Acyclic butyl nucleic acid (BuNA): A novel scaffold for A-switch

Article abstract of DOI:10.1039/c3ra41255e

The construction of an A-switch, derived from the artificial nucleic acid called acyclic Butyl Nucleic Acid (BuNA) was accomplished. The phosphoramidite building blocks of (S)-BuNA were synthesized from (R)-aspartic acid. To demonstrate its use as an A-switch, a stretch of polyadenine nucleotides of (S)-BuNA was studied by circular dichroism (CD) and ultraviolet (UV) spectroscopy under neutral and acidic conditions. Acid-base titration revealed two state transitions at pH 4.8 and highly pH-dependent structural conformation reversibility. Thermal melting (T_m) studies suggest that at neutral pH, poly BuNA(A) is a weakly organized single strand, while at low pH it adopts a highly organized and rigid structure. Furthermore, MALDI-TOF-MS data revealed intermolecular interactions which led to the formation of an A-motif composed of a double helical structure. Since BuNA does not suffer from depurination under acidic conditions, this allowed us to determine the thermodynamic parameters of the A-motif. This is the first report of the construction of an A-switch using artificial nucleic acids.

Full text of DOI:10.1039/c3ra41255e

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Acyclic butyl nucleic acid (BuNA): a novel scaffold for
A-switch3
Cite this: RSC Advances, 2013, 3,
19330
Vipin Kumar and Venkitasamy Kesavan*
The construction of an A-switch, derived from the artificial nucleic acid called acyclic Butyl Nucleic Acid
(BuNA) was accomplished. The phosphoramidite building blocks of (S)-BuNA were synthesized from (R)-
aspartic acid. To demonstrate its use as an A-switch, a stretch of polyadenine nucleotides of (S)-BuNA was
studied by circular dichroism (CD) and ultraviolet (UV) spectroscopy under neutral and acidic conditions.
Acid–base titration revealed two state transitions at pH 4.8 and highly pH-dependent structural
conformation reversibility. Thermal melting (Tm) studies suggest that at neutral pH, poly BuNA(A) is a
weakly organized single strand, while at low pH it adopts a highly organized and rigid structure.
Furthermore, MALDI-TOF-MS data revealed intermolecular interactions which led to the formation of an
A-motif composed of a double helical structure. Since BuNA does not suffer from depurination under
acidic conditions, this allowed us to determine the thermodynamic parameters of the A-motif. This is the
first report of the construction of an A-switch using artificial nucleic acids.
Received 15th March 2013,
Accepted 2nd August 2013
DOI: 10.1039/c3ra41255e
www.rsc.org/advances
acids containing negatively charged phosphodiester linkages
have an advantage over a peptide backbone as they facilitate
the solubility in aqueous medium.
Introduction
The growing field of DNA technology requires new chemically
modified nucleic acid analogues that can perform advanced
and superior functions with greater chemical stability.1
Artificial nucleotide analogues have been widely explored in
order to modulate the nucleic acids, which make them
promising building blocks for constructing molecular
devices.2 These molecular devices are made up of unusual
structures, involving non-Watson–Crick base pairing such as
in i-motif, G-quadruplex and A-motif.3 In order to modulate
the properties of these molecular devices with superior
functions, researchers have investigated various analogues of
natural nucleotides like Unlocked Nucleic Acid (UNA),4 Locked
Nucleic Acid (LNA),5 Peptide Nucleic Acid (PNA),6 methylpho-
sphonate,7 phosphoramidite,7 phosphorothioate8 and 39-
S-phosphorothiolate,9 which have been incorporated into
nucleic acid structures. In addition, DNA has been used for
construction of the nanomechanical devices based on the B-Z
conformational transition.10
Since acyclic nucleotides are structurally simplified15 so the
chemical synthesis of their phosphoramidite building blocks
can be achieved by rather simple organic transformations.
Glycol Nucleic Acid (GNA)16a reported by Eric Meggers was
used for constructing two mirror image 4-Helix Junctions.16b
This report suggests that acyclic nucleic acid can be used for
constructing nanostructures. On similar lines, Threoninol
Nucleic Acid (aTNA)17a and Serinol Nucleic Acid (SNA)17b have
been demonstrated as C-3 scaffold-based acyclic nucleic acids,
which adopt A-form and B-form-like helical structures respec-
tively. Furthermore, aTNA has been demonstrated to be an
effective scaffold for constructing photo switches.18 In addi-
tion, by using artificial nucleic acid building blocks, it might
be possible to create molecular devices with new chemical and
physical properties which are not observed in DNA/RNA-based
devices.
The molecular properties of poly r(A) at low pH were
studied by Fresco and Klemperer.19 In 1961, Rich et al.
investigated the organization of adenine moieties at low pH in
a duplex of poly r(A) by X-ray studies.20 These studies revealed
that poly r(A) adopts a parallel right handed double stranded
helical structure at low pH and strands are held together by
Hoogsteen base-pairing (A+.A+) and salt bridges between the
protonated N1 of adenine and the phosphate backbone.
Recent reports illustrated the conformational changes of poly
d(A)15 at low pH and its use as a DNA-based pH sensor.21
Therefore, low pH is essential for poly d(A) to act as an
A-switch but undermines the chemical stability of the
Chemical modification of nucleic acids can be achieved by
modifying the sugar-phosphate backbone or the nucleobase
moieties.11 Researchers have investigated various artificial
nucleic acid systems containing nondeoxyribose,12 acyclic
backbones13 and polypeptide backbones.14 Artificial nucleic
Chemical Biology Laboratory, Department of Biotechnology, Indian Institute of
Technology Madras (IITM), Chennai 600036, India. E-mail: vkesavan@iitm.ac.in;
Tel: +91-44-22574124
Electronic supplementary information (ESI) available: Synthesis,
3
characterization, NMR spectra, MALDI-TOF-MS and additional figures are
provided. See DOI: 10.1039/c3ra41255e
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Fig. 1 Chemical structures of DNA, (S)-(BuNA) and (S)-BuNA nucleoside.
N-glycosidic bond of purines under the same conditions.22
G-quadruplexes and i-switches have been developed from PNA,
but this system cannot be used for constructing an A-switch
due to its neutral peptide backbone. Hence, it is vital to
explore new artificial nucleic acids, which are chemically and
enzymatically more stable, in order to construct molecular
devices with superior functions. Recently, we have investigated
the pairing properties of acyclic Butyl Nucleic Acid (BuNA) and
its incorporation in to DNA strands.23 Herein, we report the
construction of a functional A-motif from acyclic Butyl nucleic
acid (BuNA) (Fig. 1) which behaves as an A-switch by acid–base
changes.
Scheme 1 Synthesis of phosphoramidite building blocks of (S)-BuNA. (a) HNO2/
HBr, 60% yield. (b) Me2S-BH3, THF, 87%. (c) Cs2CO3, CH2Cl2, 65%. (d) DMT-Cl,
triethylamine, CH2Cl2. 60%. (e) Thymine, 0.3 equiv. NaH, dry DMF, 110 uC, 45%.
(f) 2-Cyanoethyl N,N,N9,N9-tetraisopropylphosphordiamidite, diisopropyl ammo-
nium tetrazolide, CH2Cl2, 73%. (g) Adenine, 0.3 equiv. NaH, dry DMF, 110 uC,
45%. (h)
3 equiv. TMS-Cl, benzoyl chloride, anhydrous pyridine, 60%. (i)
2-Cyanoethyl N,N,N9,N9-tetraisopropylphosphordiamidite, diisopropyl ammo-
nium tetrazolide, CH2Cl2, 67%.
ing blocks of A and T in 63% and 73% yield respectively.23
Synthesis of oligonucleotides was carried out using an
automated solid-phase oligonucleotide synthesizer by stan-
dard protocol (Table 1). Purification of oligonucleotides was
carried out by preparative denaturing polyacrylamide gel
electrophoresis (PAGE) and product identity was confirmed
Result and discussion
by MALDI-TOF-MS (Table S5, ESI ).
3
Design and synthesis of (S)-BuNA
Incorporation of (S)-BuNA nucleotides in to DNA strand and
pairing properties of poly BuNA (A) and poly BuNA (T)
The design of the acyclic scaffold was based on the six-bonds-
per-backbone unit13e of vicinal phosphodiester groups similar
to DNA or RNA, which may enable conventional Watson–Crick
or Hoogsteen base-pairing as in unmodified nucleic acids
(Fig. 1). Accordingly, (R)-aspartic acid 1 was converted into
bromosuccinic acid 2 by the diazotization reaction in the
presence of sodium bromide. Compound 2 was reduced by
borane dimethyl sulphide in dry THF at 0 uC to give
corresponding bromodiol 3. Treatment of 3 with cesium
carbonate in dry dichloromethane afforded the epoxide 4 in
65% yield. Preparation of DMT-epoxide 5 was achieved by
treatment of 4 with DMT-Cl in the presence of triethylamine in
60% yield. DMT-epoxide underwent ring opening with the
respective nucleobases in the presence of 0.3 equivalent of
Prior to demonstrating the use of BuNA as molecular switch, it
is important to study the effect of (S)-BuNA nucleotides
incorporation in the DNA duplex stability and pairing proper-
ties to its complementary strands. In order to understand the
effect of integration of (S)-BuNA nucleotides in the DNA
duplex, we investigated conformational studies by CD spectro-
Table 1 Oligonucleotides used in the study
Entry
Sequencesa
Nucleotides
ON-1
ON-2
ON-3
ON-4
ON-5
ON-6
ON-7
ON-8
49-aaaaaaaaaaaaaaA-39 BuNA(A)14dA
49-ttttttttttttttT-39 BuNA(T)14dT
49-aaaaaaaatttttttT-39
15
15
16
16
15
15
15
15
NaH to afford DMT-alcohols
Heteronuclear multiple-bond correlation (HMBC) spectro-
scopy was used for the confirmation of regioselectivity of
6
and
8
(Scheme 1).
49-atatatatatatataT-39
nucleobase N-alkylation (Fig. S4, ESI ). To avoid side reactions
59-AAAAAAAAAAAAAAA-39
59-AAAAAAAaAAAAAAA-39
59-TTTTTTTtTTTTTTT-39
59-TTTTTTTTTTTTTTT-39
3
in the phosphitylation step, the exocyclic amine of adenine
was protected by the benzoyl group to give 9 in 67% yield.
Phosphitylation of DMT-alcohols was carried out using 1.5
equivalents N,N,N9,N9-tetraisopropylphosphordiamidite and
0.5 equivalent diisopropyl ammonium tetrazolide in anhy-
drous dichloromethane, yielding the phosphoramidite build-
a
Lower case letters represent (S)-BuNA nucleotides and upper case
letters represent DNA nucleotides. Sequences for BuNA are shown
from 49 to 39 instead of 49 to 29 because BuNA oligonucleotides were
synthesized on dA and dT CPG-solid supports.
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7.23 Fig. 2b clearly indicates that the conformation of ON-1 is
associated with a positive cotton effect at 269 nm and a
negative cotton effect at 217 nm with a crossover at 247 nm,
which revealed its preorganized structure. On the other hand,
under similar experimental conditions, the CD of ON-2
showed positive peaks at 273 nm and 219 nm, and a negative
peak at 242 nm with less cotton effect. The CD profile of the
1 : 1 mixture of ON-1 and ON-2 suggests a new conformation
adopted by the duplex resulting in positive peaks at 273 nm
and 217 nm with negative peaks at 245 nm and 203 nm
(Fig. 2b). UV-melting at 260 nm was carried out to determine
the Tm of the duplex (ON-1/ON-2), which was observed as 22 uC
in 10 mM phosphate buffer in 1 M sodium chloride at pH 7
(data not shown). We were not surprised by the low duplex
stability of ON-1 and ON-2 due to the unusual behaviour of
poly (A) and poly (T).25 To further demonstrate its pairing
properties we analysed the duplex stability of self-complemen-
tary sequences ON-3 and ON-4, which showed melting
temperatures of 39 uC and 40 uC, respectively, in 150 mM
sodium chloride (data not shown). Based on literature
evidence it can be suggested that (S)-BuNA adopts a left
handed A-form-like structure.26 These studies indicate that
BuNA nucleotides do not alter the B-form of DNA duplexes and
that BuNA oligonucleotides are capable of forming duplexes
with their complementary strands.
pH-induced structural changes of ON-1 probed by CD
(switching property)
The pH-induced structural transition of (S)-BuNA was inves-
tigated by CD. At neutral pH a positive cotton effect at 268 nm
and a negative cotton effect at 217 nm were observed for ON-1
(Fig. 3a). The melting transition observed at pH 7.0 was found
to be noncooperative (Fig. 3a inset).
Fig. 2 CD profile of modified DNA duplexes and pairing of (S)-BuNA duplex. (a)
Change in structural conformation by BuNA modified DNA strands (5 mM each
strand). (b) CD spectra of a 1 : 1 (5 mM each strand) mixture of BuNA strands
(ON-1 and ON-2) and individual strands ON-1 and ON-2 (20 mM each).
Experimental conditions: 10 mM phosphate buffer, 150 mM NaCl, pH 7.0 at
20 uC.
These results suggest that at neutral pH, ON-1 demonstrates
a weakly organized structure. On the other hand at pH 3.0 in
unbuffered solution, ON-1 exhibited
a more organized
structure, which is supported by CD observation (Fig. 3b). A
more profound peak with an increase in ellipticity up to six-
fold at 265 nm with a blue shift from 268 nm, along with a
shoulder peak at 273 nm was observed. Interestingly, the
negative cotton effect at 217 nm (neutral pH) completely
disappeared and a more prominent positive band at 217 nm
emerged. It is evident from Fig. 3b (inset) that the melting
transition is of cooperative nature at low pH, indicating a more
rigid structure for ON-1. ON-3 also showed similar conforma-
tional changes at low pH, but the secondary structure was
scopy and duplex stability by UV-melting. Two modified DNA
strands ON-6 and ON-7 were studied, where a BuNA nucleotide
was incorporated in the centre, since at this position
maximum destabilization can be observed for the acyclic
nucleotides.24 UV-melting experiment at 260 nm revealed that
integration of adenine (S)-BuNA nucleotide decreases the
melting temperature (Tm) by 6 uC per modification, while
incorporation of thymine nucleotide destabilizes the duplex by
9 uC.23 Furthermore, CD studies revealed that these duplexes
(ON-6/ON-8 and ON-5/ON-7) adopt a B-form structure, similar
to the unmodified DNA duplex (ON-5/ON-8), which clearly
indicates that integration of (S)-BuNA nucleotide does not
affect the B-form structural conformation of DNA duplex
(Fig. 2a).
found to be less stable (Fig. S6, ESI ). These observations
3
clearly indicate that a contiguous stretch of adenine nucleo-
tides of BuNA undergoes conformational changes at low pH,
which is termed as an A-motif.
Determination of apparent pKa of A-motif
In order to investigate the switching properties of a poly
BuNA (A), it is important to understand the properties of
individual strands of poly BuNA(A) and poly BuNA(T). At 20 uC
a pentadecamer BuNA(A)14dA (Table 1, ON-1) formed a left
handed double helical structure with its complementary
pentadecamer strand BuNA(T)14dT (Table 1, ON-2) in 10 mM
phosphate buffer containing 150 mM sodium chloride at pH
In order to understand the formation of the A-motif, it is
important to determine its apparent pKa.27 At 20 uC a decrease
in ellipticity at 265 nm was observed with increasing pH, with
a sharp transition point at pH 4.8 in a citrate buffer, which
indicates the two state transition of the A-motif (Fig. 4). The
change in ellipticity at wavelength 265 nm was plotted as a
function of pH, which revealed the existence of highly stable
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To prove the sequence dependency of the BuNA-based
A-motif, similar experiments were carried out using ON-3. The
conformational changes of ON-3 under acidic pH was easily
affected by a slight increase in pH unlike ON-1, hence it was
difficult to determine apparent pKa for ON-3. In order to
demonstrate the switching property of the A-motif, ON-1 was
treated with alternating acid-basic cycles demonstrating it to
be highly reversible (Fig. S7, ESI ). It is important to note that
3
no precipitation was observed for ON-1 or ON-3 at pH 2.
Effect of salt on A-motif
The A-motif is stabilized by hydrogen bonds and salt bridges
therefore, it is important to investigate the effect of strong
electrolytes on its structure. It has been reported that the DNA-
based A-motif is affected by the concentration of sodium
chloride in the system, which was investigated by CD
studies.21a On similar lines, we investigated the effect of
sodium chloride on the BuNA-based A-motif (ON-1). A solution
of 1 mM oligonucleotide (ON-1) at pH 3.0 was treated with
increasing sodium chloride concentrations resulting in a
range from 0 mM to the 531 mM final concentration. CD
was recorded after each addition, but the ellipticity at 217 nm
Fig. 3 Temperature-dependent CD profile of ON-1. (a) ON-1 with 20 mM
oligonucleotide concentration in 10 mM phosphate buffer, 150 mM NaCl at pH
7. (b) ON-1 with 10 mM oligonucleotide concentration at pH 3. Insets: CD-
melting profile of ON-1 at particular pH.
conformation in the range of pH (3 to 4.2). When the pH of the
solution was increased (pH . 4.2), a gradual conversion of the
rigid structure into a weakly organized one was observed
(Fig. 4 inset). These observations clearly indicate that an
A-motif exists at pH , 4.8, which dissociates into a single
strand at pH . 4.8.
Fig. 5 Effect of sodium chloride on the A-motif (ON-1). (a) CD spectrum of ON-1
with 1 mM solution of oligonucleotide at pH 3.0 in unbuffered solution with
different concentrations of sodium chloride. (b) Effect of sodium chloride in 10
mM acetate buffer at pH 4.
Fig. 4 pH-titration of 1 mM (ON-1) in 2 mM citrate buffer. Titration was started
at pH 3.0 and pH was increased by gradual addition of sodium citrate. Inset
shows the decrease in ellipticity at 265 nm on increasing pH of the solution. The
midpoint pH of the transition results an apparent pKa of the A-motif.
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Fig. 6 Irreversibility of thermal melting transition of the A-motif (ON-1 with 1
mM oligo concentration). (a) CD-melting monitored at wavelength 265 nm in
unbuffered aqueous solution at pH 3. (b) The CD signal was regained by
addition of 0.1 N sodium hydroxide solution to neutralize the oligonucleotide
solution followed by addition of 0.1 N hydrochloric acid to decrease the pH.
Fig. 7 Reversible CD-melting profile of A-motif (ON-1 with 1 mM oligo
concentration) in 10 mM citrate buffer, monitored at wavelength 265 nm.
It is evident from Fig. 6b, that ON-1 regained its CD signal,
whereas in the case of DNA no such observations were made.
In order to achieve the reversible melting transition, a melting
experiment at pH 3.8 was performed, where half of the
adenine nucleotides were expected to be protonated. It is clear
from Fig. 7a that the melting curve is completely reversible at
pH 3.8 with a Tm of 64 uC. After lowering the pH to 3.4 in
citrate buffer, partial reversibility of the melting curve was
observed with an increase in Tm to 73 uC (Fig. 7b). It is
important to note that folding and unfolding of the A-motif at
pH . 3.8 was completely reversible. These findings suggest
that the A-motif displays a reversible melting transition at half
or less than half protonation, while it exhibits an irreversible
melting transition at more than half protonation of
BuNA(A)14dA. pH-dependent melting experiments show that
the melting temperature increases with decreasing pH of the
solution (Fig. 8a) (Table 2).
Furthermore, we investigated the effect of oligonucleotide
concentration on melting temperature. Tm was determined
with different concentrations of ON-1 (500 nM, 5 mM and 10
mM) at pH 4 in acetate buffer. It is evident from Fig. 8b that the
melting temperature is concentration dependent. This study
suggests that the A-motif is composed of intermolecular
interactions between oligonucleotide strands.
and 265 nm remained unchanged in the tested concentration
range as evident from Fig. 5a.
A similar experiment was carried out in 10 mM sodium
acetate buffer at pH 4.0, but no change in conformation was
detected (Fig. 5b). These results clearly suggest that sodium
chloride does not affect the structure of the BuNA-based
A-motif.
Thermal stability of A-motif (ON-1) and effect of pH on
reversibility of melting transition
We have demonstrated that at pH . 4.8 the BuNA-based
A-motif exhibits a transition from highly organized to weakly
organized structure. To study the thermal stability of the
A-motif, melting studies were carried out in a pH range of 3 to
5. The melting temperature (Tm) value of ON-1 was determined
to be 70 uC at pH 3.0 in unbuffered solution (Fig. 6a). Thermal
transition was found to be irreversible at this pH. While
determining Tm, it was noticed that the oligonucleotide
solution became turbid and the intensity of CD signal was
almost zero after the cooling ramp. Interestingly, the CD signal
could be restored by increasing the pH of the turbid solution
followed by acidification to the desired pH.
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Fig. 8 Effect of pH and oligonucleotide concentrations on melting temperature
of A-motif (ON-1). (a) pH-dependent melting experiment. All the melting
profiles were generated at 265 nm with 1 mM of ON-1 in 10 mM citrate buffer
with different pH values. (b) Concentration dependent melting temperature in
acetate buffer at pH 4. Tm = 59 uC (0.5 mM); Tm = 61 uC (5 mM); Tm = 64 uC (10
mM). Solid lines represent sigmoidal curve fitting of melting curves.
Fig. 9 (a) UV-spectrum of ON-1. (b) UV-melting profile at 260 nm of ON-1.
Experiments were carried out in unbuffered solution with 4 mM oligonucleotide
solution at pH 3.
blue shift at 258 nm (Fig. 9a). This hypochromic effect
suggests that in acidic condition, ON-1 is a rigid structure
compared to neutral pH. The Tm value of the A-motif was
determined to be 77 uC in unbuffered solution at pH 3.0
(Fig. 9b). Melting curves clearly indicate a hypochromic effect
at pH 3 instead of a hyperchromic one due to the turbidity of
the solution. These results are in agreement with the
observation made by CD-melting studies at pH 3. It is
noteworthy that the UV-melting at pH 4 in acetate buffer was
observed as reversible melting transitions with hyperchromic
effect (data not shown). This study supports the reversible
thermal melting transition of A-motif at pH . 3.8 which was
investigated by CD-melting experiments. Similar UV-melting
experiments were carried out with d(A)15 (ON-5) at pH 3.0 with
Tm (51 uC), where the melting curve can be easily differentiated
UV spectroscopy
Ultra-violet (UV) light absorption spectroscopy is sensitive to
the stacking of p-bonded moieties such as nucleobases and to
hydrogen bonding between nucleotides of nucleic acids.
Therefore, it is an effective method to monitor the helix-to-
coil transition between the folded and unfolded structure. UV
spectral studies at neutral and acidic pH can provide structural
insights into the BuNA-based A-motif. At 25 uC ON-1 was
dissolved in 10 mM phosphate buffer at pH 7.0 showed a peak
at 260 nm (Fig. 9a). On the other hand, in unbuffered aqueous
solution at pH 3.0 a hypochromic effect was observed with a
from the depurination curve (Fig. S8, ESI ).
3
Table 2 Melting temperature of A-motif (ON-1) at different pH
MALDI-TOF-MS study of ON-1
Mass spectrometry (MS) has been proved as a successful
technique to investigate non-covalent intermolecular interac-
tions of DNA,28 RNA29 and DNA-RNA based molecular devices.
MALDI-TOF-MS was used to further characterize BuNA based
A-motif. Five peaks were observed at m/z [4243.692], [4243.692
+ Na+], [4243.692 + K+], [4243.692 + Na+ + K+], and [4243.692 + 2
Entry
pH
Tm (uC)a
1
2
3
4
3.4
3.8
4.4
4.8
73
64
59
49
a
Experiments were carried out in 10 mM citrate buffer.
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Fig. 10 MALDI-TOF-MS spectra of ON-1 (single strand).
K+] at neutral pH, where the first peak corresponds to
molecular weight of the ON-1 (Fig. S9a, ESI ). Next we
3
investigated MALDI-TOF-MS of ON-1 in citrate buffer at pH
3. It is evident from Fig. 10 that a peak at m/z [4256.062]
indicates [MW + 12H+] which corresponds to a protonated
single strand. Formation of the A-motif was confirmed by
interstrand interactions resulting in a peak at m/z [8513.225]
corresponding to the duplex [2MW + 25H+] (Fig. 11). Other
peaks observed in the mass spectrum belong to sodium
adducts. These results clearly suggest that the A-motif is
formed by two strands of ON-1.
Fig. 12 (a) CD profile of ON-3 at pH 3 and pH 7 at 0 uC. (b) Temperature-
dependent CD profile of ON-3 (4 mM oligo concentration) in 10 mM tris-buffer
containing 100 mM NaCl, 10 mM MgCl2 at pH 7.
Switching property of ON-3
We have demonstrated that a contiguous stretch of (S)-BuNA
adenine nucleotides shows the pH-dependent behavior of an
A-switch. To investigate the switching properties of a self-
complementary strand of BuNA, an oligonucleotide containing
a stretch of eight adenine nucleotides (ON-3) was used. CD
spectroscopy of ON-3 in 10 mM tris buffer containing 100 mM
sodium chloride and 10 mM MgCl2 at pH 7 revealed an
A-form-like helical structure with positive ellipticity at 269 nm
and 222 nm with negative peaks at 246 nm and 204 nm,
(Fig. 12a). On the other hand, ON-3 under acidic condition
adopted a different conformational structure, similar to
A-motif (ON-1), with positive CD bands at 267 nm and 217
and a negative band at 234 nm (Fig. 12a). Fig. 12a demon-
strates that the antiparallel duplex at neutral pH showed a
positive ellipticity at 222 nm, while at low pH a positive peak
with blue shift emerged at 217 nm with high intensity, which
support the switching in conformation.
Temperature-dependent CD studies of ON-3 revealed stable
duplex formation at pH 7 (Fig. 12b), while at low pH a decrease
in stability was observed (Fig. 13a). In order to determine the
stability of the A-motif of ON-3, CD-melting was carried out,
which displayed a Tm of 12 uC (Fig. 13b). It is important to note
that the melting transition of ON-3 was reversible at pH 3,
which was observed to be irreversible for ON-1. At present we
do not have experimental proof for the orientation of BuNA
strands in the A-motif, which could be oriented in either
parallel or antiparallel directions. Furthermore, ON-3 was
analyzed by MALDI-TOF-MS. The mass spectrum shows a peak
at m/z [4460.590] corresponding to a single strand [MW + 3H+]
Fig. 11 MALDI-TOF-MS spectra of ON-1 (A-motif).
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These experiments prove that the switching property of ON-1
or ON-3 is not due to flexibility of the acyclic backbone, but
due to the contiguous arrangement of BuNA adenine nucleo-
tides. This study clearly indicates that polyadenine building
blocks derived from either DNA or BuNA follow the same
physical principles for formation of an A-motif.
Thermodynamics analysis of A-motif (ON-1)
The thermally-induced reversible melting transition of folded
structures can be monitored by CD-melting.30 Additionally, by
selecting an appropriate wavelength, the decrease in ellipticity
of CD with increasing temperature can be monitored, and
converted to fraction folded versus temperature.31
Furthermore, this data can be used to obtain a Van’t Hoff
plot in order to determine thermodynamic parameters.32 The
concentration-dependent melting temperature of ON-1
revealed the intermolecular nature of A-motif. Moreover, on
the basis of MALDI-TOF-MS, a molecularity of two was used for
calculating the thermodynamic parameters of A-motif. Free
energy (DGu) of the A-motif (ON-1) was evaluated, where
enthalpy and entropy are assumed to be independent of
temperature using Gibbs equation DGu = 2RT ln(Ka). Ka is the
equilibrium constant of folding and unfolding of the A-motif.
Fig. 13 (a) Temperature-dependent CD profile of ON-3 (4 mM oligo concentra-
tion) in 10 mM citrate buffer at pH 4. (b) CD-melting profile of ON-3 at pH 3.
(Fig. S9e, ESI ). At pH 3 a peak at m/z [4469.696] corresponds to
3
[MW + 9H+] with six extra protons (Fig. S9g, ESI ) and the
3
second peak at m/z [8936.886] corresponding to [2MW + 15H+]
can be assigned to a duplex formation by two oligonucleotide
strands (Fig. S9h, ESI ). Additionally, both the peaks are
3
associated with sodium adducts as was observed for ON-1.
These results clearly indicate the switching conformations of
ON-3, from antiparallel duplex (pH 7) to A-motif, by the
stimulus of protons.
Effect of flexible backbone of (S)-BuNA on A-motif
(S)-BuNA is composed of an acyclic backbone. Therefore one
can debate that the formation of the A-motif might be due to
backbone flexibility despite the contiguous stretch of acyclic
adenine nucleotides. Therefore, we investigated other
sequences (ON-2 and ON-4) to demonstrate the sequence-
dependent formation of the A-motif. BuNA(T)14dT (ON-2)
failed to undergo such conformational changes (data not
shown), since protonation and an A-form-like structure
through the Hoogsteen hydrogen bonding is not possible for
BuNA(T)14dT under acidic condition. Next, we investigated the
self-complementary BuNA sequence ON-4, where acyclic
adenine and thymine nucleotides were arranged in an
alternative fashion. In aqueous unbuffered solution at pH
Fig. 14 Thermodynamic analysis of A-motif (ON-1) at pH 4.0 in 10 mM acetate
buffer (squares: no salt was added, circles: 150 mM sodium chloride). (a)
Fraction folded as a function of temperature. (b) Determination of DHu and DSu
from Van’t Hoff plot of ln(K) versus 1/T. DHu was calculated from slope of the
curve and DSu was calculated from intercept of the curve.
3.0, ON-3 displayed no change in conformation (Fig. S10, ESI ).
3
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Table 3 Thermodynamic data of ON-1
NaCl (mM)
DHu (kcal mol21
)
DSu (kcal mol21K21
)
DGu (kcal mol21
)
Tm (uC)
0.0
150
283.7
275.6
20.222
20.206
214.8
211.7
61
48
CD-melting was carried out for ON-1(1 mM) in 10 mM of
acetate buffer at pH 4 and heated from 10 uC to 90 uC at a
heating rate of 1 uC min21 and the Tm value was determined to
be 61 uC. It is noteworthy that no annealing is required for
performing the melting experiment because the formation of
the A-motif is an instantaneous process. The equilibrium
constant was expressed as Ka = h/(2c(1 2 h)2) for a bimolecular
arrangement involving identical strands, where c is the total
strand concentration and h is the fraction of folded oligonu-
cleotide, which was determined at each temperature from the
reversible melting curve (Fig. 14a). To extract thermodynamic
data we chose points with 0.15 ¡ h ¡ 0.85 in the van’t Hoff
plot, because this is the region where Ka values are most
precise.32b In order to calculate enthalpy (DHu) and entropy
(DSu) a plot of ln(Ka) against 1/T(K21) was plotted, which gave a
straight line under a two-state transition model (Fig. 14b). The
slope and intercept of the straight line correspond to (2DHu/R)
and (DSu/R), respectively. Heating curves were corrected for
baseline prior to evaluating the Tm and thermodynamic
parameters (Table 3). Free energy (DG) was calculated at 37 uC.
In order to investigate the effect of sodium chloride on the
thermal stability of the A-motif, similar experiments were
carried out in the presence of 150 mM sodium chloride in
acetate buffer at pH 4. The melting temperature was found to
any such decomposition, since no DNA adenine nucleotide is
present in this sequence (Fig. S11b, ESI ). This clearly indicates
3
that ON-5 was completely decomposed under acidic condi-
tions at pH 3 (Fig. 15d), which showed a major peak at 4.144
min corresponding to adenine. This study suggests that the
BuNA scaffold is chemically stable at low pH which is an
important property for constructing molecular devices.
be 48 uC with free energy of folding DGu = 211.7 kcal mol21
.
These results clearly demonstrate that the presence of sodium
chloride affects the thermal stability of the A-motif by
destabilizing salt bridges. Since curve fitting (van’t Hoff)
analysis is only applicable for reversible melting transitions,
determination of thermodynamic parameters of DNA-based
A-motif was not possible due to the irreversible melting
transition at pH 4.
Chemical integrity of A-motif
In order to investigate the chemical integrity of (S)-BuNA and
DNA oligonucleotides at low pH, we performed stability tests.
10 mM solution of each oligonucleotide in aqueous unbuffered
solution at pH 3.0 was heated to 90 uC for 20 min. Analysis was
carried out by reverse-phase HPLC by Luna C18 (100A, 250 6
4.60 mm, 5 micron) column. A buffer system of 100 mM
triethyl ammonium acetate at pH 7.0 with gradient flow (10–
30% acetonitrile in 20 min) was used for eluting the
oligonucleotides and its decomposed products. HPLC profiles
(Fig. 15) demonstrate that BuNA nucleotides do not show
depurination, while the DNA oligonucleotide (ON-5) decom-
poses, due to depurination of the deoxyadenine nucleotides.
The HPLC chromatogram showed two peaks for ON-1 at 11.172
min and 10.185 min, which was expected due to the presence
of one natural deoxyadenine nucleotide at the 39 end which
underwent depurination (Fig. 15b). ON-3, which is composed
of eight acyclic contiguous adenine nucleotides, did not show
Fig. 15 HPLC profile of oligonucleotides. (a) ON-1. (b) ON-1 heated at pH 3. (c)
ON-5. (d) ON-5 heated at pH 3.
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Conclusions
In conclusion, we report the synthesis and construction of a
BuNA-based A-switch. CD studies suggested that the (S)-BuNA
duplex forms a left handed A-form-like structure at pH 7 and
the incorporation of (S)-BuNA nucleotides does not alter the
B-form helical structure of the DNA duplexes. UV and CD-
melting studies revealed a highly stable structure of the
A-motif. Furthermore, MALDI-TOF-MS experiments proved
that the A-motif is assembled of two BuNA strands. Due to
the chemical stability of BuNA at low pH, we were able to
calculate thermodynamic parameters, where the DG value for
the formation of the A-motif structure was determined to be
214.8 kcal mol21 (pH 4.0, 37 uC). In the future these findings
might provide a new platform for developing acyclic nucleic
acids-based molecular devices. Further studies are going on to
construct BuNA-based i-switches and G-quadruplexes.
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Acknowledgements
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The authors acknowledge financial support from the DBT and
ICMR, Govt. of India (BT/PR/10064/AGR/36/30/07). The
authors gratefully thank department of Chemistry IIT
Madras, SAIF-IIT Madras for analytical data. Vipin thanks IIT
Madras for fellowship. Vipin thanks to Prof. Yamuna
Krishnan’s research group from National Center for
Biological Sciences (NCBS) India, for valuable discussion
about DNA-based A-motif. Vipin thanks to Dr David Schaffert
from Interdisciplinary Nanoscience Center-INANO-MBG,
Aarhus University for proof reading of manuscript.
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