ABSTRACT
Pyrimidine oligonucleotides containing alternating anionic and stereo-uniform cationic
N
-(dimethylaminopropyl)phosphoramidate linkages [e.g. d(T+T-)
7
T, d(T+T-)
2
(T+C-)
5
T and (U
'
+U
'
-)
7
dT, where U
'
is 2
'
-
O
- methyluridine)] are shown to bind to complementary double-stranded DNA segments in 0.1 M NaCl at pH 7 to form triple-stranded complexes with the pyrimidine.purine.pyrimidine motif. For each of the sequences investigated, one stereoisomer bound with higher affinity,
and the other stereoisomer with lower affinity, than the corresponding all-phosphodiester oligonucleotide. The stereoisomer of d(T+T-)
7
T that interacted weakly with a dT.dA target in 0.1 M NaCl formed a novel
dA.dA.dT triple-stranded complex with poly(dA) or d(A
15
C
4
A
15
) in 1 M NaCl; in contrast, the stereoisomer that bound strongly to the dT.dA
target failed to form a dA.dA.dT triple-stranded complex.
Current research on triple-stranded polynucleotides has been stimulated by a general interest in
molecular recognition and by the potential applications for triple-stranded complexes in processing DNA chemically (
1
) and in modulating biological processes dependent on DNA (
2
). Two major structural motifs for these complexes have been recognized. One,
designated pyr.pur.pyr, involves binding a pyrimidine oligonucleotide to a duplex segment, with the
added pyrimidine strand oriented parallel to the purine strand of the duplex (
3
,
4
). In the other motif, pur.pur.pyr (
5
), a purine oligonucleotide binds to a duplex such that the two purine strands
are antiparallel (
6
-
8
). Since formation of a triplex brings three polyanionic strands into proximity,
the stability of these complexes is very dependent on salt concentration. High
salt concentrations (e.g. 1.0 M NaCl) favor triplex formation. In 0.1 M NaCl at
pH 7 triplexes containing a moderate size oligonucleotide (e.g. 15mer) have low
stability.
Recently we reported that an oligonucleotide containing alternating phosphodiester and stereo-uniform cationic
N
-(dimethylaminopropyl)phosphoramidate linkages, d(T+T-)
7
T, binds to poly(dA) and poly(A) targets with unusually high affinity in low
salt solutions (
9
). Under the same conditions, the binding affinity of the stereoisomer with opposite chirality at the phosphoramidate linkages is very low. These findings suggest that a zwitterionic oligonucleotide with appropriate stereochemistry at the P-N internucleoside linkages might serve as a superior probe for double-stranded DNA sequences in solutions of low ionic strength, as in
biological media. We report here the results of a study to test this
possibility. The zwitterionic probes and the targets investigated are listed in
Chart 1, where `
a
' represents the stereoisomer with the upfield, and `
b
' the isomer with the downfield, signal in the
31
P NMR spectrum for the phosphoramidate groups. The symbol `+' signifies a protonated [3'-OP(O)(NHCH
2
CH
2
CH
2
NMe
2
)-O-5'] internucleoside link; `-', a phosphodiester link; and U', a 2'-
O
-methyluridine unit in an oligonucleotide. For comparison, the all-phosphodiester probes dT
15
, U'
14
dT and d[T
4
(TC)
5
T] were also prepared.
Probes
Targets
d(T+T-)
7
T
(U'+U'-)
7
dT
d(T
15
C
4
A
15
), d(A
15
C
4
A
15
), Poly(dA)
1a
and
1b
2
a
and
2b
4
5
d(T+T-)
2
(T+C-)
5
T
d[A
5
(GA)
5
T
4
(TC)
5
T
5
]
3a
and
3b
6
Reagents grade chemicals were used throughout. Pyridine, DMF and CH
3
CN were dried over calcium hydride. THF was freshly distilled over sodium/benzophenone prior to use. 5'-
O
-Dimethoxytritylthymidine (5'-O-DMT-dT) was purchased from Chem Impex, IL, and 5'-
O
-dimethoxytrityl-2'-
O
-methyluridine was purchased from Monomer Sciences (Huntsville, AL). Column
chromatography was performed on silica gel (70-230 mesh, 60 A; Aldrich). Reversed phase (RP) HPLC was carried out on a Supelco LC-18 column (25 cm * 4.6 mm) with a 0-50% gradient (2%/min) of CH
3
CN in 5% CH
3
CN in 0.1 M TEAA buffer, pH 7.5; flow rate 1 ml/min or on a Dionex chromatograph
with a Hewlett Packard Hypersil ODS-5 column (4.6 * 200 mm) using a 1%/min gradient of CH
3
CN in 30 mM triethylammonium acetate buffer at pH 7.0 with a flow rate of 1
ml/min. A Dionex DX 500 chromatograph system equipped with a P40 gradient pump,
an AD 20 absorbance detector, and a NucleoPack PA-100 column (4 * 250 mm) was used for ion-exchange (IE) chromatography. The eluant was 10 mM aqueous
NaOH, and a 2%/min gradient of 1.0 M NaCl in 10 mM NaOH was used with a flow
rate of 1.5 ml/min. TLC was performed using Whatman analytical silica gel
plates (60 Å) or Merck Silica Gel 60 F
254
precoated TLC aluminum plates. The TLC plates were prerun in 8% CH
3
OH/2%TEA/CH
2
Cl
2
, then spotted and developed in the same system.
Circular dichroism (CD) measurements were carried out on a Hitachi Joel 500
instrument. Temperature was controlled with a refrigerated water bath, and
nitrogen was continuously circulated through the cuvette compartments. Melting curves were determined using either a Varian Cary 3E UV-Visible spectrophotometer or a Perkin Elmer Lambda 2 UV spectrophotometer
equipped with a Peltier 2 temperature programmer for automatically increasing
the temperature at the rate of 0.5oC/min. Unless stated otherwise, dissociation curves and CD spectra were obtained in a 0.1 M NaCl, 10 mM
phosphate buffer at pH 7.0 with each oligomer 3.3 [mu]M for the
T
m
measurements and 1.7 [mu]M for the CD spectra. Concentrations were calculated using [epsilon] = 8.1 and 8.4 A
260
U/[mu]mol of residue for dT
15
and poly(dA), respectively. The extinction coefficients for the mixed oligomers
were calculated using a formula based on nearest neighbors (
10
). The
31
P NMR spectra were run in CH
3
CN (dimer blocks) or D
2
O (oligomers) with d
6
DMSO for a lock and aq. 85% H
3
PO
4
as an external reference. Ion-spray MS (ESI) measurements were run by Dr Frank Masiarz at Chiron on a
Perkin-Elmer PE SCIEX API III electrospray quadrupole instrument. LSI MS were
obtained by Dr Doris Hung at Northwestern University on a VG 70SE instrument.
3'-
O
-
t
-Butyldimethylsilylthymidine, dT(TBDMS), (10.4 mmol) was treated with 5'-
O
-dimethoxytritylthymidine-3'-
O
-(methyl
N
,
N
-diisopropylphosphoramidite) (10 mmol) in 50 ml dry CH
3
CN containing 20 mmol tetrazole. The reaction was complete after stirring for 5 min at room temperature, as indicated by TLC (silica gel;
5% MeOH and 1% Et
3
N in CH
2
Cl
2
); the triester product migrated slightly faster than dT(3'-
O
-TBDMS). The reaction mixture was diluted with CH
2
Cl
2
and extracted with 400 ml 5% aq. NaHCO
3
/NaCl. The organic layer was dried (Na
2
SO
4
) and concentrated under reduced pressure. After coevaporation of the residue
with several portions of dry toluene, the triester was dissolved in 100 ml dry CH
3
CN and treated with 3-dimethylaminopropylamine (10 ml, 16 mmol), followed by dropwise addition of iodine
(2.53 g, 10 mmol) in 50 ml dry CH
3
CN (
11
). After 30 min, the reaction mixture was poured into 300 ml 5% aqueous sodium
bisulfite and extracted with CH
2
Cl
2
. The organic layer was washed repeatedly with 5% aq. NaHCO
3
and sat. NaCl, then dried (Na
2
SO
4
) and concentrated under reduced pressure. The resulting diastereomers of d(DMT)T+T(TBDMS) (7.7 g, 7.4 mmol) were separated by silica gel chromatography as the `fast' and the `slow'
eluting isomers using a 2-6% MeOH gradient in 2% Et
3
N in CH
2
Cl
2
:
.
`fast isomer',
a
, 2.9 g (29%), Rf 0.62,
31
P NMR 10.4 p.p.m., ESI MS, MW calcd for C
52
H
71
N
6
O
13
PSi 1047.2, found 1046.6; `slow isomer',
b
, 3.1 g (30%), Rf 0.40,
31
P NMR 10.6 p.p.m., ESI MS, MW calcd 1047.2, found 1046.9. The middle fraction containing both
isomers amounted to 1.7 g (16%). Efforts to separate the two isomers after,
rather than before, desilylation were unsuccessful.
The amidate dinucleotide block d(DMT)T+C
Bz
(TBDMS) was prepared from 5'-
O
-dimethoxytritythymidine-3'-
O
-(methyl
N
,
N
- diisopropylphosphoramidite) and dC
Bz
(3'-
O
-TBDMS) on a 20 mmol scale essentially as described above for the preparation of
(DMT)T+T(TBDMS). Separation of the stereoisomers by silica gel chromatography
yielded,
a
, 1.78 g (8%), Rf 0.75,
31
P NMR 10.4 p.p.m., and
b
, 1.94 g (8.5%), Rf 0.60,
31
P NMR 10.6 p.p.m. The 2'-
O
-methyluridine derivatives, (DMT)U'+U' (TBDMS), were obtained in the same way:
a
, 1.66 g (16%), Rf 0.50,
31
P NMR 10.6 p.p.m., ESI MS, MW calcd for C
52
H
71
N
6
O
15
PSi 1079.2, found 1078.6;
b
, 1.94 (19%), Rf 0.39,
31
P NMR 11.1 p.p.m.; ESI MS calcd MW 1079.2, found 1078.6.
Desilylation of the `fast isomer',
a
, of d(DMT)T+T(TBDMS) (0.7 mmol) was effected by treatment with
tetrabutylammonium fluoride (3.5 mmol) in dry CH
3
CN (20 ml) for 3 h. After dilution with CH
2
Cl
2
and washing with water (2 * 200 ml), the organic layer was dried (Na
2
SO
4
) and evaporated under reduced pressure. Purification by silica gel
chromatography using a 0-18% gradient of MeOH in 2% Et
3
N in CH
2
Cl
2
afforded d(DMT)T+T (isomer
a
) as a white solid: 0.57 g (80%),
31
P NMR 10.4 p.p.m., ESI MS, MW calcd for C
46
H
57
N
6
O
13
P 933.0, found 933.3. Desilylation of the `slow isomer' was achieved in a
similar fashion and gave a comparable yield of the diastereomeric dimer block,
b
:
31
P NMR 10.6 p.p.m., ESI MS, MW calcd. 933.0, found 933.5.
The other dimer blocks were desilylated in the same way. The % yield and
31
P NMR shifts for the products were: d(DMT)T+C
Bz
, isomer
a
, 77%, 10.4 p.p.m.; isomer
b
, 75%, 10.6 p.p.m.; (DMT)U'+U' isomer
a
, 82%, 10.6 p.p.m.; isomer
b
, 56%, 11.1 p.p.m.
(DMT)T+T isomer
a
(0.61 g, 0.57 mmol) in 5 ml dry of CH
2
Cl
2
and 0.75 ml (6.5 mmol) of diisopropylethylamine was treated with 1.5 eq. of 2-cyanoethyl
N
,
N
-diisopropylchlorophosphoramidite (Cl-BCE) at room temperature. The reaction was complete in 3 h as
indicated by TLC (8% MeOH in 2% Et
3
N in CH
2
Cl
2
). After dilution with CH
2
Cl
2
(200 ml), the organic layer was washed successively with 250 ml 5% aq. NaHCO
3
and 250 ml sat. NaCl. The organic layer was dried (Na
2
SO
4
) and concentrated under reduced pressure. Chromatography on silica gel afforded
the phosphoramidite derivative as a white solid: 0.52 g (79%);
31
P NMR, 150.0 and 149.7 p.p.m. (stereoisomeric phosphoramidite groups) and 10.4
p.p.m. (internucleoside phosphoramidate group) (the ratio of the integrated
areas for the phosphoramidite/phosphoramidate peaks was 1/1); LSI MS, MW calcd
1133.2, found 1133.
Isomer
b
was prepared similarly:
31
P NMR, 150.0,
149.7 and 10.6 p.p.m. (phosphoramidite/phosphoramidate, 1/1).
The other phosphoramidite dinucleotide blocks were prepared in the same manner,
except that for the 2'-
O
-methyluridine derivatives 2.2 equiv. of 2-cyanoethyl
N
,
N
,-diisopropylphosphoramidite was used and the phosphitilation reaction was
allowed to proceed for 18 h. Each exhibited signals for phosphoramidite and
phosphoramidate in a 1/1 ratio. The yields (%) and
31
P signals (p.p.m.) were: DMT(T+C
Bz
)BCE
a
, 68% and 150.1, 149.7, 10.56, 10.47 p.p.m.;
b
, 68% and 150.1, 149.7, 10.86, 10.80 p.p.m.; DMT(U'+U')BCE
a
, 95% and 151.9, 151.5, 10.7, 10.6 p.p.m.;
b
, 95% and 151.8, 151.3, 11.1 p.p.m.
Dimer blocks were used to synthesize pentadecanucleotide derivatives by
conventional phosphoramidite chemistry using cyanoethyl phosphoramidite
reagents as previously described (
9
). The RP-HPLC elution times in minutes for oligomers with the DMT group `on', and
the DMT group `off', were, respectively:
1a
, 19.8, 11.6;
1b
, 19.3, 11.4;
2a
, 19.0, 11.2;
2b
, 18.3, 10.8;
3a
, 19.0, 9.6;
3b
, 18.3, 9.4. The
31
P NMR (D
2
O solvent, DMT `off') [delta] values in p.p.m. were:
1a
, 9.0 (phosphoramidate) and -2.2 (phosphodiester), area ratio, 1/1;
1b
, 9.35, -2.2 (plus a minor peak at -2.0), phosphoramidate/phosphodiester, 1/1. The spectra of the
other oligomers were similar (solvent, 1:5 H
2
O/D
2
O):
2a
, 8.95 and -2.4 (ratio, 1/1);
2b
, 9.8 and -2.4 (ratio 1/1);
3a
, 8.95 and -2.3 (ratio 1/1);
3b
, 9.2 and -2.3 (ratio 1/1).
The stereo-uniform zwitterionic oligomers,
1a
,
1b
,
2a
,
2b
,
3a
and
3b
, were prepared by a general procedure outlined previously (
9
) which utilized sequential coupling of stereo-uniform dimer blocks on a solid support. The key intermediates were the fully protected
dinucleotide phosphoramidate derivatives blocks represented by formula I. These
compounds were separated into the component stereoisomers by chromatography on
silica gel. Subsequent removal of the silyl protecting group and
phosphitilation afforded the phosphoramidite reagents employed in the polymer
synthesis. Comparison of properties of
1a
,
b
,
2a
,
b
and
3a
,
b
to properties of oligonucleotide analogues containing aminoethylphosphonate (
12
), uncharged phosphormorpholidate (
13
-
15
) and methyl phosphonate (
16
,
17
) linkages suggests that, for a given pair of stereoisomers in our series, the
compound exhibiting the greater upfield shift in the
31
P NMR spectrum for the amidate P, and designated here as `
a
', probably has the R configuration. In each case the compounds in the `
a
' series came from the stereoisomer of the fully protected dimer block that
eluted first on silica gel chromatography.
We used d(T
15
C
4
A
15
),
4
, to test for hybridization of thymidine and 2'-
O
-methyluridine zwitterionic derivatives to a duplex target. In absence of other oligomers,
4
forms a self-complementary structure with a dT.dA stem of high stability (
T
m
62oC in 0.1 M NaCl and 78oC in 1 M NaCl). We found that the affinities of the oligothymidylate
derivatives for this target depend strongly on the charge and stereochemistry
of the probe and the ionic strength of the solution (Table
1
). Of special interest was the observation that
one of the zwitterionic isomers,
1a
, formed a relatively stable triple-stranded complex with target
4
even in 0.1 M NaCl.
The melting curve for an equimolar mixture of
1a
and
4
showed a transition (
T
m
24oC) for dissociation of the zwitterionic strand from the duplex segment, as well as a transition (
T
m
68oC) for denaturation of
4
(Fig.
1
); and a plot of A
260
versus
titrant for titration of
1a
with
4
at 0oC displayed a break at equimolar concentrations of the two oligomers (data
not shown), in accord for a complex with the dT.dA.dT motif. Under the same
conditions, neither dT
15
nor the zwitterionic isomeric oligomer (
1b
) interacted significantly with
4
(Fig.
1
). In a high salt solution (1.0 M NaCl) dT
15
did bind to
4
(
T
m
30oC). The stability of the complex formed by
1a
also increased with an increase in the salt concentration (
T
m
32oC in 1.0 M NaCl); however, the rise in
T
m
was less than in the case of the all anionic probe. Oligomer
1b
did not bind significantly to
4
even in 1 M NaCl.
We previously noted an unusual feature in the melting curve obtained at 260 nm
for a 1/1 dT/dA mixture of
1b
and poly(dA) in 1 M NaCl solution (
9
). The melting curve showed two transitions, differing in
T
m
by ~20oC. On the basis of further work we now propose that the first
transition stems from a reversible disproportionation of two
1b
.poly(dA) duplex segments to give a triplex with the dA.dA.dT motif plus a
strand of
1b
, and that the second transition represents reversible dissociation of the
triplex to give free poly(dA) and
1b
. Several lines of evidence support this conclusion. (i) Melting curves for this
system are given in Figure
2
. Two transitions appear in the curve measured at 280 nm as well as the one at
260 nm. An unusual feature, however, is that the higher temperature transition
in the 280 nm curve is hypochromic. Characteristically, transitions for
dissociation of dT.dA duplexes and dT.dA.dT triplexes are hyperchromic at this
wavelengths. This results points to dissociation of a complex with a novel
structure. (ii) Melting curves obtained in low salt solutions (0-0.1 M NaCl) showed a single transition (~
T
m
22oC) that was independent of the salt concentration. As the salt
concentration was increased incrementally, a second transition appeared at
progressively higher temperatures. The salt dependence for the second
transition is consistent with expectations for dissociation of a triple-stranded complex containing a zwitterionic and two anionic strands. (iii)
Association curves obtained by cooling solutions of the oligomers slowly from
80 to 0oC demonstrated that the transitions are fully reversible. As a further
test, a 1dT/1dA mixture of
1b
and poly(dA) in 1 M NaCl was allowed to cool from 80 to 25oC and held at the lower temperature for 2 h to permit a slow equilibration to take place. No change in absorbance
occurred during the 2 h, indicating that the system was stable at this
temperature. When the temperature was then further reduced through the range
for the lower transition to 18oC, the absorbance at 260 nm dropped rapidly and equilibrium was reached
within 2 min. (iv) A single transition (
T
m
42oC; 1 M NaCl) was observed on heating a mixture of
1b
and poly(dA) at a 1dT/2dA nucleotide ratio from 0 to 80oC, demonstrating that the triple-stranded complex was stable at 0oC in absence of excess
1b
. (v) In agreement with these results, titration of
1b
in 1 M NaCl (pH 7.0) with poly(dA) consumed twice as much poly(dA) at 30oC as at 0oC, and the breaks corresponded to formation of a 1dT/2dA complex at 30oC and a 1dT/1dA complex at 0oC. As controls, dT
15
was also titrated with poly(dA) under the same conditions. These experiments
showed breaks corresponding to 2dT/1dA, 1dT/1dA and 1dT/1dA, as expected, for
titrations at 0oC in 1.0 M NaCl, 30oC in 1.0 M NaCl and 0oC in 0.1 M NaCl, respectively. Also, titration of
1b
at 0oC in 0.1 M NaCl gave a 1dT/1dA ratio. (vi) A single transition (
T
m
26oC) was found on heating a mixture of
1b
with either one or two equivalents of d(CCA
15
CC) in 1.0 M NaCl. This result suggested that appearance of the second
transition in the case of poly(dA) might be related to the fact poly(dA) could
fold to a hairpin structure that would stabilize a dA.dA.dT triple-stranded complex. We therefore examined hybridization of
1b
with a shorter, well defined oligomer, d(A
15
C
4
A
15
), which also could fold to a hairpin structure. This target indeed simulated
poly(dA) in behavior, although the complexes formed were somewhat less stable (
T
m
~ 17oC for the disproportionation reaction and ~32oC for dissociation of the triplex in 1.0 M NaCl; see Fig.
3
). A comparison with the heating curve for d(A
15
C
4
A
15
) alone (curve B in Fig.
3
) shows that these breaks indeed reflect interaction of
1b
with this target oligonucleotide. As in the case with poly(dA), the transitions
were reversible.
Three pairs of stereoisomeric zwitterionic 15mers were prepared for this study.
Pair
1
was derived from thymidine, pair
2
from 2'-
O
-methyluridine and pair
3
from thymidine and deoxycytidine. In each case, all phosphoramidate linkages in a given oligomer had the same
configuration. Thermal denaturation experiments showed that participation of
these alternating cationic-anionic oligonucleotides in formation of triple-stranded complexes is highly dependent on chirality at the modified
phosphate linkages.
The most significant finding with respect to potential applications is that in each case one of the stereoisomeric probes, designated as the `
a
' isomer, binds more effectively to a complementary double-stranded DNA target than the corresponding all phosphodiester probe does.
In 0.1 M aqueous NaCl at pH 7, the enhancement in
T
m
is of the order of 20oC for the dT and the dT,dC zwitterionic 15mers (
1a
and
3a
) and ~35oC for the 2'-
O
-methyluridine derivative (
2a
). The enhancement in
T
m
in 1 M NaCl solutions is less, but still significant. It is noteworthy that the
affinity of
3a
is relatively high at pH 7, even though the probe contains several dC units.
Strong binding of this unsymmetrical, mixed base probe to the double-stranded target is consistent with a structure in which the third strand
is oriented parallel to the purine strand, as in other triple-stranded complexes derived from two pyrimidine and one purine strand.
In contrast to the results with the `
a
' stereoisomeric oligomers, the phosphoramidate oligomers with the opposite
configuration at phosphorus (`
b
') exhibited very low affinity for the double-stranded targets. Since the absolute configuration of the isomers has not
yet been definitively assigned, and structural information on the geometry of
the triple strand complex is limited, speculation on the reasons for these
differences is premature. It is interesting, however, that the configuration at the phosphoramidate links that favors binding of an oligonucleotide probe to double-stranded DNA is the same that favors binding to a single-stranded target to give a double-stranded complex (
9
).
Gryaznov
et al
. have shown that oligodeoxyribonucleotide N3'-P5' phosphoramidate derivatives bind to DNA duplexes to give
unusually stable pyr.pur.pyr and pur.pur.pyr complexes (
20
,
21
), and Nielsen
et al
. have found that `peptide nucleic acids' (PNAs) interact with appropriate DNA
sequences by strand invasion to give PNA.pur.PNA triple-stranded segments (
22
-
24
). The stereo-uniform cationic phosphoramidate derivatives comprise another family of oligonucleotide analogues that bind strongly to double-stranded DNA targets. Since these three families differ from one
another in physical and chemical properties and, no doubt, in behavior in
biological systems as well, they offer the chemist rich opportunities for
tailoring DNA binding agents for specific applications.
A surprising set of equilibria was found for systems containing d(T+T-)
7
T isomer
1b
and poly(dA) or d(A
15
C
4
A
15
) in 1 M NaCl solution. A model consistent with the data is indicated schematically in Chart 2 for a system containing
1b
and poly(dA) in a 1dT/1dA ratio. According to this, at 0oC the components form a double-stranded complex; at 30oC they exist as a dA.dA.dT triple-stranded complex and an equivalent of free
1b
; and at 50oC the complex is fully dissociated. The transitions between the states are
relatively sharp (
T
m
22 and 42oC) and equilibrium is established rapidly both on heating and cooling the
solution. The system involving d(A
15
C
4
A
15
) fits the same scheme, but with a dC
4
strand serving as the linker to facilitate alignment of two dA
15
segments. The importance of the configuration of the phosphoramidate linkages
is shown by the fact that oligomer
1a
, in contrast to
1b
, did not form triple stranded complexes of this type.
Two features in these equations are unusual, though not without some related
precedents: (i) formation of a stable triple-stranded complex containing exclusively dA.dA.dT triads, and (ii) formation of a triple-stranded complex by a thermally induced disproportionation of a
double-stranded complex. Although a number of examples of pur.pur.pyr triplexes
have been reported (see
5
-
8
for representative cases), the stable complexes containing dA.dA.dT triads that
have been described have also contained dG units in one of the purine strands.
Pilch
et al
. have noted that [rdquor]...the presence of guanine residues appears to be crucial for stabilization of short pur.pur.pyr triplexes" (
8
). For the ribonucleotide family, however, Broitman
et al
. have described a triple-stranded complex, poly(A).poly(A).poly(U), which can form when the degree of polymerization
of poly(A) falls in the range of ~28-150 (
25
), and Lauceri
et al
. (
26
) have shown that cationic porphyrins in low concentration induce formation of
poly(A).poly(A).poly(U) triple-stranded complexes from high molecular weight poly(A) (>400 bp). With
respect to thermally induced disproportions, examples affording pyr.pur.pyr
complexes have been reported for both ribonucleotide, [poly(A).poly(U)] (
27
,
28
,
25
), and deoxyribonucleotide polymers, [poly(dTdC).poly(dGdA)] (
29
-
31
).
We conclude that the unusual stability of the dA.dA.dT complexes containing oligonucleotide
1b
, and the disproportionation reaction leading to their formation, must reflect the presence of the stereo-uniform side chains in the probe. The properties of zwitterionic oligomers
with this chirality may prove useful in designing novel self-assembly systems based on oligonucleotide hybridization.
The research at Northwestern University was supported by a research grant from
the Chiron Corp. We thank Dr Paul Loach for use of the CD spectrophotometer.
+
Present address: Chemsyn Science Laboratory, Lenexa, KS 66215-1297, USA
REFERENCES
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