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2
ip
ip
2
Notes and references
7
10
2
2
5
5
5
2
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ip
ip
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6 H
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ip
ip
1
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ip
ip
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284 | RSC Adv., 2013, 3, 7282–7284
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