Artificial DNA made exclusively of nonnatural C-nucleosides with four types of nonnatural bases

Article abstract of DOI:10.1021/ja801058h

We describe a new class of DNA-like oligomers made exclusively of nonnatural, stable C-nucleosides. The nucleosides comprise four types of nonnatural bases attached to a deoxyribose through an acetylene bond with the β-configuration. The artificial DNA fo

Full text of DOI:10.1021/ja801058h

Published on Web 06/11/2008
Artificial DNA Made Exclusively of Nonnatural C-Nucleosides
with Four Types of Nonnatural Bases
Yasuhiro Doi, Junya Chiba,* Tomoyuki Morikawa, and Masahiko Inouye*
Graduate School of Pharmaceutical Sciences, UniVersity of Toyama, Sugitani 2630,
Toyama 930-0194, Japan
Received February 12, 2008; E-mail: chiba@pha.u-toyama.ac.jp; inouye@pha.u-toyama.ac.jp
Abstract: We describe a new class of DNA-like oligomers made exclusively of nonnatural, stable
C-nucleosides. The nucleosides comprise four types of nonnatural bases attached to a deoxyribose through
an acetylene bond with the ꢀ-configuration. The artificial DNA forms right-handed duplexes and triplexes
with the complementary artificial DNA. The hybridization occurs spontaneously and sequence-selectively,
and the resulting duplexes have thermal stabilities very close to those of natural duplexes. The artificial
DNA might be applied to a future extracellular genetic system with information storage and amplifiable
abilities.
Introduction
exceptions for entirely modified oligonucleotide analogues
composed of nonnatural bases and various sugars. Furthermore,
Apart from its biological significance, the beauty of the DNA
duplex structure has been inspiring chemists to create artificial
analogues in their own right. This type of research is primarily
motivated by pure scientific exploration and eventually directed
toward biomedical applications. Creation of the artificial
analogues is roughly categorized into the following two
modifications of natural DNA: the sugar-phosphodiester back-
bone and the Watson-Crick base pairing. The former is
represented by peptide nucleic acids (PNA),1 locked nucleic
acids (LNA),2 threo-furanosyl nucleic acids (TNA),3 and 2′,5′-
linked nucleic acids.4 These analogues usually possess natural
bases because of their potential use in antisense and antigene
strategies. On the other hand, examples of the latter are properly
aimed at their applications to nanotechnology,5 biotechnology,6
and chemical biology.7 Until recently, however, those analogues
have contained only one or a few nonnatural bases linked with
deoxyribose. Eschenmoser and co-workers8 reported notable
Kool and co-workers9 disclosed artificial duplexes in which all
of the natural base pairs are replaced by pairs of nonnatural
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Lett. 2008, 10, 1091–1094. (b) Clever, G. H.; Kaul, C.; Carell, T.
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2006, 1, 190–194. (h) Miyake, Y.; Togashi, H.; Tashiro, M.;
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9
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10.1021/ja801058h CCC: $40.75
2008 American Chemical Society

Completely Artificial DNA
A R T I C L E S
and natural bases. The question, however, still remains whether
structural variations for phosphodiester-based duplexes can be
further extended. Here we describe a new class of such base-
pairing right-handed duplexes and triplexes, which can be
formed only from artificial DNA strands containing only
nonnatural nucleoside residues possessing four types of non-
natural bases through nonnatural C-glycoside linkers. The
hybridization occurs spontaneously and sequence-selectively,
and the resulting duplexes have net thermal stabilities very close
to those of natural duplexes in spite of their differences for the
geometries and hydrogen-bonding patterns. These findings
provide further proof that a genetic phosphodiester-based mole-
cular framework is not limited to the natural DNA one and can
be extended to many types of nonbiological entities.
Results and Discussion
The molecular design for a new three-point hydrogen-bonding
pair was based on our previous studies of molecular recognition
for 2-amino-3H-pyrimidin-4-one derivatives.10 These hetero-
cyclic compounds have the hydrogen-bonding array of donor-
donor-acceptor (DDA). Although the skeleton can tautomerize
to its 1H form, bearing a DAA hydrogen-bonding pattern, our
results showed that the skeleton selectively interacts with a
cytosine derivative (AAD type) among all kinds of natural bases
in less polar organic media. This finding led us to choose this
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Soc. 2008, 130, 412–413. (b) Tsai, C.-H.; Chen, J.; Szostak, J. W.
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Geierstanger, B. H.; Wemmer, D. E.; Schultz, P. G.; Spraggon, G.;
Romesberg, F. E. J. Am. Chem. Soc. 2007, 129, 10466–10473. (d)
Murata, S.; Mizumura, Y.; Hino, K.; Ueno, Y.; Ichikawa, S.; Matsuda,
A. J. Am. Chem. Soc. 2007, 129, 10300–10301. (e) Taniguchi, Y.;
Kool, E. T. J. Am. Chem. Soc. 2007, 129, 8836–8844. (f) Seio, K.;
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Figure 1. Chemical structures and synthesis of our artificial DNA. (a)
Chemical structures of two types of natural and nonnatural base pairs. (b)
Synthetic strategy for the artificial DNA.
heterocycle as a skeleton for a G base analogue. Its actual
structure is depicted in Figure 1a and referred to as G* for the
free nonnatural base (similarly, C*, A*, and T* for the remaining
three nonnatural bases). The counterpart of G* is C*, which
possesses an AAD hydrogen-bonding pattern. The structure of
C* is derived from 4-amino-1H-pyrimidin-2-one by replacing
the hydrogen atom at the N1 position by a methyl group. This
substitution inhibits the original skeleton from tautomerizing
to its 3H form that can ambiguously behave as an ADD
hydrogen-bonding module as G* does. The position at the bases
connected to deoxyribose was selected to be on carbon and not
to significantly alter the natural DNA framework. This decision
necessarily results in a reversed hydrogen-bonding pattern for
the G*/C* combination relative to that of the natural G/C pair.
Therefore, the abbreviations of iG* (isoG*) and iC* (isoC*)
are used instead of G* and C* when the artificial bases are
incorporated into nucleoside units. The structures of another
pair of nonnatural bases, A* and T*, were determined in order
that the pair has a two-point, DA-type hydrogen-bonding
complementarity and has the same geometry as the iG*/iC* pair.
The pattern of hydrogen bonds between A* and T* must be
incompatible with that between iG* and iC* when the A*/T*
pair is superimposed on the iG*/iC* one, being set the glycoside
bonds to the same directions.
(8) For homo-DNA, P-RNA, and hexitol nucleic acids: (a) Egli, M.; Pallan,
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O.; Jaun, B.; Krishnamurthy, R.; Eschenmoser, A. HelV. Chim. Acta
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A R T I C L E S
Doi et al.
All of the halogen-attached nonnatural bases were synthesized
from appropriate commercial materials and connected to deox-
yribose through an acetylene bond to afford nonnatural, stable
C-nucleosides in good overall yields (Figure 1b and Scheme
S1 in Supporting Information). Our laboratory already reported
the synthesis of 4,4′-dimethoxytrityl- (DMTr-) protected ethynyl
C-2-deoxy-ꢀ-D-ribofuranoside as a key component of nonnatural
nucleosides used in this study.11 By means of the synthetic
versatility of the acetylenic function, a variety of candidates
for nonnatural bases can be attached to the deoxyribose with
the ꢀ-configuration of natural nucleosides. The C-nucleosides
thus prepared were further protected on the bases and converted
into phosphoramidites. The phosphoramidites were then sub-
jected to automated DNA synthesis to give oligomeric non-
natural nucleotides, hereafter called simply “artificial DNA”.
After having been released from the solid support, the artificial
DNAs were deprotected, purified by reverse-phase HPLC, and
characterized by matrix-assisted laser desorption ionization time-
of-flight (MALDI-TOF) mass spectrometry (Figure S1). Pre-
liminary modeling studies revealed that artificial DNA forms a
similar duplex as natural DNA and that the deoxyribose-
phosphodiester backbone of artificial DNA appears to
scarcely be distorted by adopting suitable conformations. This
motion may be assisted by increased puckering flexibilities
of the deoxyribose with the aid of the acetylene spacers.
First, homooligomers [abbreviated as d(T*)y, where y is
the number of nucleoside residues] of artificial DNA were
investigated solely on the basis of UV-vis and circular
dichroism (CD) spectra in a buffer solution (10 mM Hepes,
10 mM MgCl2, and 100 mM NaCl, pH 7.0). The UV-vis
spectra of d(T*)16 and d(iC*)8 hardly changed in the
temperature range from 0 to 70 °C, and their CD spectra
were almost silent at 20 °C (Figure S2). Although d(A*)16
also showed temperature-insensitive UV-vis absorption,
strong Cotton effects were seen for the CD spectrum in the
wavelength region where A* absorbs (Figure S3). The CD
intensities somewhat weakened by about 40% when the
temperature of d(A*)16 was raised to 70 °C. Therefore, the
emerging Cotton effects would simply be attributed to the
chiral arrangement of the A* chromophores and not to the
formation of a higher-order structure. On the other hand, the
behavior of d(iG*)8 is quite interesting, as in the case for
natural G homooligomers (Figure 2). Significant Cotton
effects of d(iG*)8 at room temperature began to weaken upon
heating with isodichroic points and finally almost disap-
peared, accompanied by hyperchromicity of 45% at 310 nm
Figure 2. CD and UV-vis data for d(iG*)8 (2.0 µM) in 10 mM Hepes
(pH 7.0), 10 mM MgCl2, 100 mM NaCl. (a) CD spectra at 10 (blue), 20,
30, 40, 50, 60, and 70 °C (red). (b) UV-vis spectra at 10 (blue) and 70 °C
(red). (c) UV melting curve monitored at 310 nm.
in the UV-vis spectra. A cooperative, sigmoidal transition
was definitely observed at 40 °C for d(iG*)8 by thermal
denaturation studies. Cooling of d(iG*)8 to room temperature
restored the Cotton effects and the hypochromism to the same
level of the previous state. This finding means that the iG*
homooligomer of artificial DNA forms a higher-order struc-
ture by itself, possibly resembling the G-quartet structure of
natural DNA.
Table 1 summarizes the complementary sequences of artificial
and natural DNA used in this study and their melting temper-
ature (Tm) values. The addition of d(iC*)8 to a solution of
d(iG*)8 caused drastic changes in the CD spectra, resulting from
the transformation of the self-ordered structure of d(iG*)8 into
the hybridized one between d(iG*)8 and d(iC*)8 (entry 3). The
Cotton effects of the mixture weakened when the temperature
of the mixture was raised to 70 °C and approached that of the
sum of the two oligomers at the same temperature (Figure 3a).
Thermal denaturation profile of the mixture showed a typical
two-state, sigmoidal transition with hyperchromicity of 25% at
305 nm in UV-vis spectra, from which Tm for the combination
was estimated to be 59 °C (Figure 3b,c). The 1:1 stoichiometry
for this complexation was confirmed on the basis of the CD
changes by adding CD-silent d(iC*)8 to a solution of d(iG*)8,
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Completely Artificial DNA
A R T I C L E S
Table 1. Artificial and Natural DNAs in This Study and Their
Melting Temperatures
a
entry
duplexes and triplexes
Tm (°C)
1
2
3
4
5
6
7
8
5′-d(iG*)8
5′-d(iG*)6/3′-d(iC*)6
5′-d(iG*)8/3′-d(iC*)8
5′-d(iG*)10/3′-d(iC*)10
5′-d(iG*)6/3′-d(C)6
5′-d(iG*)8/3′-d(C)8
5′-d(iG*)10/3′-d(C)10
5′-d(G)6/3′-d(C)6
40b
41
59
70
33
50
58
30
55
58
59
9
10
11
5′-d(G)8/3′-d(C)8
5′-d(G)10/3′-d(C)10
5′-d(iG*iC*iG*iC*iG*iG*iC*iC*)/
3′-d(iC*iG*iC*iG*iC*iC*iG*iG*)
3′-d(iG*iC*iG*iC*iG*iG*iC*iC*)/
3′-d(iC*iG*iC*iG*iC*iC*iG*iG*)
5′-d(GCGCGGCC)/3′-d(CGCGCCGG)
5′-d(iG*iC*iG*iC*A*iG*iG*iC*iC*)/
3′-d(iC*iG*iC*iG*T*iC*iC*iG*iG*)
5′-d(GCGCAGGCC)/3′-d(CGCGTCCGG)
5′-d(T*iG*iC*iG*iC*iG*iG*iC*iC*T*)/
3′-d(A*iC*iG*iC*iG*iC*iC*iG*iG*A*)
5′-d(TGCGCGGCCT)/3′-d(ACGCGCCGGA)
5′-d(T*iG*iC*iG*iC*iG*iG*iC*iC*T*)/
3′-d(A*iC*iG*iC*T*iC*iC*iG*iG*A*)d
5′-d(TGCGCGGCCT)/3′-d(ACGCTCCGGA)d
5′-d(T*iG*iC*iG*iC*A*iG*iG*iC*iC*T*)/
3′-d(A*iC*iG*iC*iG*T*iC*iC*iG*iG*A*)
5′-d(TGCGCAGGCCT)/3′-d(ACGCGTCCGGA)
5′-d(A*)16/3′-d(T*)16
12
c
13
14
55
56
15
16
55
66
17
18
64
46
19
20
35
64
21
22
23
24
25
63
22e
30e
35e
c
5′-d(A*)20/3′-d(T*)20
5′-d(A*)24/3′-d(T*)24
5′-d(A*A*T*T*A*A*T*T*A*A*T*T*A*A*T*T*
A*A*T*T*)
a Tm values were obtained from the maxima of the first derivatives of
the melting curves measured in the buffer solution: 2.0 µM duplex, 10
mM Hepes (pH 7.0), 10 mM MgCl2, and 100 mM NaCl. Errors were
estimated at ( 1.0 °C. b Tm value of self-ordered structure. c No
apparent sigmoidal transition. d iC*T* and CT mismatch base pairs are
underlined. e Exclusive triplex formation was observed: 1.0 µM A*
homooligomer and 2.0 µM T* homooligomer.
Figure 3. CD and UV-vis measurements were carried out at 2.0 µM
duplex in 10 mM Hepes (pH 7.0), 10 mM MgCl2, and 100 mM NaCl unless
otherwise noted. (a) CD spectra at 10 (blue), 20, 30, 40, 50, 60, and 70 °C
(red) of the duplex of entry 3. Dotted line indicates the sum of the CD
spectra at 70 °C for each single strand of d(iG*)8 and d(iC*)8. (b) UV-vis
spectra at 10 °C (blue) and 70 °C (red) of the duplex of entry 3. Dotted
line indicates the sum of the UV-vis spectra at 70 °C for each single strand
of d(iG*)8 and d(iC*)8. (c) UV melting curves of entry 3 (305 nm, black
solid line), entry 16 (305 nm, black dashed line), entry 18 (305 nm, black
dotted line), entry 22 (295 nm, red solid line), entry 23 (295 nm, red dashed
line), and entry 24 (295 nm, red dotted line). In the cases of entries 22-24,
[A* homooligomer] ) 1.0 µM and [T* homooligomer] ) 2.0 µM.
which showed an apparent saturation at the molar ratio of 1
(Figure 4a). These observations indeed suggest the formation
of a complementary hydrogen-bonding duplex of d(iG*/iC*)8
between the two strands of the artificial DNAs with π-stacking
interactions. Furthermore, the positive-negative pair of the CD
bands from the longer wavelength is structurally analogous to
that of the natural DNA of d(G/C)8 of entry 9, implying the
right-handedness of the artificial duplex (Figure S4).12 The
artificial DNA d(iG*)8 also forms a similar two-stranded
complex with the natural DNA d(C)8, giving rise to a Tm value
of 50 °C, while the higher-order structure for this heterotype
duplex remains to be elucidated (entry 6 and Figure S5). Thus,
the relative stabilities of the higher-order structures decreased
in the following order: duplex of d(iG*/iC*)8 > duplex of d(G/
C)8 > duplex of d(iG*/C)8. The highest stability for the
artificial-artificial combination is also observed in the cases
for d(iG*)6 and d(iG*)10.
Duplex formation of artificial DNA proved to have a
generality as shown in entries 11-21. Thus, artificial DNA
of various mixed sequences formed artificial duplexes with
complementary artificial DNA. The thermal denaturation
profile for artificial DNA of a mixed sequence was illustrated
with the combination of entry 16 (Figure 3c and Figure S6),
and its 1:1 stoichiometry was also corroborated (Figure S7).
The antiparallel orientation of artificial duplexes was deter-
mined by comparing entries 11 (antiparallel complementarity)
and 12 (parallel complementarity): no characteristic sigmoidal
transition was seen in entry 12. Artificial duplexes revealed
a similar relationship between the Tm values and the sequence
contexts as for natural duplexes. The Tm values consecutively
rose with increasing number of nucleoside residues when the
same base pair was added to the duplexes of a single base
pair (entries 2-4 vs 8-10). Duplexes containing two types
of base pairs behaved in a complicated manner. When an
A*/T* base pair was added into the middle of a iG*/iC*
duplex sequence, the Tm values were faintly influenced
(entries 11 vs 14 and 16 vs 20). On the other hand, the Tm
values of the duplexes substantially increased by adding A*/
T* base pairs at the ends of the sequences (entries 11 vs
16). It was also found that the artificial duplexes of mixed
sequences have thermal stabilities near the stabilities of
9
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A R T I C L E S
Doi et al.
artificial and natural base pairs. The artificial base pair has
a pyrimidine/pyrimidine plane that is smaller than a pyrimi-
dine/purine one of the natural base pair. On the other hand,
acetylene linkers in the artificial pair may contribute to add
an extra stabilization for the stacking as reported in other
cases.15 Thus, the insufficiency for the interaction caused by
the smaller plane should be compensated by the presence of
the acetylene linkers in the artificial system. These factors
appear to play a role in the observation that the artificial
duplexes have thermal stabilities near those of the natural
duplexes of analogous sequences.
An important feature of natural DNA is its discerning
ability between matched and mismatched sequences even in
the presence of only a single mismatched base if the duplex
is short. Entry 18 represents the combination of such artificial
DNA, in which a single mismatched base pair of iC*/T*
exists near the center of the 10-mer duplex. The Tm value
for the mismatched duplex largely decreased by 20 °C
compared to that for the corresponding fully matched duplex
in entry 16 (Figure 3c and Figure S8). This drop in Tm is
noticeable because the size of the mismatched iC*/T* base
pair is formally identical to that of the matched iC*/iG* base
pair in our artificial duplexes. Pairing selectivity in artificial
DNA comes only from complementary hydrogen bonding,
not from complementary size. Nevertheless, the ∆Tm is
comparable to that of the natural counterpart, which may
cause a significant backbone distortion (entries 17 and 19).
This finding demonstrates the preciseness of our molecular
design for the two types of nonnatural, incompatible base
pairs as shown in Figure 1a.
Homooligomers composed of A* and T* were subjected
to hybridization experiments (entries 22-24 and Figure S9)
and found to behave somewhat unlike the corresponding
natural DNA does. The Job’s plot between d(A*)24 and
d(T*)24 was conducted on the basis of UV-vis spectra at 0
°C after annealing processes (Figure 4b). Mole fraction of
d(A*)24 was raised incrementally from 0% to 100% under
conditions where the total concentration of d(A*)24 and
d(T*)24 was maintained constant at 2.0 µM. In this experi-
ment, we observed that one set of isosbestic points changed
to the other set strictly when a mole fraction of d(A*)24
increased beyond 0.33. After that, the absorption bands of
d(A*)24 simply grew in the spectra (Figure S10a). This
behavior was also seen in the CD Job’s (Figure S10b) and
titration (Figure S11) experiments. The thermal denaturation
profiles revealed a similar two-state sigmoidal transition even
when the molar ratio of d(A*)24 was varied (Figure 3c).
Furthermore, the slope around the inflection point on the
sigmoidal curve is much steeper than any other combination,
indicating a strongly cooperative two-state transition (Figure
S12). These data unambiguously suggest the exclusive
formation of d(T*/A*/T*)24-type artificial triplex without
d(A*/T*)24-type duplex even at the low concentration applied.
Figure 4. Evaluation of stoichiometry for hybridization events. (a) Titration
curve monitored by CD (288 nm, 25 °C) for the duplex of entry 3 at
[d(iG*)8] ) 2.0 µM in 10 mM Hepes (pH 7.0), 10 mM MgCl2, and 100
mM NaCl. (b) Job’s plot monitored by UV (295 nm, 0 °C) for the duplex
of entry 24 at [d(A*)24] + [d(T*)24] ) 2.0 µM in 10 mM phosphate buffer
(pH 7.0), 0.1 mM EDTA, and 100 mM NaCl.
natural duplexes with analogous sequences (entries 14 vs 15,
16 vs 17, and 20 vs 21). To shed light on this similarity, the
thermodynamic parameters were measured for these artificial
DNAs by van’t Hoff analysis (Figure S13).13 The ratios of
∆H/T∆S for the duplex formation are about 1.3 and are
almost equal to those in natural DNA of the analogous
sequences. Artificial DNA was found to compensate a
disadvantageous entropy change with an advantageous en-
thalpy one in the duplex formation, equally as well as natural
DNA does.
The thermodynamic stability of duplexes should principally
result from two factors: hydrogen-bonding and stacking (and/
or hydrophobic) interactions. Although the hydrogen-bonding
pattern for the iG*/iC* combination (DDA/AAD type) in the
artificial duplex is reversed from that of the natural G/C one
(ADD/DAA type), the total number and the interaction type
(i.e., DDA/AAD or DAD/ADA or DDD/AAA) of hydrogen
bonds are the same.14 With respect to the stacking interaction,
two different aspects must be considered for comparing the
(12) (a) Johnson, W. C. In Circular Dichroism: Principles and Applications,
2nd ed.; Berova, N., Nakanishi, K., Woody, R. W., Eds.; Wiley-
VCH: New York, 2000; pp 703-718. (b) Maurizot, J. C. In Circular
Dichroism: Principles and Applications, 2nd ed.; Berova, N., Nakan-
ishi, K., Woody, R. W., Eds.; Wiley-VCH: New York, 2000; pp 719-
739.
(13) Marky, L. A.; Breslauer, K. J. Biopolymers 1987, 26, 1601–1620.
(14) (a) Chen, G.; Kierzek, R.; Yildirim, I.; Krugh, T. R.; Turner, D. H.;
Kennedy, S. D. J. Phys. Chem. B 2007, 111, 6718–6727. (b) Johnson,
S. C.; Sherrill, C. B.; Marshall, D. J.; Moser, M. J.; Prudent, J. R
Nucleic Acids Res. 2004, 32, 1937–1941. (c) Chaput, J. C.; Switzer,
C. J. Am. Chem. Soc. 2000, 122, 12866–12867. (d) Robinson, H.;
Gao, Y.-G.; Bauer, C.; Roberts, C.; Switzer, C.; Wang, A. H.-J.
Biochemistry 1998, 37, 10897–10905. (e) Roberts, C.; Bandaru, R.;
Switzer, C. J. Am. Chem. Soc. 1997, 119, 4640–4649. (f) Roberts, C.;
Bandaru, R.; Switzer, C. Tetrahedron Lett. 1995, 36, 3601–3604. (g)
Piccirilli, J. A.; Krauch, T.; Moroney, S. E.; Benner, S. A. Nature
1990, 343, 33–37. (h) Switzer, C.; Moroney, S. E.; Benner, S. A. J. Am.
Chem. Soc. 1989, 111, 8322–8323.
(15) (a) Chaput, J. C.; Sinha, S.; Switzer, C. Chem. Commun. 2002, 1568–
1569. (b) Ogawa, A. K.; Wu, Y.; McMinn, D. L.; Liu, J.; Schultz,
P. G.; Romesberg, F. E. J. Am. Chem. Soc. 2000, 122, 3274–3287.
(c) McMinn, D. L.; Ogawa, A. K.; Wu, Y.; Liu, J.; Schultz, P. G.;
Romesberg, F. E. J. Am. Chem. Soc. 1999, 121, 11585–11586. (d)
´
´
Sa´gi, J.; Szemozo¨, A.; Ebinger, K.; Szabolcs, A.; Sa´gi, G.; Ruff, E.;
¨
Otvo¨s, L. Tetrahedron Lett. 1993, 34, 2191–2194. (e) Froehler, B. C.;
Jones, R. J.; Cao, X.; Terhorst, T. J. Tetrahedron Lett. 1993, 34, 1003–
1006.
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Completely Artificial DNA
A R T I C L E S
a matrix (see Figure S1). d(iG*)6: calcd for MH+ (C66H73N18O34P5),
1817.33; found, 1817.92. d(iG*)8: calcd for MH+ (C88H97N24O46P7),
2444.43; found, 2444.64. d(iG*)10: calcd for MH+ (C110H121N30-
O58P9), 3070.52; found, 3070.88. d(iC*)6: calcd for MH+ (C72H85N18-
O34P5), 1901.42; found, 1902.44. d(iC*)8: calcd for MH+ (C96H113N24O46-
P7), 2556.55; found, 2556.28. d(iC*)10: calcd for MH+ (C120H141N30O58P9),
3210.68; found, 3210.97. d(A*)16: calcd for MH+ (C176H193N48O78P15),
4693.88; found, 4694.84. d(A*)20: calcd for MH+ (C220H241N60O98P19),
5882.09; found, 5884.77. d(A*)24: calcd for MH+ (C264H289N72O118P23),
7071.30; found, 7071.71. d(T*)16: calcd for MH+ (C192H209N32O110P15),
5189.79; found, 5191.35. d(T*)20: calcd for MH+ (C240H261N40O138P19),
6502.98; found, 6503.89. d(T*)24: calcd for MH+ (C288H313N48O166P23),
7815.17; found, 7816.01. 5′-d(iG*iC*iG*iC*iG*iG*iC*iC*)-3′: calcd for
MH+ (C92H105N24O46P7), 2500.49; found, 2500.60. 3′-d(iC*iG*iC*iG*iC*i-
C*iG*iG*)-5′: calcd for MH+ (C92H105N24O46P7), 2500.49; found,
This strong tendency for the formation of triplexes over
duplexes should primarily be due to the symmetry of A*
along the axis from the amino nitrogen to the carbon
connected to the acetylene linker. Therefore, interaction of
the third d(T*)24 strand with d(A*/T*)24 produces no energetic
loss and rather adds a further stabilization arising from the
enforced π-stacking interaction of the enlarged π-plane. This
situation should cause the equilibrium constant between
d(T*)24 and d(A*/T*)24 to be larger than that of the d(A*/
T*)24 duplex formation, resulting in the observed two-state
transition. In the cases of natural A and T homooligomers, the
relative magnitudes of the two equilibrium constants are strongly
depend on the concentrations of the oligomers and the existing
salts.16 The selectivity for the formation of triplex over duplex is
so high that the thermal denaturation study of the palindromic 20-
mer in entry 25 displayed little hyperchromism and no apparent
sigmoidal transition. This finding reconfirms that triplexes pre-
dominantly are formed rather than duplexes from the artificial
DNAs composed only of A* and T*.
2500.50. 3′-d(iG*iC*iG*iC*iG*iG*iC*iC*)-5′: calcd for MH+ (C92H105
-
N24O46P7), 2500.49; found, 2500.66. 5′-d(iG*iC*iG*iC*A*iG*iG*iC*iC*)-
3′: calcd for MH+ (C103H117N27O51P8), 2797.54; found, 2798.50. 3′-d(iC*-
iG*iC*iG*T*iC*iC*iG*iG*)-5′: calcd for MH+ (C104H118N26O53P8),
2828.53; found, 2828.88, 5′-d(T*iG*iC*iG*iC*iG*iG*iC*iC*T*)-3′:
calcd for MH+ (C116H131N28O60P9), 3156.58; found, 3156.72. 3′-d(A*iC*-
iG*iC*iG*iC*iC*iG*iG*A*)-5′: calcd for MH+ (C114H129N30O56P9),
3094.59; found, 3093.61. 3′-d(A*iC*iG*iC*T*iC*iC*iG*iG*A*)-5′: calcd
for MH+ (C115H130N29O57P9), 3109.59; found, 3110.05. 5′-d(T*iG*iC*iG*-
iC*A*iG*iG*iC*iC*T*)-3′: calcd for MH+ (C127H143N31O65P10), 3453.63;
found, 3453.34. 3′-d(A*iC*iG*iC*iG*T*iC*iC*iG*iG*A*)-5′: calcd for
MH+ (C126H142N32O63P10), 3422.64; found, 3421.71. 5′-d(A*A*T*T*)5-
3′: calcd for MH+ (C230H251N50O118P19), 6130.04; found, 6129.23.
UV and Tm Measurements. UV-vis spectra and Tm melting
curves (1.0 °C/1.0 min) were obtained on a Jasco V-560 UV/vis
spectrophotometer with a Peltier and a temperature controller in a
temperature range from 0 to 90 °C. The Tm values were determined
from the maxima of the first derivatives of the melting curves
measured in a buffer solution: 10 mM Hepes (pH 7.0), 10 mM
MgCl2, and 100 mM NaCl. Errors were estimated at (1.0 °C.
Concentrations of the solutions containing artificial DNAs were
determined on the basis of molar extinction coefficients at 260 nm
(ε260) of the artificial nucleoside monomers 9, 13, 17, and 20 (see
Supporting Information).
Conclusion
Artificial DNA made exclusively of nonnatural nucleosides
with four types of nonnatural bases represents a new class
of DNA-like synthetic oligomers. The iG*/iC*-rich artificial
DNA forms right-handed duplexes with the complementary
artificial DNA in a sequence-specific manner with antiparallel
orientation. The thermal stabilities and thermodynamic
parameters of the artificial duplexes are very close to those
of the natural duplexes in spite of differences in their
geometries and hydrogen-bonding patterns. On the other
hand, the artificial homooligomeric DNAs consisted only of
A* and T* were found to exclusively form triplexes. By
means of the synthetic feasibility of the artificial nucleoside
unit, a variety of candidates for nonnatural bases can be
incorporated into the artificial DNA. Therefore, the present
molecular framework has a potential for storing genetic
information and for application to enzymatic replication
directed toward engineered genetics. Furthermore, the arti-
ficial DNA may be a superior building scaffold for construct-
ing nanostructures of materials interest because of the stable
C-nucleosides against ubiquitous naturally occurring enzymes
such as DNase.
CD Measurements. CD spectra were recorded on a Jasco-J-
720WI spectropolarimeter with a temperature controller at 0, 10,
20, 30, 40, 50, 60, and 70 °C in a buffer solution: 10 mM Hepes
(pH 7.0), 10 mM MgCl2, and 100 mM NaCl.
Titration Experiments. Titration curves for artificial DNAs
were obtained by monitoring a specified wavelength of CD or
UV. In entry 3, for example, 3.0 mL of a d(iG*)8 solution (2.0
µM with 10 mM Hepes (pH 7.0), 10 mM MgCl2, and 100 mM
NaCl) was prepared, and CD measurement of the solution was
carried out at 25 °C in a quartz cell of 1 cm path length.
Separately, 150 µL of a d(iC*)8 solution (100 µM in the same
buffer) was then prepared, and 12.0 µL of the d(iC*)8 solution
[0.2 equiv relative to d(iG*)8] was added to the d(iG*)8 solution
in the quartz cell. The mixed solution was stirred for 15 min at
room temperature, and then CD measurement was performed.
A series of the operations was repeated for all the ratios of
[d(iC*)8]/[d(iG*)8] ) 0, 0.2, 0.4, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2,
1.3, 1.4, 1.6, 1.8, and 2.0. The normalized CD changes at 288
nm were plotted against [d(iC*)8]/[d(iG*)8] (Figure 4a).
Job’s Plots. Job’s plots for artificial DNAs were obtained by
monitoring a specified wavelength of CD or UV. In entry 24, for
example, 20 mL of a d(A*)24 solution [2.0 µM with 10 mM
phosphate (pH 7.0), 0.1 mM EDTA, and 100 mM NaCl] and 20
mL of a d(T*)24 solution (2.0 µM in the same buffer) were prepared.
The d(A*)24 solutions of 0, 50, 100, 150, 200, 250, 300, 350, 400,
450, 500, 550, 600, 650, 700, 750, 800, 900, and 1000 µL were
mixed in micro test tubes with 1000, 950, 900, 850, 800, 750, 700,
650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 100, and 0 µL
of the d(T*)24 solutions, respectively. All the mixed solutions were
heated to 80 °C, followed by slow cooling to 25 °C over 60 min,
Experimental Section
Synthesis of Artificial DNA Oligomers. Artificial DNA oli-
gomers were synthesized by use of phosphoramidites 8, 12, 16,
and 19 (see Supporting Information and Scheme S1) on an
Applied Biosystems 392 synthesizer by standard ꢀ-cyanoeth-
ylphosphoramidite chemistry with a coupling reaction time of
15 min. The solid support (Universal Support II), which allows
for 3′ placement of nonnatural nucleosides, was purchased from
Glen Research. After automated synthesis, the oligomers were
removed from the solid support with 2 M ammonia methanol
solution at 30 °C for 30 min and deprotected with concentrated
NH4OH at 40 °C for 8 h. The oligomers were then purified by
reverse-phase HPLC on a Chemcobond 5-ODS-H column (10
× 150 mm) with an eluent of 5 mM ammonium formate and a
linear gradient of acetonitrile (0-30 min, 0-15% CH3CN) at a
flow rate of 3.0 mL/min.
MALDI-TOF Mass Measurements. MALDI-TOF mass spectra
were recorded on a Bruker-Daltonics-Autoflex mass spectrometer
operating in the positive ion mode with 3-hydroxypicolinic acid as
(16) Roberts, R. W.; Crothers, D. M. Proc. Natl. Acad. Sci. U.S.A. 1996,
93, 4320–4325.
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A R T I C L E S
Doi et al.
and then kept in a refrigerator (4 °C) for 1 h. UV-vis measurements
of all of the solutions were carried out at 0 °C in a quartz cell of
1 cm path length. Against [d(A*)24]/([d(A*)24] + [d(T*)24]) were
plotted the normalized UV changes for the mixtures of d(A*)24
and d(T*)24 at 295 nm. The changes were corrected by subtracting
the sum of the intensities for each strand at the same concentrations
(Figure 4b).
Ministry of Education, Culture, Sports Science and Technology,
the Japanese Government.
Supporting Information Available: Synthetic details, Scheme
S1, and Figures S1-13. This material is available free of charge
via the Internet at http://pubs.acs.org.
Acknowledgment. This study was performed through Special
Coordination Funds for Promoting Science and Technology of the
JA801058H
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8768 J. AM. CHEM. SOC. VOL. 130, NO. 27, 2008