Unexpectedly stable artificial duplex from flexible acyclic threoninol

Article abstract of DOI:10.1021/ja105539u

A new foldamer, acyclic threoninol nucleic acid (aTNA), has been synthesized by tethering each of the genetic nucleobases A, G, C, and T to d-threoninol molecules, which were then incorporated as building blocks into a scaffold bearing phosphodiester link

Full text of DOI:10.1021/ja105539u

Published on Web 10/01/2010
Unexpectedly Stable Artificial Duplex from Flexible Acyclic Threoninol
Hiroyuki Asanuma,* Takasuke Toda, Keiji Murayama, Xingguo Liang, and Hiromu Kashida
Graduate School of Engineering, Nagoya UniVersity, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan
Received June 24, 2010; E-mail: asanuma@mol.nagoya-u.ac.jp
(2-amino-1,3-butanediol, known as threoninol) to covalently in-
Abstract: A new foldamer, acyclic threoninol nucleic acid (aTNA),
has been synthesized by tethering each of the genetic nucleobases
A, G, C, and T to D-threoninol molecules, which were then
incorporated as building blocks into a scaffold bearing phosphodi-
ester linkages. We found that with its fully complementary strand in
an antiparallel fashion, the aTNA oligomer forms an exceptionally
stable duplex that is far more stable than corresponding DNA or
corporate various functional dyes into natural DNA.4 These base
surrogates derived from D-threoninol can be incorporated into
natural DNA without destabilizing the duplex.5 The high compat-
ibility of D-threoninol with natural DNA as well as the stable GNA
duplex prompted us to design a new foldamer with this more flexible
acyclic scaffold, threoninol.
Herein we propose a new foldamer, acyclic threoninol nucleic
RNA duplexes, even though single-stranded aTNA is rather flexible
acid (aTNA),6 prepared using D-threoninol as building block
and thus does not take a preorganized structure.
tethered to one of the genetic nucleobases A, G, C, and T. Although
there have been several reports of modified DNA involving several
“thymidine” aTNAs,7 a fully changed aTNA oligomer has not been
synthesized to date.8 We found that the aTNA oligomer forms an
A key feature of natural DNA is its spontaneous hybridization
of two strands that are complementary with each other in an
antiparallel fashion. Although nature uses ribose or deoxyribose
as a scaffold for nucleic acids to carry genetic codes, investigation
of DNA hybridization has revealed that deoxyribose is not a
prerequisite for this supramolecular property.1 Recently, surprising
results were reported by Meggers et al.,2 who demonstrated that
the glycol nucleic acid (GNA: see Figure 1a) bearing phosphodiester
linkages consisting of artificial oligonucleotides synthesized from
an acyclic propylene glycol forms a more stable duplex in an
antiparallel fashion than does natural DNA. This result proved that
simple acyclic diols can form excellent scaffolds for new “foldam-
ers” that can spontaneously be folded into a double-helical structure
in a programmed manner.3
exceptionally stable duplex in an antiparallel fashion with its fully
complementary strand.
Figure 2. Melting curves of antiparallel aTNA-M(t)/aTNA-N(a) (black),
single-stranded aTNA-M(t) (blue) and aTNA-N(a) (yellow), parallel aTNA-
M(t)/aTNA-N(a) (green), native DNA-M(t)/DNA-N(a) (magenta), and
native RNA-M(t)/RNA-N(a) (purple). Conditions: [oligonucleotide] ) 2
µM, [NaCl] ) 100 mM, 10 mM phosphate buffer (pH 7.0).
All of the aTNA oligomers listed in Figure 1b were synthesized
from the corresponding phosphoramidite monomers (see Scheme
S1 in the Supporting Information). The melting profiles of aTNAs
were first examined by analyzing the change of absorbance at 260
nm with temperature. As shown by the solid black line in Figure
2, the 1:1 mixture of 8-mer aTNA-M(t) and aTNA-N(a), which
are complementary to each other in an antiparallel fashion, exhibited
a typical sigmoidal curve. The melting temperature (Tm) was
determined to be as high as 62.7 °C, which is remarkably higher
than that measured for the corresponding natural DNA (29.0 °C;
magenta line in Figure 2) and RNA duplex (38.9 °C; purple line).
In contrast, neither aTNA-M(t) nor aTNA-N(a) in a single-stranded
state showed such a sigmoidal curve (blue and orange lines) above
20 °C. Furthermore, a parallel combination of complementary
aTNA-M(t) and aTNA-N(a) also did not display sigmoidal melting
(green line). These results demonstrate that two complementary
aTNAs could form a remarkably stable duplex in an antiparallel
fashion. Similarly, aTNA-N(a) displayed a sigmoidal curve only
with an antiparallel counterstrand of aTNA-M(t) and not with
Figure 1. (a) Structures of natural DNA and aTNA synthesized from
D-threoninol. (b) Sequences of oligonucleotides synthesized in this study.
Over the past decade, we have developed unique base surrogates
based on an acyclic compound with three carbons in its main chain
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14702 J. AM. CHEM. SOC. 2010, 132, 14702–14703
10.1021/ja105539u  2010 American Chemical Society

C O M M U N I C A T I O N S
mM NaCl (see Table S2), which was ∼40 °C higher than that of the
corresponding DNA duplex (25.1 °C). The Tm of a GNA duplex
involving 18 A-T base pairs was reported to be 63 °C, which also
exceeded the stability of the corresponding DNA duplex by 22.5 °C
in the presence of 200 mM NaCl.2a Although we could not compare
the stability of aTNA with GNA precisely, the aTNA duplex appears
to be much more stable than the GNA duplex.9
According to Meggers,2a even single-stranded GNA showed a
significant Cotton effect, indicating that helical preorganization of the
GNA backbone had already formed in the single strand, allowing
formation of a duplex that was highly stable in comparison with native
ones. However, single-stranded aTNA did not form such a helical
preorganized structure, as shown in Figure 3c,d. It should be noted
that although aTNA is more flexible than DNA or GNA, it still formed
a remarkably stable duplex in an antiparallel manner.10
In conclusion, we have developed from threoninol a new artificial
oligonucleotide, aTNA, that forms an unexpectedly stable duplex
in an antiparallel fashion. Even nucleobases tethered on a flexible
scaffold that did not take a preorganized structure in the single-
stranded state could form a duplex that was remarkably more stable
than DNA or RNA duplexes by inducing formation of a right-
handed double-helical structure. Our findings might allow for the
new design of artificial duplexes that do not rely on rigid
preorganization. Furthermore, by combining the “threoninol
nucleotides”4,5 that tether functional molecules with the present
aTNA, functional foldamers can be prepared.
Figure 3. Temperature dependence of the CD spectra of (a) aTNA-M(t)/
aTNA-N(a) and (b) native DNA-M(t)/DNA-N(a) duplexes and single-
stranded (c) aTNA-M(t) and (d) aTNA-N(a). Conditions: [oligonucleotide]
) 5 µM, [NaCl] ) 100 mM, 10 mM phosphate buffer (pH 7.0).
parallel aTNA-N(a) (see Figure S1 in the Supporting Information).
In order to substantiate that canonical Watson-Crick base-pairing
dominates the stable association of aTNAs, the effect of mismatches
on Tm was examined with aTNA-M(t). As listed in Table S1 in
the Supporting Information, we found that introduction of a T-C
mismatch [aTNA-M(t)/aTNA-N(c)] significantly lowered the Tm
from 62.7 to 49.2 °C. Similarly, T-T and T-G mismatches
destabilized the duplex by 8 and 3.7 °C, respectively.
Acknowledgment. This work was supported by a Grant-in-Aid
for Scientific Research from the Ministry of Education, Culture,
Sports, Science, and Technology, Japan.
Supporting Information Available: Experimental procedures for
the syntheses of aTNAs and spectroscopic measurements, melting
profiles and temperatures of duplexes, and NMR spectra. This material
is available free of charge via the Internet at http://pubs.acs.org.
Next, the aTNA duplex was analyzed by circular dichroism (CD)
spectroscopy. As shown in Figure 3a, hybridization of aTNA-M(t)
and aTNA-N(a) below Tm for the duplex induced a symmetrical
positive-negative Cotton effect similar to that seen for the typical
B-type DNA-M(t)/DNA-N(a) duplex (compare panel b with panel
a in Figure 3), demonstrating that hybridization of aTNA allowed
the formation of a right-handed helix. However, the induced CD
decreased with increasing temperature and became very weak at
80 °C, where the duplex was completely dissociated. CD analysis
of single-stranded aTNA-M(t) and aTNA-N(a) revealed that the
shapes of their CD spectra in the 200-300 nm region were entirely
different from that of the duplex (compare panels c and d with
panel a in Figure 3). Although the individual strands are chiral
oligomers, their CD signals were very small at all temperatures
examined here, indicating that single-stranded aTNA did not take
a particularly preorganized structure but existed as a random coil.
Nevertheless, hybridization of these strands induced formation of
a right-handed double-helical structure.
Comparison of the thermal stability of aTNA with those of other
natural duplexes revealed that the aTNA duplex is exceptionally stable.
As described above, the Tm of the aTNA-M(t)/aTNA-N(a) duplex
was 62.7 °C. However, the corresponding DNA and RNA duplexes
(DNA-M(t)/DNA-N(a) and RNA-M(t)/RNA-N(a)) yielded Tm values
of only 29.0 and 38.9 °C, respectively, which are far lower than that
of the corresponding aTNA duplex. We also examined other sequences
and found that the aTNA duplex was remarkably stable irrespective
of the sequence. For example, a 12-mer aTNA duplex composed of
only A and T gave a Tm as high as 64.3 °C in the presence of 100
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