Molecular recognition of a thymine bulge by a high affinity, deazaguanine-based hydrogen-bonding ligand

Article abstract of DOI:10.1039/b817733n

7-Deazaguanine (7-DeG) was developed as a hydrogen-bonding module capable of enhanced recognition of uracil (U) and thymine (T); a water-soluble derivative displayed high affinity and selectivity toward DNA and RNA duplexes containing single T- and U-bulg

Full text of DOI:10.1039/b817733n

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Molecular recognition of a thymine bulge by a high affinity,
deazaguanine-based hydrogen-bonding ligandw
Hugo C. Ong, Jonathan F. Arambula, Sreenivasa Rao Ramisetty, Anne M. Baranger
and Steven C. Zimmerman*
Received (in Cambridge, UK) 8th October 2008, Accepted 4th November 2008
First published as an Advance Article on the web 8th December 2008
DOI: 10.1039/b817733n
7-Deazaguanine (7-DeG) was developed as a hydrogen-bonding
module capable of enhanced recognition of uracil (U) and
thymine (T); a water-soluble derivative displayed high affinity
and selectivity toward DNA and RNA duplexes containing
single T- and U-bulges.
Single-base bulges in DNA are irregularities that may arise
from replication errors or from recombination between
Fig. 1 Hydrogen-bonded complex of ligand 5 to thymine (T).
imperfectly homologous sequences.1 Owing to their inherent
thermodynamic instability, bulges are susceptible targets for
small molecules and proteins with recognition capabilities. A
number of ligands have been developed that can recognize
adenine (A), guanine (G), or cytosine (C) at a DNA bulge or
mismatch site. These ligands consist of a bicyclic ring system
for intercalation and hydrogen-bonding groups for base-
specificity.2 To our knowledge, there are no known ligands
that bind a thymine (T) bulge in preference to the other base
bulges.3 This may in part be attributed to its acceptor–
donor–acceptor (ADA) hydrogen-bonding motif pairing
weakly.4
Schematic illustration of T-bulge recognition by 5.
Table 1 Equilibrium association constants of various 7-DeG and
DAP derivatives in CDCl3. The association constants are averages of
at least two experiments by titration or dilution at 20 1C in which
values agreed within Æ15%
Herein we introduce 7-deazaguanine (7-DeG) as a module
capable of binding thymine and uracil with higher affinity than
most modules containing the donor–acceptor–donor (DAD)
motif. The 7-DeG unit with an intramolecularly hydrogen-
bonded amide group at N-2 (purine numbering) contains a
rigid bicyclic structure that preorganizes the hydrogen-bond
donor and acceptor groups.5 The rigidification occurs partly
through the formation of an intramolecular hydrogen-bond
with NH-1 (Fig. 1) and is clearly seen in the X-ray analysis of
1.6 Possibly as a result of the greater degree of preorganization,
1H NMR binding studies in CDCl3 reveal a comparatively high
stability for the 1ÁU complex (Ka = 5800 MÀ1) (Table 1).
Typical diamidopyridine derivatives such as 3–4 exhibit weaker
Compound
X
Y
Z
R2
Ka/MÀ1
1 (7-DeG)
t-Bu
CH2CO2Et
—
–H
5800
2 (7-DeG)
2 (7-DeG)
a
a
—
—
–H
2860
2560
–CH3
affinity toward thymine and uracil (Ka E 101–103
M
À1).4c,7
3 (DAP)
4 (DAP)
C7H9
—
—
C7H9
C7H9
–H
–H
160
40
Other factors that may play a role in the enhanced binding
exhibited by 1 include enhanced acidity or basicity of the
hydrogen-bond donor and acceptor sites and the orientation
of the NH-7 group.
To illustrate the utility of 7-DeG, ligand 5 was synthesized6
as a water-soluble derivative for recognition of thymine and
uracil bulges in oligonucleotides (Fig. 1). A naphthoyl unit was
incorporated to introduce a hydrophobic aromatic group
extending the ligand’s intercalative ability while still retaining
affinity toward the target thymine or uracil. The design was
validated by our studies in chloroform, which showed
comparable affinity of 2 towards thymine and uracil (Table 1).
Department of Chemistry, University of Illinois at Urbana-
Champaign, 600 S. Mathews Avenue, Urbana, IL 61801, USA.
E-mail: sczimmer@illinois.edu; Fax: +1 (0)217 244 9919;
Tel: +1 (0)217 233 6655
w Electronic supplementary information (ESI) available: Synthesis of
ligands, chloroform binding studies, thermal denaturation studies, and
ITC profiles. CCDC 704965. For ESI and crystallographic data in CIF
or other electronic format, see DOI: 10.1039/b817733n
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Fig. 3 ITC binding isotherms 5 to (a) DNA A and (b) RNA J. In (b)
one anomalous point resulting from an air bubble has been removed
(see ESIw). For (a) and (b) ligand was added to 5–40 mM solution of
DNA/RNA in 10 mM sodium cacodylate pH 7.0, 100 mM NaCl and
1.0 mM EDTA.
values. It is proposed that the K1 value (henceforth called Kd)
represents specific binding at the bulge, whereas the second
complexation event represents nonspecific complexation,
including potentially at the bound bulge site. Similar findings
have been reported by others.8 Using a 2-aminopurine con-
taining oligonucleotide with fluorescence titration, it was
confirmed that the binding with 5 was 1:1 with a Kd similar
to that determined by ITC.6
Fig. 2 Graphical summary of thermal denaturation studies with
ligands 5–9 with various single base bulges flanked with purines and
pyrimidines. 20 mM ligand, 5 mM duplex, 10 mM sodium cacodylate
pH 7.0, 100 mM NaCl, 1 mM EDTA.
Ligand 5 was determined to bind deoxyoligonucleotide A
with a Kd of 4.0 Æ 0.6 mM. Ligand 5 showed a 5-, 19-, and
13-fold decrease in affinity towards duplexes containing
C- (B), A- (C), and G- (D) bulged bases flanked by guanines,
respectively (Table 2). To examine the effect of flanking
pyrimidines (C), which can increase the equilibrium of a
T-bulge towards its extrahelical state,9 ITC studies were
performed with duplexes E–H. High affinity of 5 towards
E was observed in addition to the same selectivity pattern
towards other base bulges (F–G) indicating that selectivity
towards a T-bulge containing DNA duplex is accomplished
regardless of whether the bulge is flanked by guanine or
cytosine. No binding was detected for H or the normal duplex
d(TCCACCCAAC) indicating the Kd to be 4100 mM.
Diamidopyridine derivative 9 binding duplex A provided an
B25-fold decrease in affinity (Kd = 95 mM) when compared to
A qualitative structure–function relationship was established
by thermal denaturation studies of DNA oligonucleotides
containing a single T-bulge (Fig. 2). In the presence of ligand
5, DNA duplex A containing a single T-bulge flanked by
guanines was stabilized by +7.0 1C. Control ligands, lacking
one or more features of ligand 5, were also studied by thermal
denaturation. Control 6 lacks the intercalative naphthoyl
group, while retaining its hydrogen-bonding capability. Control
7 lacks the 7-DeG unit entirely. Neither 6 nor 7 stabilized
duplex A, suggesting that both aromatic groups are essential for
stabilization. Alkyl substitution of NH-9 (8) stabilizes duplex A
by +2.9 1C, suggesting that blocking the DAD motif weakens
the stabilization. Compound 9 provided no stabilization,
despite having both the naphthalene and a recognition group
that is fully complementary to T; however, an increased melting
temperature was observed at higher concentrations. It should
be noted that 9 is not a perfect comparison because it contains
only a single basic amino group. No stabilization of fully
complementary duplex d(TCCACCCAAC) was observed in
the presence of 5.
Table 2 Equilibrium dissociation constants of ligand 5 to various
nucleic acid secondary structures determined by ITC
Base bulge
Duplex
Flanking bases
Kd/mMa
T
C
A
G
T
C
A
G
A
B
C
D
E
F
G
H
I
G
G
G
G
C
C
C
C
—
G
4.0
21
74
51
7.4
37
61
Quantitative determination of the affinity of ligand 5 for the
single-base bulge sequences A–J was carried out by isothermal
titration calorimetry (ITC) (Fig. 3). Each dataset was fit to
several models and in each case it was found that a sequential
two-site model gave the best fit,6 whereas a single-site model fit
the data poorly. The K2 values varied considerably in magni-
tude, but, with one exception, were always less than the K1
4100
4100
9.9
Duplex
U (RNA)
J
a
Average of 2–4 measurements. Repeat experiments were within 20%
error.
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5 binding to A. These results are in accordance with both the
DTm and chloroform studies and indicate that the special
features of the 7-DeG unit are partially responsible for the
observed affinity. Lastly, ITC binding analysis showed that
5 could bind RNA J with a Kd of 9.9 Æ 1.2 mM (Fig. 3b,
Table 2), thus confirming the ligand’s ability to bind both
T- and U-bulges.
Chem. Lett., 2004, 14, 3431; (e) H. Suda, A. Kobori, J. Zhang,
G. Hayashi and K. Nakatani, Bioorg. Med. Chem., 2005, 13, 4507;
(f) F. Takei, H. Suda, M. Hagihara, J. Zhang, A. Kobori and
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7530.
3 There are ligands that are pyrimidine selective, binding both C and T:
e.g., see ref. 2d.
4 (a) J. Pranata, S. G. Wierschke and W. L. Jorgensen, J. Am. Chem.
Soc., 1991, 113, 2810; (b) T. J. Murray and S. C. Zimmerman,
J. Am. Chem. Soc., 1992, 114, 4010; (c) S. C. Zimmerman and
T. J. Murray, Tetrahedron Lett., 1994, 35, 4077; (d) J. R. Quinn,
S. C. Zimmerman, J. E. Del Bene and I. Shavitt, J. Am. Chem.
Soc., 2007, 129, 934.
5 A urea of 7-deazaguanine (7-DeUG) has been used as a component
in a highly stable ADDAÁDAAD quadruply hydrogen-bonded
complex: H. C. Ong and S. C. Zimmerman, Org. Lett., 2006, 8,
1589.
In summary, we have introduced 7-DeG as a high affinity
recognition unit for the selective binding of thymine and
uracil. Selective recognition of single base bulges in DNA
could provide use in single nucleotide polymorphism (SNP)
analysis,10 and in RNA, where bulges are ubiquitous and play
functional roles in cellular regulatory processes. The 7-DeG
unit may also find application in supramolecular chemistry,
where stronger DADÁADA motifs are needed. Structural
elucidation studies of 5ÁA and 5ÁJ are currently underway.
We thank the National Science Foundation and the
National Institutes of Health for their generous support of
this work.
6 See ESIw for details.
7 (a) A. D. Hamilton and D. Van Engen, J. Am. Chem. Soc., 1987,
109, 5035; (b) TriaminotriazineÁcyanuric acid (ADAÁDAD)
complexes are more stable: A. G. Bielejewska, C. E. Marjo,
L. J. Prins, P. Timmerman, F. de Jong and D. N. Reinhoudt,
J. Am. Chem. Soc., 2001, 123, 7518; (c) Adenine itself binds
T weakly, Ka = 130 MÀ1 (see ref. 3c for this and many more
examples).
8 (a) M. Kaul and D. S. Pilch, Biochemistry, 2002, 41, 7695;
(b) M. Kaul, C. M. Barbieri and D. S. Pilch, J. Mol. Biol., 2005,
346, 119; (c) J. R. Thomas, X. Liu and P. J. Hergenrother,
Biochemistry, 2006, 45, 10928.
9 Pyrimidine bulges have a propensity to be in an intra- and
extrahelical equilibrium when flanked by purines (temperature
dependent), and extrahelical when flanked by pyrimidines:
(a) M. W. Kalnik, D. G. Norman, M. G. Zagorski, P. F. Swann
and D. J. Patel, Biochemistry, 1989, 28, 294; (b) M. W. Kalnik,
D. G. Norman, B. F. Li, P. F. Swann and D. J. Patel, J. Biol.
Chem., 1990, 265, 636; (c) K. M. Morden, B. M. Gunn and
K. Maskos, Biochemistry, 1990, 29, 8835; (d) J. Zhu and
R. M. Wartell, Biochemistry, 1999, 38, 15986.
Notes and references
1 (a) E. C. Friedberg, G. C. Walker and W. Siede, DNA Repair and
Mutagenesis, ASM Press, Washington, 1995; (b) Y. Ionov,
M. A. Peinado, S. Malkhosyan, D. Shibata and M. Perucho,
Nature, 1993, 363, 558; (c) G. Streisinger, Y. Okada, J. Emrich,
J. Newton, A. Tsugita, E. Terzaghi and M. Inouye, Cold Spring
Harbor Symp. Quantum Biol., 1966, 31, 77.
2 Selected examples: (a) K. Nakatani, S. Sando and I. Saito, J. Am.
Chem. Soc., 2000, 122, 2172; (b) L. S. Kappen, Z. Xi and
I.
H.
Goldberg,
Biochemistry,
2001,
40,
15378;
(c) B. T. Patterson, J. G. Collins, F. M. Foley and F. R. Keene,
J. Chem. Soc., Dalton Trans., 2002, 4343; (d) A. Kobori,
T. Murase, H. Suda, I. Saito and K. Nakatani, Bioorg. Med.
10 K. Nakatani, ChemBioChem, 2004, 5, 1623.
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This journal is The Royal Society of Chemistry 2009
670 | Chem. Commun., 2009, 668–670