ABSTRACT
Homopurine sequences of duplex DNA are binding sites for triplex-forming oligodeoxyribopyrimidines. The interactions of synthetic duplex
DNA targets with an oligodeoxyribopyrimidine containing
N
4-(6-amino- 2-pyridinyl)deoxycytidine (1), a nucleoside designed to
interact with a single C[middot]G base pair interruption of the purine target tract, was studied by UV
melting, circular dichroism spectroscopy and dimethylsulfate alkylation
experiments. Nucleoside 1 supports stable triplex formation at pH 7.0 with
formation of a 1[middot]Y[middot]Z triad, where Y[middot]Z is a base pair in the homopurine tract of the target.
Selective interaction was observed when Y[middot]Z was C[middot]G, although A[middot]T and, to a lesser extent, T[middot]A and G[middot]C base pairs were also recognized. The
circular dichroism spectra of the triplex having a 1[middot]C[middot]G triad were similar to those of a triplex having a C+[middot]G[middot]C triad, suggesting that the overall structures
of the two triplexes are quite similar. Removal of the 6-amino group from 1 essentially eliminated triplex formation. Reaction of a
triplex having the 1[middot]C[middot]G triad with dimethylsulfate resulted in a 50% reduction of
methylation of the G residue of this triad. In contrast, the G of a similar
triplex containing a U[middot]C[middot]G triad was not protected from methylation by dimethylsulfate.
These results are consistent with a binding mode in which the 6-amino-2-pyridinyl group of 1 spans the major groove of the target
duplex at the 1[middot]C[middot]G binding site and forms a hydrogen bond with the O6 of G. An
additional stabilizing hydrogen bond could form between the N4 of the imino tautomer of 1 and the N4 amino group of C.
Oligodeoxyribonucleotides can interact with double-stranded DNA to form triple-stranded complexes. There is considerable interest in the structure
of these triplexes and in the possibility of using triplex-forming oligonucleotides as novel reagents to control gene expression (
1
). Currently, sequence recognition of DNA by triplex-forming oligonucleotides is limited to interactions between oligopyrimidines or oligopurines and homopurine tracts within the DNA target. Although purine sequences of considerable length are frequently found in human and eukaryotic genes (
2
,
3
), these sequences are often interrupted by one or a few pyrimidine bases. Such
py[middot]pu
base pair interruptions essentially act as mismatches and reduce the stability of the triplex or prevent triplex formation altogether (
4
-
10
). The range of sequences recognized by triplex-forming oligonucleotides could be expanded considerably if means could be found to deal with these interruptions.
Various strategies have been developed to accommodate isolated
py[middot]pu
interruptions of purine tracts. One approach is to simply skip the
interruption. However, incorporation of an abasic site into an oligopyrimidine at a site opposite a single
py[middot]pu
interruption resulted in considerable destabilization of the triplex (
11
). Apparently, maintenance of continuous stacking interactions along the third
strand is an important contributor to triplex stability.
Natural nucleic acid bases can interact with a
py[middot]pu
interruption. Thus guanine can interact with a T
[middot]
A base pair to form a G
[middot]
T
[middot]
A triad (
6
,
8
,
12
) and thymine can form a T
[middot]
C
[middot]
G triad (
6
-
10
). Both the G
[middot]
T
[middot]
A and T
[middot]
C
[middot]
G triads involve the formation of a single hydrogen bond between the third
strand base and the pyrimidine interruption (
13
-
15
). This and distortions of the triplex at the site of the G
[middot]
T
[middot]
A or T
[middot]
C
[middot]
G triad may account for the generally observed reduced stability of triplexes
involving these base triads.
Nucleoside analogs have also been shown to interact with C
[middot]
G interruptions.When incorporated into an oligopurine, 2'-deoxynebularine interacts selectively with C
[middot]
G and A
[middot]
T base pairs in a purine
[middot]
purine
[middot]
pyrimidine triplex motif (
16
). Similar recognition was observed for 5-fluoro-2'-deoxyuridine, an analog of thymidine (
17
). The
N
-7-benzoyl derivative of 2'-deoxyformycin A was also found to allow triplex
formation involving C
[middot]
G interruptions (
18
).
Non-natural nucleosides which recognize
py[middot]pu
interruptions have been synthesized. Thus 1-(2-deoxy-[beta]-D-ribofuranosyl)-4- (3-benzamidophenyl)imidazole
when incorporated into an oligopyrimidine specifically recognizes T
[middot]
A and C
[middot]
G interruptions (
19
). This nucleoside was found to interact by sequence-specific intercalation, rather than by hydrogen bonding to the base pair
interruption (
20
). Azole-2'-deoxyribonucleosides incorporated into triplex-forming oligopurines allow formation of triplexes with
duplex targets containing T
[middot]
A or C
[middot]
G interruptions (
21
,
22
). The manner in which these nucleosides interact with the interruption remains to be characterized. Recently, 2-methyl-8-(
N
'-
n
-butylureido)naphth[1,2-
d
]imidazole was shown to hydrogen bond to a C
[middot]
G base pair in chloroform solution (
23
).
We have reported in a preliminary communication that an oligopyrimidine containing
N
4
-(6-amino-2-pyridinyl)deoxycytidine forms a triplex with a duplex target containing a
single C
[middot]
G interruption (
24
). In this report we describe the syntheses of oligonucleotides containing this and other
N
4
-(aryl)deoxycytidine derivatives and studies which further characterize triplex formation by these oligonucleotides.
Reagent grade chemicals were used unless otherwise noted. HPLC grade
acetonitrile was dried over calcium hydride. Anhydrous pyridine and methylene
chloride were from Aldrich Chemical Co. Inc. Protected nucleoside 3'-(2-cyanoethyl-
N
,
N
-diisopropylphosphoramidites) and nucleoside-derivatized controlled pore glass supports were from Glen Research. Flash chromatography
was carried out using EM Science Kieselgel 60 (230-400 mesh). Thin layer chromatography (TLC) was performed on EM Reagents
silica gel plates (0.2 mm). Analytical and preparative reversed phase HPLC were carried out on 4.6 * 150 mm and 10 * 250 mm Microsorb 5 [mu]m C-18 columns (Rainin Instrument Co.) respectively at flow
rates of 1.0 or 2.0 ml/min. Proton NMR spectra were recorded on a Brucker AMX
300 spectrometer. Chemical shifts are reported in p.p.m. Mass spectra were
obtained using electron ionization (EI) or fast atom bombardment (FAB)
techniques. Circular dichroism spectra were recorded on an Aviv circular
dichroism spectropolarimeter fitted with a thermostatted cell compartment.
A rapidly stirred suspension of 8.4 g 1,2,4-triazole in 120 ml dry acetonitrile was treated at 0oC by slow addition of 2.4 ml phosphorus oxychloride followed by
dropwise addition of 18 ml triethylamine. The suspension was left stirring for
30 min and a solution of 936 mg 3',5'-di-
O
-acetyl-2'-deoxyuridine (
25
) in 15 ml dry acetonitrile was added over a period of 20 min. Stirring was continued for 1.5 h until TLC showed no starting material remained. The reaction was stopped by addition of 150 ml saturated sodium bicarbonate.
The aqueous solution was extracted with methylene chloride and the methylene
chloride was removed by rotatory evaporation. The product, which was crystallized from ethyl acetate, was obtained in 73% yield as 800 mg of a pale yellow solid. Silica gel TLC
R
f
: 0.25, ethyl acetate/hexane (3:1 v/v).
1
H NMR (DMSO-d
6
): d p.p.m. 1.92 (s, 3H, CH
3
), 1.87 (s, 3H, CH
3
), 2.33 (m, 2H, H2', H2''), 4.13 (m, 2H, H5'), 4.22 (m, 1H, H4'), 5.07 (m, 1H, H3'), 5.99 (t, 1H, H1'), 6.89 (d, 1H, H5), 8.03 (s,
1H, triazole), 8.25 (d, 1H, H6), 9.21 (s, 1H, triazole).
A solution of 804 mg, 1 mmol, nucleoside
4
and 804 mg, 8 mmol, 2,6-diaminopyridine in 10 ml dry pyridine was refluxed for 3 days. TLC showed that no starting material remained. Pyridine was removed by evaporation, the residue was extracted into methylene chloride and the organic layer was washed with water. The methylene
chloride was evaporated and the residue was subjected to silica gel flash
chromatography using chloroform/methanol (10:1 v/v) as solvent. Nucleoside
5a
was obtained in 53% yield as a pale yellow glassy solid. Silica gel TLC
R
f
: 0.22, chloroform/methanol (10:1 v/v). FAB MS(+): 404 (M+H)
+
, 204 (M-ribose+H)
+
.
1
H NMR (DMSO-d
6
): d p.p.m. 1.92 (s, 3H, CH
3
), 1.93 (s, 3H, CH
3
), 2.19 (m, 2H, H2', H2''), 4.06~4.11 (m, 3H, H4', H5'), 5.04 (m, 1H, H3'), 5.65 (s, 2H, NH
2
), 6.03 (m, 2H, H1', Hpyr), 6.45 (br, 1H, H5), 7.16 (m, 2H, Hpyr), 7.06 (d, 1H, H6), 9.67
(br, 1H, NH).
Sixty milligrams, 0.15 mmol, nucleoside
5a
was dissolved in 6 ml water/methanol (1:1 v/v) solution containing 0.2 N sodium
hydroxide. The solution was stirred at room temperature for 20 min and was then neutralized by addition of 6 N hydrochloric acid at 0oC. The solvents were removed and the residue was taken up in methanol. The
resulting precipitate was removed by filtration and the filtrate was
evaporated. The residue was subjected to silica gel column chromatography using
chloroform/methanol (3:1 v/v) as solvent. The product was obtained as a white
solid. Silica gel TLC
R
f
: 0.15, chloroform/methanol (4:1 v/v). Reversed phase HPLC retention time, 19.3
min, using a gradient of 2-3% acetonitrile, 0-12 min, and 3-30% acetonitrile in 50 mM sodium phosphate buffer, pH 5.8.
1
H NMR (DMSO-d
6
): d p.p.m. 1.85 (m, 1H, H2'), 2.00 (m, 1H, H2''), 3.41 (br, 2H, H5'), 3.64 (m, 1H, H4'), 3.92 (m, 1H, H3'), 4.86 (d, 1H, 3'OH), 5.06 (t, 1H, 5'OH), 5.73 (br, 2H, NH
2
), 5.99 (m, 2H, H1', H pyr), 6.41 (br, 1H, H5), 7.00 (br, 1H, H pyr), 7.20 (t, 1H, H pyr),
7.84 (d, 1H, H6), 9.71 (br, 1H, NH).
A solution of 200 mg, 0.5 mmol, nucleoside
5a
in 5 ml dry pyridine was treated by dropwise addition of 0.17 ml, 1.5 mmol,
benzoyl chloride at 0oC. The solution was stirred at room temperature for 2.5 h. Saturated sodium
bicarbonate was added at 0oC, the solution was extracted with methylene chloride and the organic layer
was washed with water. The organic layer was evaporated and the residue was
washed with ethanol. Nucleoside
5b
was obtained in 71% yield as a white precipitate. Silica gel TLC
R
f
: 0.33, ethyl acetate/hexane (5:1 v/v).
1
H NMR (DMSO-d
6
): p.p.m. 2.05 (s, 3H, CH
3
), 2.11 (s, 3H,CH
3
), 2.37 (m, 2H, H2', H2''), 4.25~4.43 (m, 3H, H4', H5'), 5.25 (m, 1H, H3'), 6.13 (t, 1H, H1'), 6.56 (d, 1H, H5), 7.66
(m, 14H, Ar), 11.0 (s, 1H, NH).
Two hundred milligrams, 0.3 mmol, nucleoside
5b
was dissolved in 20 ml water/methanol (1:1 v/v) solution containing 0.2 N
sodium hydroxide. The solution was stirred at room temperature for 20 min and
then neutralized by addition of 6 N hydrochloric acid. The product was obtained
as a white precipitate which was removed by filtration, washed several times
with ice water and dried under vacuum in the presence of phosphorous pentoxide.
Product
6a
was obtained in 70% yield as 89 mg of a white solid.
1
H NMR (DMSO-d
6
): p.p.m. 2.08 (m, 1H, H2'), 2.28 (m, 1H, H2''), 3.52 (m, 2H, H5'), 3.80 (m, 1H, H4'), 4.30 (m, 1H, H3'), 5.09 (t, 1H, 5'OH), 5.30 (d, 1H, 3'OH), 6.25 (t, 1H, H1'), 7.24 (br, 1H,
H pyr), 7.60~8.04 (m, 8H, H5, H Ar), 8.16 (d, 1H, H6), 10.24 (s, 1H, NH), 10.55 (s, 1H,
NH).
A solution of 80 mg, 0.19 mmol, nucleoside
6a
in 2 ml dry pyridine was treated with 80 mg dimethoxytrityl chloride and 3 mg 4-
N
,
N
-dimethylaminopyridine for 2 h at room temperature. A 0.5 ml aliquot of methanol was added and stirring was continued for 20 min. The solvents were evaporated, the residue was dissolved in methylene
chloride and the solution was extracted with water. The organic layer was evaporated and the residue subjected to silica gel column
chromatography using a solvent which contained 5% triethylamine in ethyl
acetate/methanol (15:1 v/v). The product,
6b
, was obtained in 72% yield as a white glassy solid. Silica gel TLC
R
f
: 0.38, 5% triethylamine in ethyl acetate/methanol (15:1 v/v).
1
H NMR (DMSO-d
6
): p.p.m. 1.88 (s, 6H, CH
3
), 2.40 (m, 2H, H2', H2''), 3.92 (m, 3H, H4', H5'), 4.12 (m, 1H, H3'), 5.23 (d, 1H, 3'OH), 6.07 (t, 1H, H1'), 6.55 (br, 1H, H5),
7.26 (m, 21H, Ar), 7.85 (d, 1H, H6), 10.06 (s, 1H, NH), 10.31 (s, 1H, NH).
Nucleoside
6b
was dried under vacuum for 48 h in the presence of phosphorous pentoxide.
Eighty milligrams
6a
were dissolved in a solution containing 1 ml dry methylene chloride and 0.061
ml dry triethylamine. The stirred solution was treated by dropwise addition of 0.037 ml
N
,
N
-
bis
-(diisopropylamino)-2-cyanoethoxy-phosphine and stirring was continued for 15 min.
Methanol was added and stirring was continued for an additional 10 min. The
solution was diluted with ethyl acetate and then washed several times with
saturated sodium bicarbonate. The organic layer was evaporated and the residue
was subjected to silica gel column chromatography using ethyl acetate/methylene chloride/triethylamine (1:1:0.1 v/v) as solvent. The product was obtained in 70% yield as a glassy
solid after evaporation of solvents. It was dried under vacuum in the presence
of phosphorous pentoxide. Silica gel TLC
R
f
: 0.38, ethyl acetate/methylene chloride/triethylamine (1:1:0.1 v/v).
31
P NMR (CDCl
3
): d p.p.m. 148.03, 148.62.
A solution of 726 mg nucleoside
4
and 1.7 g 1,3-phenylenediamine in 20 ml dry pyridine was refluxed for 4 h. The solvents were
evaporated, the residue was dissolved in methylene chloride and the solution
was washed with water. The organic layer was evaporated and the residue was
subjected to silica gel column chromatography using chloroform/methanol (20:1 v/v) as solvent. The product, 3',5'-di-
O
-acetyl-
N
4
-(3-aminophenyl)-2'-deoxycytidine, was obtained in 51% yield as 420 mg of a pale
yellow glassy solid. MS FAB (+): 403.2 (M+H)
+
, 203.1 (M-ribose+H)
+
. TLC
R
f
: 0.21 (CHCl
3
/MeOH 20:1).
1
H NMR (DMSO-d
6
): p.p.m. 1.93 (s, 3H, CH
3
), 1.96 (s, 3H, CH
3
), 2.22 (m, 2H, H2', H2''), 4.12 (m, 3H, H4', H5'), 4.98 (s, 2H, NH
2
), 5.07 (m, 1H, H3'), 5.89 (d, 1H, H5), 6.10 (t, 1H, H1'), 6.19 (d, 1H, H
Ar
), 6.80 (br, 1H, H
Ar
), 6.83 (t, 2H, H
Ar
), 7.64 (d, 1H, H6), 9.39 (s, 1H, NH).
A 70 mg portion of the nucleoside was dissolved in 10 ml of a solution
containing 0.2 N sodium hydroxide in methanol/water (1:1 v/v). The solution was
stirred at room temperature for 20 min and then neutralized by addition of 6 N
hydrochloric acid at 0oC. The solvents were evaporated, the residue was taken up in methanol and
the resulting precipitate was removed by filtration. The filtrate was
evaporated and the residue was subjected to silica gel column chromatography
using chloroform/methanol (6:1 v/v) as solvent. The product,
2
, was obtained as a white solid. Silica gel TLC
R
f
: 0.30, chloroform/methanol (5:1 v/v). Reversed phase HPLC retention time, 18.4
min using a gradient of 2-3% acetonitrile, 0-12 min, and 3-30% acetonitrile in 50 mM sodium phosphate buffer, pH 5.8.
1
H NMR (DMSO-d
6
): p.p.m. 2.17 (m, 1H, H2'), 2.32 (m, 1H, H2''), 3.75 (m, 2H, H5'), 3.96 (m, 1H, H4'), 4.41 (m, 1H, H3'), 5.27 (s, 2H, NH
2
), 6.24 (d, 1H, H5), 6.35 (t, 1H, H1'), 6.40 (d, 1H, H
Ar
), 7.12 (m, 3H, H
Ar
), 8.10 (d, 1H, H6), 9.82 (s, 1H, NH).
A solution of 726 mg nucleoside
4
and 1.4 g 2-aminopyridine in 20 ml dry pyridine was refluxed for 6 days. The solvents
were evaporated, the residue was dissolved in methylene chloride and the
organic solution was washed with water. The organic layer was evaporated and
the residue subjected to silica gel column chromatography using chloroform/methanol (20:1 v/v) as solvent. The product, 3',5'-di-
O
-acetyl-
N
4
-(2-pyridinyl)-2'-deoxycytidine, was obtained in 63% yield as 470
mg of a pale yellow glassy solid. Silica gel TLC
R
f
: 0.51, chloroform/methanol (20:1 v/v). MS FAB(+): 388 (M+H)
+
, 189 (M-ribose+H)
+
.
1
H NMR (DMSO-d
6
): p.p.m. 2.03 (s, 3H, CH
3
), 2.06 (s, 3H, CH
3
), 2.31 (m, 2H, H2', H2''), 4.23 (m, 3H, H4', H5'), 5.18 (m, 1H, H3'), 6.18 (t, 1H, H1'), 6.48 (br, 1H, H5), 7.07 (t,
1H, H
Ar
), 7.79 (m, 2H, H
Ar
), 8.31 (m, 2H, H
Ar
, H6), 10.40 (s, 1H, NH).
A 420 mg portion of the nucleoside was dissolved in 50 ml of a solution
containing 0.2 N sodium hydroxide in methanol/water (1:1 v/v). The solution was
stirred at room temperature for 20 min and then neutralized by addition of 6 N
hydrochloric acid at 0oC. The solvents were evaporated, the residue was taken up in methanol and
the resulting precipitate was removed by filtration. The filtrate was
evaporated and the residue was subjected to silica gel column chromatography
using chloroform/methanol (6:1 v/v) as solvent. The product,
3
, was obtained as a white solid. Silica gel TLC
R
f
: 0.25, chloroform/methanol (6:1 v/v). Reversed phase HPLC retention time, 18.6
min using a gradient of 2-3% acetonitrile, 0-12 min, and 3-30% acetonitrile in 50 mM sodium phosphate buffer, pH 5.8.
1
H NMR (DMSO-d
6
): p.p.m. 1.90 (m, 2H, H2', H2''), 3.62 (m, 1H, H4'), 4.05 (m, 1H, H3'), 5.97 (t, 1H, H1'), 6.29 (br, 1H, H5), 6.88 (t, 1H, H
Ar
), 7.60 (t, 1H, H
Ar
), 7.92 (d, 1H, H
Ar
), 8.14 (m, 2H, H
Ar
, H6), 10.20 (br, 1H, NH).
These were synthesized on an Applied Biosystems Model 392 DNA/RNA synthesizer using standard phosphoramidite chemistry (
26
). The 5'-terminal dimethoxytrityl group was removed from the support-bound protected oligomer by the synthesizer. The oligomers
were cleaved from the support and deprotected by treating the controlled pore
glass support with a solution containing concentrated ammonium
hydroxide/pyridine (1:1 v/v) at 55oC for 6 h. In the case of oligomers containing
N
4
-(6- amino-2-pyridinyl)-2'-deoxycytidine, treatment was extended to 18 h to ensure complete removal
of the 6-benzamido protecting group from this nucleoside. Deprotected oligomers
were purified by preparative C-18 reversed phase HPLC using a 40 ml linear gradient of 5-15% acetonitrile in 50 mM sodium phosphate, pH 5.8. The column was operated at a flow rate of 2.0 ml/min and was monitored at 280 nm. Oligomers were desalted on C-18 SEP PAK cartridges (Water Associates) or on a small
Microsorb (Rainin Instruments) C-18 reversed phase HPLC guard column cartridge. The oligomers gave the
expected products after digestion to their component nucleosides using a
combination of snake venom phosphodiesterase and bacterial alkaline phosphatase. Their extinction coefficients were determined as previously described (
27
).
Solutions containing 1.8 [mu]M [
32
P]
IV
and 2.2 [mu]M
V
or 2.2 [mu]M
IV
and 1.8 [mu]M [
32
P]
V
in 50 mM MOPS, pH 7.0, 20 mM MgCl
2
, 0.1 M NaCl were prepared. A 5 [mu]l aliquot of the preformed duplex, which contained 12 000 d.p.m. of
32
P-labeled oligonucleotide, was mixed with a 5 [mu]l aliquot of buffer or buffer containing 2, 10 or 20 [mu]M
I
at room temperature. These solutions were incubated overnight at 4oC. The solutions were then incubated for 30 min at 0oC. Each solution was treated with 1 [mu]l dimethylsulfate for 3 min at 0oC. The reaction was quenched by addition of 2 [mu]l 2-mercaptoethanol. Each solution was diluted with 3 [mu]l 3 M NaOAc, 10 [mu]g in 1 [mu]l salmon sperm DNA and 100 [mu]l ethanol. The solutions were placed
on dry ice for 1 h and then centrifuged at 4oC at 16 000 r.p.m. for 20 min. The supernatant was removed and the
precipitate was washed with 100 [mu]l 90% ethanol. The pellet was dried under vacuum, dissolved in 50 [mu]l 1 M aqueous piperidine and the solution was incubated at 90oC for 30 min. The solvent was removed by evaporation and the
residue was evaporated once with 20 [mu]l 50% acetonitrile/water. The residue was then dissolved in 10 [mu]l 90% formamide loading buffer. The solution was heated for 1 min at 90oC, loaded onto a 40 cm, 20% denaturing polyacrylamide gel and the
samples were electrophoresed at 800 V. The gels were dried and subjected to
autoradiography at -80oC and were also scanned by a phosphorimager.
Triplex formation was studied in the intermolecular systems
I[middot]II[middot]III(X[middot]Y[middot]Z)
and
I[middot]IV[middot]V(X[middot]Y[middot]Z)
, shown in Figure
1
. The duplex targets contained the four possible Watson-Crick base pairs at position
Y[middot]Z
. The third strand oligopyrimidine
I(X)
, whose backbone is parallel to the purine-rich strands of the duplexes, contained two 5-methyldeoxycytidines (
The deoxycytidine derivatives were prepared as outlined in Figure
2
and described in Materials and Methods. The oligomers used the corresponding
phosphoramidites following standard procedures.
With the exceptions noted below, the A
260
vs tempeature profiles of triplex
I[middot]II[middot]III(X[middot]Y[middot]Z)
and triplex
I[middot]IV[middot]V(X[middot]Y[middot]Z)
showed two sigmoidal transitions corresponding first to dissociation of the
third strand and second dissociation of the duplex. Only small linear increases
in absorbance were seen when the individual strands of these triplexes were
heated, suggesting that these strands do not undergo self-interactions (data not shown). The melting temperatures at pH 7.0 for
dissociation of oligomer
I(X)
from these triplexes are shown in Table
1
and
2
. Oligomer
I(1)
interacted to various extents with all eight duplex targets. The most stable
triplexes were formed with duplex targets which contained
C[middot]G
or
A[middot]T
base pairs at position
Y[middot]Z
.
Table 1
Melting temperatures of
I[middot]II[middot]III(X[middot]Y[middot]Z)
triplexes containing base analogs
As shown in Figure
3
, triplex
I[middot]II[middot]III(1[middot]C[middot]G)
melted in a unique manner. When the triplex was heated at a rate of 0.5oC/min, two transitions corresponding to dissociation of
I(1)
from target duplex
II[middot]III(C[middot]G)
were observed with
T
m
values of 25 and 38oC. These transitions occured prior to dissociation of the target duplex,
whose
T
m
is 48oC.
Only a single transition was seen at 25oC when the sample was cooled. When the sample was heated at a slower rate,
0.2oC/min, a single broad transition was observed whose inflection point is 28oC. Prolonged incubation of
I[middot]II[middot]III(1[middot]C[middot]G)
at 4oC prior to melting resulted predominantly in the appearance of the
transition at 38oC.
Reactions of duplex
IV[middot]V
with dimethylsulfate in the presence of increasing concentrations of
I(1)
or
I(U)
were carried out at 0oC. This temperature is well below the
T
m
values for the dissociation of the third strands of these triplexes.
Autoradiograms of the gels from reactions run in the presence of
I(U)
or
I(1)
are shown in Figure
5
A and B respectively. The topmost band observed in these gels is undissociated
duplex
IV[middot]V
. Lanes 1-4 of Figure
5
A show that G19 of the upper strand of the duplex (strand
IV
) was protected from methylation by
I(U)
. Protection was almost complete at a 1:1 stoichiometry of
I(U)
to duplex. This result is consistent with formation of a triplex between
I(U)
and the duplex target. Although G16 is also part of the binding site for
I(U)
, it was not protected by the oligomer. In addition, G31 and G32, which are
located outside the oligomer binding site, appeared to be hypermethylated.
Similar hypermethylation of guanines outside a triplex binding site has been
observed in other systems (
28
). No decrease in methylation is observed for G20 of the lower strand (strand
V
), even in the presence of a 10-fold excess of
I(U)
, as shown in lanes 5-8.
The pyrimidine base of a
py[middot]pu
interruption provides only one site for hydrogen bond formation with a base of
a triplex-forming oligonucleotide. Base analogs which can potentially form hydrogen
bonds with both bases of the
py[middot]pu
base pair might enhance triplex stability and/or specificity at the site of interruption. Previously, we showed that oligomer
I(X)
, where
X
is
N
4
-(3-acetamidopropyl)deoxycytidine, formed a stable, but rather weak, triplex with
II[middot]III(C[middot]G)
(
29
). The flexible acetamidopropyl arm of this deoxycytidine derivative is sufficiently long to span the major
groove of the duplex and potentially hydrogen bond to the guanine base of the
C[middot]G
base pair.
N
4
-(6-Amino-2-pyridinyl)deoxycytidine (
1
) has a more rigid arm and two potential hydrogen bonding sites for interaction
with a
C[middot]G
base pair (
24
). Molecular models indicate that the pyridine ring of
1
is capable of spanning the major groove of the target duplex. As shown in
Figure
6
, this would place the 6-amino group of
1
within hydrogen bonding proximity to the O6 or N7 of G. Formation of the imino
tautomer of
1
would allow additional hydrogen bonding between the N4 of
1
and the
N
4
-amino group of the C
[middot]
G base pair. In addition to providing a rigid platform to position the hydrogen
bonding groups, the planar aromatic ring of
1
could also participate in stabilizing stacking interactions with bases
neighboring
1
. Alternatively, the pyridine ring of
I
could intercalate into the duplex in a manner analogous to the binding mode of 1-(2-deoxy-[beta]-D-ribofuranosyl)-4-(3-benzamidophenyl) imidazole (D
3
) (
20
).
The authors wish to thank Ms Sarah Kipp for help in synthesizing the
oligonucleotides, Dr Tina L.Trapane for help in obtaining the circular
dichroism spectra and Dr David Shortle for use of the circular dichroism spectropolarimeter. This research was supported by a grant from the National Institutes of Health (GM45012). NMR studies were
performed in the Biochemistry NMR Facility at the Johns Hopkins University,
which was established by a grant from the National Institutes of Health
(GM27512) and a Biomedical Shared Instrumentation Grant (RR06262). C-YH was a GENTA Inc. post-doctoral fellow in the Department of Biochemistry.
+
Present address: Chiron Inc., 4560 Houton Street, Emeryville, CA 94608, USA
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