Kinetic study of the binding of triplex-forming oligonucleotides containing partial cationic modifications to double-stranded DNA

Article abstract of DOI:10.1016/j.bmcl.2014.05.031

Several triplex-forming oligonucleotides (TFOs) partially modified with 2′-O-(2-aminoethyl)- or 2′-O-(2-guanidinoethyl)-nucleotides were synthesized and their association rate constants (k_on) with double-stranded DNA were estimated by UV spectrophotometry. Introduction of cationic modifications in the 5′-region of the TFOs significantly increased the k_on values compared to that of natural TFO, while no enhancement in the rate of triplex DNA formation was observed when the modifications were in the middle and at the 3′-region. The k_on value of a TFO with three adjacent cationic modifications at the 5′-region was found to be 3.4 times larger than that of a natural one. These results provide useful information for overcoming the inherent sluggishness of triplex DNA formation.

Full text of DOI:10.1016/j.bmcl.2014.05.031

Bioorganic & Medicinal Chemistry Letters 24 (2014) 3046–3049
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Kinetic study of the binding of triplex-forming oligonucleotides
containing partial cationic modifications to double-stranded DNA
⇑
⇑
Yoshiyuki Hari , Shin Ijitsu, Masaaki Akabane-Nakata, Takuya Yoshida, Satoshi Obika
Graduate School of Phamaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita 565-0871, Japan
a r t i c l e i n f o
a b s t r a c t
Several triplex-forming oligonucleotides (TFOs) partially modified with 20-O-(2-aminoethyl)- or 20-O-
(2-guanidinoethyl)-nucleotides were synthesized and their association rate constants (kon with
Article history:
Received 15 April 2014
Revised 8 May 2014
Accepted 10 May 2014
Available online 17 May 2014
)
double-stranded DNA were estimated by UV spectrophotometry. Introduction of cationic modifications
in the 50-region of the TFOs significantly increased the kon values compared to that of natural TFO, while
no enhancement in the rate of triplex DNA formation was observed when the modifications were in the
middle and at the 30-region. The kon value of a TFO with three adjacent cationic modifications at the 50-
region was found to be 3.4 times larger than that of a natural one. These results provide useful informa-
tion for overcoming the inherent sluggishness of triplex DNA formation.
Keywords:
Kinetic studies
Modified nucleotides
Oligonucleotides
Triplex DNA
Ó 2014 Elsevier Ltd. All rights reserved.
Triplex-forming oligonucleotides
Triplex-forming oligonucleotides (TFOs) bind to double-stranded
DNA (dsDNA) forming triplex DNA. They can be used as reagents for
gene modification, control of gene expression, gene detection etc.1
However, triplex DNA formation by TFO has some drawbacks. For
instance, triplexes are formed by two Hoogsteen hydrogen bonds
between T and C in the TFO with AT and GC base pairs in dsDNA,
respectively, which is less stable than the Watson–Crick base pairing
in the duplex DNA. Moreover, the sequence recognizable by natural
TFO is limited to homopurine/homopyrimidine tracts within dsDNA.
Several strategies, including introduction of modified nucleotides in
the TFO, to stabilize triplex DNA or to expand the recognizable
sequence have been reported.2 These reports mainly deal with the
thermodynamic evaluations of triplex DNA formation using melting
temperature (Tm), binding/dissociation constant (Kd) etc.
However, the kinetic studies are limited to examples of 50-trimeth-
ylpsoralen-conjugated 20-methoxy-TFOs. Moreover, to the best of
our knowledge, there are no other reports where the effects of
the number and distribution of modified nucleotides in TFOs were
examined in detail.3,6–8
Under such background, we considered that it is worth explor-
ing the effects of modifications in TFOs without any conjugation on
the rate of triplex DNA formation. Cationic modifications in the
TFO are expected to enhance the rate of triplex formation by
suppressing the electrostatic repulsion between phosphate
backbones of TFO and dsDNA. Therefore, 20-O-(2-aminoethyl)-6 or
20-O-(2-guanidinoethyl)-nucleotides9 were chosen as cationic
modifications (Fig. 1), and in this study, we aimed at exploration
of the rate of triplex DNA formation using their modified TFOs.
Kinetic studies of triplex formation revealed that the rate of for-
mation of triplex DNA was approximately 103 times slower than
that of duplex DNA.3 In 2004, Seidmen and coworkers studied
the effects of partial 20-O-(2-aminoethyl) modification within
50-trimethylpsoralen-conjugated 20-methoxy-TFOs, on kinetics of
triplex DNA formation.4 Interestingly, they found that an increase
in the rate of formation of triplex led to a significant increase in
the gene targeting efficacy, which strongly suggests the signifi-
cance of the evaluation of kinetic parameter of triplex formation.4,5
(a)
(b)
O
O
Base
Base
O
O
NH2
NH2
O
P
O
O
P
O
NH3
amine unit
N
H
O
O
O
O
guanidine unit
alleviation of electrostatic repulsion
between phosphate backbones in
triplex DNA formation
protonation at neutral pH
⇑
Corresponding authors. Tel./fax: +81 6 6879 8201 (Y.H.); tel.: +81 6 6879 8200;
fax: +81 6 6879 8204 (S.O.).
E-mail addresses: hari@phs.osaka-u.ac.jp (Y. Hari), obika@phs.osaka-u.ac.jp
Figure 1. Structures of 20-O-(2-aminoethyl)nucleotide (a) and 20-O-(2-guanidino-
(S. Obika).
ethyl)nucleotide (b).
http://dx.doi.org/10.1016/j.bmcl.2014.05.031
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

Y. Hari et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3046–3049
3047
GCC-30 (spacer = hexaethylene glycol), was examined at various
concentrations of KCl and MgCl2 at pH 6.8 (Table 1).15,16 In general,
at a given MgCl2 concentration, the Tm values decreased with
increase in KCl concentration (e.g., entries 2, 6 and 9). On the
contrary, increase in MgCl2 concentration significantly stabilized
the triplex DNA (e.g., entries 8–11), and in the absence of MgCl2
no triplex formation was observed (entries 4 and 11). These
findings coincide with that reported by Dervan’s group.17 Eventu-
ally, a buffer with 10 mM KCl and 10 mM MgCl2, in which the most
stable triplex DNA was formed (Tm = 43 °C, entry 8), was chosen for
kinetic experiments.
N
O
N
N
N
N
Me
NH
Me
DMTrO
O
DMTrO
O
N
O
N
(ii)
O
O
O
OR
O
N
O
P
O
i-Pr2N
OCE
O
R = H (1)
O
3
(i)
N(i-Pr)2
P
2
)
R =
(
OCE
All kinetic experiments were carried out at 20 °C, where
complete triplex formation was observed (Fig. 2).18 The measure-
ments were made on a UV spectrophotometer equipped with a
rapid mixing stopped-flow accessory.19 This method can observe
formation of triplex DNA itself without conjugation to any mole-
cules or solid-supports. As representative results, the decay curves
obtained for the triplex formation between TFOs 7–9 and 14 and
dsDNA 15 are shown in Figure 3. The association rate constants,
kon, calculated are summarized in Table 2. As it can be seen from
the table, kon values depends on the number of 20-O-(2-amino-
ethyl) modification at 50-region of the TFO and increased with
increase in the number of modifications (entries 1–4). TFO 7
including three adjacent modifications formed a triplex 3.4 times
faster than natural TFO 14. On the other hand, TFO 8 with three
adjacent modifications in the middle showed only a slightly higher
kon value than natural 14, whereas TFO 9 with the modification at
the 30-region, showed a lower kon value than that of 14 (entries 5
and 6). The result of TFO 9 might imply that modification of
5-methylcytidine negatively affects the rate of triplex formation
though the further investigation is required to clarify it. Interest-
ingly, although triplexes formed by TFOs 7 and 8 had the same
thermal stability (Tm = 54 °C), the kon of TFO 7 is apparently larger
than that of TFO 8. Similar trend in kon and Tm values were
observed for the corresponding 20-O-(2-guanidinoethyl) modified
TFOs 10–13 (entries 7–10).
The kinetic data shown in Table 2 revealed that the rate of tri-
plex formation increased significantly when the cationic modifica-
tions were introduced at the 50-region. Interestingly, this result is
in contrast to the report by Seidman’s group4 where they observed
an increase in the association rate, even with a single modification
at the 30-region. Although the reason is unclear, the presence of tri-
methylpsoralen at the 50-terminus is considered to have an effect
in triplex formation.
The mechanism of triplex formation could be explained by
nucleation-zipping model.7 Here, the nucleation step in which a
small number of base triads are formed can be assumed to be
the rate-limiting step. It is possible that the formation of the
O
N
Me
O
5'-TTTTTCTTTCTCTCT-3' (5)
5'-TTTTTCTTTCTCTCT-3' (6)
5'-TTTTTCTTTCTCTCT-3' (7)
5'-TTTTTCTTTCTCTCT-3' (8)
5'-TTTTTCTTTCTCTCT-3' (9)
5'-TTTTTCTTTCTCTCT-3' (10)
5'-TTTTTCTTTCTCTCT-3' (11)
NH
DMTrO
2, 3 and
O
O
(iii)
N
O
OCE
O
P
O
N
H
NH
OCE
TTT
12
13
5'-T
TCTTTCTCTCT-3'
(
(
)
)
i-Pr2N
OCE
TTT
5'-TTTTTC
CTCTCT-3'
4
O
N
NH2
O
Me
Me
Me
NH
N
NH
O
O
O
O
N
O
N
O
O
O
O
T =
C =
T =
NH2
NH2
O
P
O
O
P
O
O
P
O
NH3
NH3
N
O
O
O
O
O
O
H
Scheme 1. Reagents and conditions: (i) i-Pr2NP(Cl)OCH2CH2CN, i-Pr2NEt, CH2Cl2, rt,
2 h, 95%; (ii) POCl3, Et3N, 1,3,4-triazole, MeCN, 0 °C, 1 h, 77%; (iii) DNA synthesis
(DMTr = 4,40-dimethoxytrityl, CE = 2-cyanoethyl, C = 20-deoxy-5-methylcytidine).
The synthesis of modified TFOs used in this study is shown in
Scheme 1. Phosphitylation of the phthalimide-protected
compound 110 prepared in reference to an alternative method11
gave the desired phosphoramidite 2 in 95% yield.12 The compound
3 which was converted into 20-O-(2-aminoethyl)-5-methylcytidine
in TFO synthesis was prepared by commonly used triazolylation.13
TFOs 5–13 with partial modifications were synthesized using phos-
phoramidites 2 and 3 for 20-O-(2-aminoethyl) modification, and 49
for 20-O-(2-guanidinoethyl) modification on an automated DNA
synthesizer, and purified by reversed phase HPLC.14
To optimize the conditions for kinetic experiments, thermal
stability of triplex formed between natural TFO 14, 50-TTTTTC
TTTCTCTCT-30 (C = 20-deoxy-5-methylcytidine), and dsDNA target
15, 50-GGCAAAAAGAAAGAGAGACGC-spacer-GCGTCTCTTTCTTTTT
Table 1
40
: TFO 14
Thermal stability of the triplex formed between TFO 14 and dsDNA 15a
: TFO 7
8
: TFO
Entry
[KCl] (mM)
[MgCl2] (mM)
Tm (°C)
30
20
10
0
: TFO 9
1
2
3
4
5
6
7
8
100
100
100
100
50
10
1
0.5
0
10
1
40
35
34
ndb
41
37
35
43
41
40
ndb
50
50
10
10
0.5
10
1
9
10
11
10
20
30
40
50
60
70
80
90
10
10
0.5
0
Temperature (ºC)
a
Conditions: Sodium cacodylate buffer (10 mM, pH 6.8), KCl (100, 50 or 10 mM)
and MgCl2 (10, 1, 0.5 or 0 mM). The final concentration of each oligonucleotide used
was 1.89 M.
No triplex DNA formation was detected.
Figure 2. UV-melting curves of representative triplexes formed between TFOs and
dsDNA 15. [Conditions: Sodium cacodylate buffer (10 mM, pH 6.8), KCl (10 mM)
and MgCl2 (10 mM). The final concentration of each oligonucleotide used was
l
b
1.89 lM.]

3048
Y. Hari et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3046–3049
J. Clin. Invest. 2003, 112, 487; (d) Sasaki, S.; Nagatsugi, F. Curr. Opin. Chem. Biol.
0
– 0.02
– 0.04
– 0.06
2006, 10, 615; (e) Duca, M.; Vekhoff, P.; Oussedik, K.; Halby, L.; Arimondo, P. B.
Nucleic Acids Res. 2008, 36, 5123; (f) Mukherjee, A.; Vasquez, K. M. Biochimie
2011, 93, 1197.
: TFO 14
: TFO 7
: TFO 8
: TFO 9
2. Reviews: (a) Doronina, S. O.; Behr, J.-P. Chem. Soc. Rev. 1997, 26, 63; (b) Luyten,
I.; Herdewijn, P. Eur. J. Med. Chem. 1998, 33, 515; (c) Gowers, D. M.; Fox, K. R.
Nucleic Acids Res. 1999, 27, 1569; (d) Purwanto, M. G. M.; Weisz, K. Curr. Org.
Chem. 2003, 7, 427; (e) Fox, K. R.; Brown, T. Quart. Rev. Biophys. 2005, 38, 311;
(f) Malnuit, V.; Duca, M.; Benhida, R. Org. Biomol. Chem. 2011, 9, 326; (g) Fox, K.
R.; Brown, T. Biochem. Soc. Trans. 2011, 39, 629; (h) Hari, Y.; Obika, S.; Imanishi,
T. Eur. J. Org. Chem. 2012, 2875; (i) Hari, Y. Yakugaku Zasshi 2013, 133, 1201.
3. Xodo, L. E. Eur. J. Biochem. 1995, 228, 918.
– 0.08
– 0.10
4. Puri, N.; Majumdar, A.; Cuenoud, B.; Miller, P. S.; Seidman, M. M. Biochemistry
2004, 43, 1343.
0
300
600
900
Time (s)
1200
1500
1800
5. In addition to the association rate, importance of affinity for nonspecific targets
was also reported. Alam, M. R.; Majumdar, A.; Thazhathveetil, A. K.; Liu, S.-T.;
Liu, J.-L.; Puri, N.; Cuenoud, B.; Sasaki, S.; Miller, P. S.; Seidman, M. M.
Biochemistry 2007, 46, 10222.
Figure 3. UV absorbance decay curves for the triplex formation between TFOs and
dsDNA 15 in kinetic experiment.
6. Cuenoud, B.; Casset, F.; Hüsken, D.; Natt, F.; Wolf, R. M.; Altmann, K.-H.; Martin,
P.; Moser, H. E. Angew. Chem., Int. Ed. 1998, 37, 1288.
7. Alberti, P.; Arimondo, P. B.; Mergny, J.-L.; Garestier, T.; Hélène, C.; Sun, J.-S.
Nucleic Acids Res. 2002, 30, 5407.
Table 2
Association rate constants at 20 °C and thermal stability of triplexes formed between
TFOs 5–14 and dsDNA 15a
8. Reports on kinetic study of triplex DNA: (a) Maher, L. J.; Dervan, P. B.; Wold, B. J.
Biochemistry 1990, 29, 8820; (b) Rougée, M.; Faucon, B.; Mergny, J. L.; Barcelo,
F.; Giovannangeli, C.; Garestier, T.; Hélène, C. Biochemistry 1992, 31, 9269; (c)
Torigoe, H.; Hari, Y.; Sekiguchi, M.; Obika, S.; Imanishi, T. J. Biol. Chem. 2001,
276, 2354; (d) Sugimoto, N.; Wu, P.; Hara, H.; Kawamoto, Y. Biochemistry 2001,
40, 9396; (e) García, B.; Leal, J. M.; Paiotta, V.; Ruiz, R.; Secco, F.; Venturini, M. J.
Phys. Chem. B 2008, 112, 7132; (f) Torigoe, H.; Rahman, S. M. A.; Takuma, H.;
Sato, N.; Imanishi, T.; Obika, S.; Sasaki, K. Chem. Eur. J. 2011, 17, 2742.
9. Prakash, T. P.; Püschl, A.; Lesnik, E.; Mohan, V.; Tereshko, V.; Egli, M.;
Manoharan, M. Org. Lett. 2004, 6, 1971.
Entry
TFO
kon (ꢀ103 Mꢁ1 sꢁ1
)
kon-modified/kon-natural
Tm (°C)
1
2
3
4
5
6
7
8
9
10
14
5
6
7
8
3.62 0.32
5.91 0.10
7.87 0.38
12.38 0.91
4.72 0.36
2.70 0.63
5.75 0.25
8.14 0.46
11.63 0.72
4.65 0.36
—
43
46
50
54
54
47
44
49
53
53
1.6
2.2
3.4
1.3
0.7
1.6
2.2
3.2
1.3
9
10. Manoharan, M.; Prakash, T. P.; Barber-Peoc’h, I.; Bhat, B.; Vasquez, G.; Ross, B.
S.; Cook, P. D. J. Org. Chem. 1999, 64, 6468.
11. Smicius, R.; Engels, J. W. J. Org. Chem. 2008, 73, 4994.
10
11
12
13
12. Synthesis of compound 2. Under
Pr2NP(Cl)OCH2CH2CN (36 L, 0.163 mmol) was added to
compound (100 mg, 0.136 mmol) and i-Pr2NEt (121 L, 0.680 mmol) in
a
nitrogen atmosphere, i-
l
a
solution of
a
1
l
Conditions: Sodium cacodylate buffer (10 mM, pH 6.8), KCl (10 mM) and MgCl2
(10 mM). The final concentration of each oligonucleotide used was 1.89 M.
anhydrous CH2Cl2 (2 mL) at 0 °C, and the mixture was stirred at room
temperature for 2 h. After addition of saturated NaHCO3 aq, the mixture was
extracted with CH2Cl2. The organic extracts were washed with saturated
NaHCO3 aq, water and brine, dried over Na2SO4, and concentrated under
reduced pressure. The residue was purified by flash silica gel column
chromatography (n-hexane/AcOEt = 2:3) to give compound 2 (121 mg, 96%, a
3:2 mixture of diastereoisomers based on the chiral phosphorus atom) as a
white foam: 1H NMR (CDCl3) d 0.98 (3.0H, d, J = 6.5 Hz), 1.11–1.14 (9.0H, m),
1.34 (3.0H, s), 2.33 (1.0H, t, J = 6.5 Hz), 2.67–2.69 (1.0H, m), 3.26–3.31 (1.0H,
m), 3.46–3.63 (5.0H, m), 3.79–3.80 (6.0H, m), 3.79–4.04 (4.0H, m), 4.16–4.26
(2.0H, m), 4.43 (0.4H, td, J = 4.5 and 11 Hz), 4.48 (0.6H, td, J = 5.0 and 9.5 Hz),
5.98 (0.6H, d, J = 4.5 Hz), 6.01 (0.4H, d, J = 6.0 Hz), 6.82–6.87 (4.0H, m), 7.22–
7.33 (7.0H, m), 7.39–7.43 (2.0H, m), 7.57, (0.4H, d, J = 1.0 Hz), 7.68–7.73 (2.6H,
m), 7.79–7.83 (2.0H, m), 7.95 (0.4H, s), 8.05 (0.6H, s). 31P NMR (CDCl3) d 150.16,
150.43. HRMS (MALDI) m/z calcd for C50H56N5NaO11P [M+Na]+: 956.3606;
found 956.3614.
l
nucleus at 50-region induce effective stacking stabilization in both
processes of nucleation and zipping. In this study, since introduc-
tion of multiple cationic modified nucleotides at the 50-region of
TFO reduced the level of local charge repulsion between the TFO
and dsDNA, the formation of nucleus at the 50-region might be
accelerated.
In conclusion, TFOs partially modified with cationic nucleotides,
20-O-(2-aminoethyl)- or 20-O-(2-guanidinoethyl)-nucleotides, were
synthesized, and their association rate constants in triplex DNA
formation were evaluated using UV spectrophotometer. Introduc-
tion of modifications at the 50-region resulted in an increase in
the association rate whereas modifications in the middle and at
the 30-region did not induce a significant change. However, we con-
sider that further improvement of the association rate is required
towards practical applications of TFOs. Slow rate of triplex forma-
tion has been a major impediment in the development of TFOs;
hence, in the design of TFOs with modified nucleotides, the focus
will probably be to improve the association rates of triplex forma-
tion. The results presents in this paper would provide helpful infor-
mation for the design of practical TFOs.
13. Synthesis of compound 3. Under
a
nitrogen atmosphere, POCl3 (45
lL,
0.483 mmol) was slowly added to
a
solution of compound (150 mg,
2
0.161 mmol), Et3N (0.55 mL, 3.94 mmol) and 1,3,4-triazole (266 mg,
3.86 mmol) in anhydrous MeCN (2.5 mL) at 0 °C, and the mixture was stirred
at 0 °C for 1 h. After addition of AcOEt and water, the mixture was extracted
with AcOEt. The organic extracts were washed with saturated NaHCO3 aq,
water and brine, dried over Na2SO4, and concentrated under reduced pressure.
The residue was purified by reprecipitation from n-hexane/AcOEt to give
compound 3 (140 mg, 88%, a 1:1 mixture of diastereoisomers based on the
chiral phosphorus atom) as a white powder: 1H NMR (CDCl3) d 0.94 (3.0H, d,
J = 6.5 Hz), 1.04–1.09 (9.0H, m), 1.72 (1.5H, s), 1.73 (1.5H, s), 2.34 (1.0H, t,
J = 6.5 Hz), 2.57–2.72 (1.0H, m), 3.35–3.54 (4.0H, m), 3.64–3.71 (0.5H, m), 3.72–
3.76 (1.0H, m), 3.79 (1.5H, s), 3.80 (1.5H, s), 3.80 (1.5H, s), 3.81 (1.5H, s), 3.82–
3.89 (0.5H, m), 3.95–4.13 (3.0H, m), 4.21–4.35 (3.0H, m), 4.52 (0.5H, dt, J = 4.5
and 12.5 Hz), 4.58 (0.5H, dt, J = 5.0 and 13 Hz), 6.04 (0.5H, d, J = 1.0 Hz), 6.07
(0.5H, s), 6.82–6.87 (4.0H, m), 7.23–7.36 (7.0H, m), 7.42–7.47 (2.0H, m), 7.68–
7.70 (2.0H, m), 7.80–7.83 (2.0H, m), 8.06 (0.5H, s), 8.07 (0.5H, s), 8.42 (0.5H, s),
8.47 (0.5H, s), 9.23 (0.5H, s), 9.25 (0.5H, s). 31P NMR (CDCl3) d 149.70, 150.83.
HRMS (MALDI) m/z calcd for C52H57N8NaO10P [M+Na]+: 1007.3827; found
1007.3861.
Acknowledgments
This work was supported in part by a Grand-in-Aid for Scientific
Research on Innovative Areas (No. 22136006) from the Ministry of
Education, Culture, Sports, Science and Technology, Japan (MEXT),
the Mochida Memorial Foundation for Medical and Pharmaceutical
Research, and a Grant-in-Aid for JSPS Fellows of Japan Society for
the Promotion of Science(JSPS).
14. The synthesis of TFOs 5–13. The syntheses were performed on a 0.2 lmol scale
on an automated DNA synthesizer (Gene Design nS-8) using the common
phosphoramidite protocol. TFOs synthesized on DMTr-ON mode were cleaved
from the CPG resin by treatment with 28% NH3 aq at room temperature for 2 h
and all the protecting groups on TFOs were removed by treatment with 28%
NH3 aq at 55 °C for 24 h. The obtained crude TFOs were purified on Sep-PakÒ
Plus C18 cartridges (Waters) followed by reversed-phase HPLC (Waters
XBridgeÒ OST C18 column 2.5
lm, 10 mm ꢀ 50 mm). The composition of the
References and notes
TFOs was confirmed by MALDI-TOF-MS analysis. MALDI-TOF-MS data
([MꢁH]ꢁ) for TFOs 5–13: 5, found 4556.60 (calcd 4556.06); 6, found 4614.43
(calcd 4615.12); 7, found 4674.08 (calcd 4674.19); 8, found 4675.14 (calcd
4674.19); 9, found 4575.04 (calcd 4674.19); 10, found 4597.98 (calcd
1. Reviews: (a) Praseuth, D.; Guieysse, A. L.; Hélène, C. Biochim. Biophys. Acta Gene
Struct. Expression Biochim. Biophys. Acta 1999, 1489, 181; (b) Knauert, M. P.;
Glazer, P. M. Hum. Mol. Genet. 2001, 10, 2243; (c) Seidman, M. M.; Glazer, P. M.

Y. Hari et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3046–3049
3049
4598.10); 11, found 4698.60 (calcd 4699.21); 12, found 4799.38 (calcd
4800.32); 13, found 4799.44 (calcd 4800.32).
18. Kinetic experiments. The experiments were carried out on SHIMADZU UV-
1800 spectrophotometer equipped with a rapid mixing stopped-flow accessory
RX2000 (Applied Photophysics Ltd). A solution of 10 mM sodium cacodylate
15. General method of UV-melting experiments. The experiments were carried out
on SHIMADZU UV-1650 and SHIMADZU UV-1800 spectrophotometers
equipped with Tm analysis accessory. Equimolecular amounts of the hairpin-
loop dsDNA 15 and TFOs were dissolved in 10 mM sodium cacodylate buffer
(pH 6.8) containing 10 mM KCl and 10 mM MgCl2 to give a final strand
buffer (pH 6.8) containing 10 mM KCl, 10 mM MgCl2 and 3.98
was prepared as solution A. A solution of 10 mM sodium cacodylate buffer (pH
6.8) containing 10 mM KCl, 10 mM MgCl2 and 3.98 M TFO was prepared as
lM dsDNA 15
l
solution B. At 20 °C, same volumes of solution A and solution B were mixed
using RX2000, and the absorbance (260 nm) of was monitored for
approximately 30 min at 20 °C. All kon values were calculated by curve-
fitting the UV data as shown in Ref. 3. SigmaPlot (Systat Software, Inc) was
used for the curve-fitting. Final kon value was an average of more than three
measurements.
concentration of 1.89 lM. The samples were annealed by heating at 100 °C
followed by slow cooling to 5 °C. The melting profiles were recorded at 260 nm
from 5 °C to 90 °C at a scan rate of 0.5 °C/min. A two-point average method was
used to obtain the Tm values and the final values were determined by averaging
three independent measurements which were accurate to within 1 °C.
16. The sequence of TFO and dsDNA is often used for evaluation of thermal
stability of triplex DNA.2i
19. Mixing of samples in UV cell using a micropipette, instead of use of rapid
mixing unit could not give reproducible results, probably due to inconsistent
and low mixing efficiency.
17. Singleton, S. F.; Dervan, P. B. Biochemistry 1993, 32, 13171.