Evaluation of pyrimidine PNA binding to ssDNA targets from nonequilibrium
melting experiments
Evaluation of pyrimidine PNA binding to ssDNA targets from nonequilibrium melting experiments
Elena A.
Lesnik*
,
Lisa M.
Risen
,
David A.
Driver
+
,
Michael C.
Griffith
}
,
Kelly
Sprankle
and
Susan M.
Freier
ISIS Pharmaceuticals, 2292 Faraday Avenue,
Carlsbad
, CA 92008,
USA
Received October 7, 1996;
Revised and Accepted December 5, 1996
ABSTRACT
Slow kinetics of homopyrimidine PNA binding to single stranded DNA and RNA
targets is manifested in significant hysteresis in thermal UV absorption
experiments. We have compared temperatures of dissociation (T
dis
) and reassociation (T
ass
) for triplexes formed by DNA and single or bis PNAs with K
50
derived from gel mobility experiments. Results indicated there was no
correlation between T
dis
and K
50
while reasonable correlation between T
ass
and K
50
was found. This correlation enabled use of easy thermal UV absorption
experiments for evaluation of PNA binding to DNA/RNA targets.
INTRODUCTION
Some years ago, peptide-nucleic acid (PNA), a novel nuclease resistant DNA analog, was synthesized
and described (
1
). Single homopyrimidine PNAs bind single stranded DNAs and RNAs in triplex
fashion (
2
,
3
) while mixed purine-pyrimidine PNAs bind them in duplex fashion (
4
). UV melting experiments are a routine, fast, and convenient method for
evaluation of oligonucleotide binding. However, slow kinetics of triplex
formation reported for single homopyrimidine PNA binding are manifested in
significant hysteresis for UV melting-cooling curves (
5
-
8
). The question arose whether we could use data derived from melting experiments
for evaluation of oligonucleotide binding and if we could compare them with K
50
derived from gel mobility or filter binding assays usually used for evaluation
of slow binding processes (
9
-
12
).
In this paper, we compare K
50
obtained from gel mobility assay and data obtained from dissociation and
reassociation nonequilibrium thermal experiments for a set of pyrimidine PNAs
binding single stranded DNA and RNA targets. No correlation was observed
between temperature of triplex dissociation (T
dis
) determined as the temperature of the main maximum in the first derivative of
the dissociation (heating) curve and K
50
. On the other hand, reasonable correlation between temperature of association
(T
ass
) determined as the temperature of the maximum in the first derivative of the
reassociation (cooling) curve and K
50
was found. The correlation enabled us to evaluate binding of the pyrimidine
PNAs to single stranded targets using easy thermal experiments and to shed some
light on the mechanism of triplex formation and stability.
MATERIALS AND METHODS
PNA and oligonucleotide synthesis
PNAs were synthesized and purified as described elsewhere (
5
) and were analyzed by high performance liquid chromatography (HPLC) and mass
spectrometry. By analogy to peptides, PNA sequences are written from amino (N)
to carboxy (C) terminus (
4
). Oligonucleotides were synthesized using an Applied Biosystems 380B automated
DNA synthesizer and standard phosphoramidite chemistry (
13
).
Thermal melting-cooling experiments
Absorbance versus temperature curves were measured in a Gilford Response II spectrophotometer. Samples were prepared in the buffers indicated
at 8 [mu]M total strand concentration. The ratio of PNA to target (single stranded
DNA or RNA) was 2:1 for single pyrimidine PNAs and 1:1 for bis PNAs. Prior to
each experiment, samples were heated at 90-95oC for 15 min and then incubated at room temperature for 2 h. Unless
otherwise indicated, absorbance at 260 nm was measured as a function of
temperature as samples were heated from 15 to 98oC at 0.7oC/min. After 10 min incubation at 98oC, samples were cooled at the same rate. Reported temperatures
for T
dis
and T
ass
are the maxima in the first derivatives [dA(260)/dT] for heating and cooling
respectively. Reported halfwidth of the peaks in first derivative curves were
measured at half height of the peaks. Reproducibility in peak position was +-0.5oC. Pattern of `minor' peaks were reproducible for melts performed
under identical conditions.
Gel mobility assays
PNA binding was measured using a gel mobility assay (
11
). Radiolabeled target (~10 pM DNA or RNA) was incubated with increasing concentrations of PNA at 37oC for 20-24 h in 100 mM Na
+
, 10 mM phosphate buffer (pH 7.0), 0.1 mM EDTA. An increase in incubation time
did not result in a decrease of K
50
values suggesting equilibrium was reached within 20 h (data not shown).
Reactions were resolved at 4oC in a 12% native polyacrylamide gel containing 44 mM tris-borate, 1 mM MgCl
2
. We define K
50
as the PNA concentration at which 50% of the target is bound (
11
).
RESULTS
Single stranded pyrimidine PNAs with and without positively charged
lysines
at the C-terminus and bis pyrimidine PNAs were evaluated for hybridization to DNA
and RNA targets (see Table
1
for a list of sequences studied). Pyrimidine PNAs hybridize to single stranded
DNA and RNA targets in triplex fashion and can bind them in two orientations:
parallel (N-terminus of PNA binds to 5'-end of DNA/RNA target) and antiparallel (N-terminus of PNA binds to 3'-end of DNA/RNA target) (
3
,
14
). We synthesized both parallel and antiparallel targets for each PNA (Table
1
). Mixed purine-pyrimidine PNA 11 was included in the study to compare the pH effect on
duplex and triplex formation. PNAs 2, 3 and bis-PNAs 8 and 9 had the same base sequence but differed in the presence of
lysine
and the direction of sequence from N to C terminus (PNAs 2 and 3) or in the
chemistry of linkers in bis-PNAs 8 and 9. PNAs 5 and 6 had the same base sequence oriented in opposite
directions from N to C terminus. PNA 7 and bis PNA 10 had the same symmetrical
base sequence and therefore one DNA target (
22
, Table
1
) was synthesized for them. Thus, we had five different base sequences arranged
differently in ten PNAs to test the effect of PNA structure peculiarities on K
50
and UV-melting data.
a
PNA base sequences are written from N to C terminus. PNA structures are shown in
(25).
b
bis PNA linkers: 3PEG designates H
2
N(CH
2
CH
2
O)
3
CH
2
COOH and Aha designates 6-aminohexanoic acid.
c
Bold letters denote the target sequence if it consists of only a part of the
oligonucleotide.
Dissociation curves for single pyrimidine PNA
2
-DNA triplexes
Examples of dissociation (heating) curves are presented in Figure
1
(plots a, b, e, f, i, and j). For two triplexes with antiparallel PNA
orientation to DNA (PNAs 4 and 6), we observed only one transition in the
dissociation curves (e.g. Fig.
1
f). For all other triplexes with single PNAs, dissociation curves exhibited more
than one transition. Comparison of the melting curves indicated the different
orientation of the same PNA toward DNA target resulted in different patterns of
`minor' peaks while `main' high temperature peaks in the differential melting
curves remained similar. Preincubation of PNA/DNA mixtures from 1 h to 3 days
at 20oC or 37oC resulted in some changes in size, shape, and positions of minor
peaks but no changes were observed for the main peaks (data not shown).
Reassociation curves for single PNA
2
-DNA triplexes
In contrast to dissociation curves, reassociation (cooling) curves always
exhibited a single cooperative transition manifested as one peak in the first
derivative plot (Fig.
1
c, d, g, h, k, l). For most PNA complexes, the maximum in the reassociation
curve was at a lower temperature than the `minor' peaks in the dissociation
curve. Hysteresis between main peaks in the dissociation and reassociation
curves ranged from 8 to 36oC. The halfwidths of reassociation transitions were 6-8oC compared to 5-7oC for equilibrium dissociation-reassociation curves of similar length
oligonucleotide duplexes.
Dissociation and reassociation curves for bis PNAs
Bis PNAs form triplexes with single stranded DNA and RNA targets binding them in
1:1 ratio with each `arm' of the bis PNA binding in opposite orientation (
5
,
8
). Hybridization rates for bis PNAs are reported to be faster than for single
PNAs (
8
). The hysteresis observed for bis PNAs 8-10 (Table
1
) was reduced 3-30 times compared to single PNAs depending on PNA base composition,
linker chemistry, and target. Bis PNA 10 comprising 11% cytosines in each arm
and three positively charged
lysines
in the linker exhibited very cooperative transitions with high T
dis
and T
ass
values and negligible hysteresis (Fig.
2
a). In contrast, bis PNA 8 with 40% cytosines and neutral poly(ethylene glycol) linker exhibited broad transitions and almost 10oC hysteresis
(Fig.
2
b).
Effect of acidic pH on PNA
2
-DNA triplex dissociation and reassociation curves
Protonation of PNA cytosines at pH 5.5 shifted both the major and minor peaks in
the dissociation curves to higher temperatures (compare Fig.
1
a, b and Fig.
3
a, b). Moreover, for PNA 1 and 2 without
lysine
at the C terminus, the size of the lower temperature peak became equal or even
larger than the higher temperature peak (Fig.
3
a, b). Association curves at pH 5.5 revealed a single cooperative transition
similar to that observed in neutral solution (Fig.
3
c, d). T
ass
for three PNAs with parallel and antiparallel DNA targets measured at pH 7.0
and 5.5 are listed in Table
2
. T
ass
increased by 4.5-17.5oC in acidic solution compared to that at neutral pH. At pH 5.5, T
ass
for triplexes with PNAs 2, 4, and 5 were similar at both PNA orientations
whereas at pH 7.0, T
ass
for triplexes 4 and 5 depended on PNA orientation. PNA 11 with mixed purine-pyrimidine base composition was used as a control demonstrating cytosine
protonation resulted in insignificant decrease of T
ass
for PNA-DNA duplex formation.
.
Temperatures of reassociation (T
ass
) for PNA
2
-DNA triplexes in neutral (10 mM cacodilate buffer, pH 7.0) and acidic (10 mM
cacodilate buffer, pH 5.5) buffers containing 100 mM Na
+
and 0.1 mM EDTA.
#
Both PNA strands parallel to target
Both PNA strands antiparallel to target
T
ass
T
ass
[Delta]T
a
[Delta]T/C
+a
T
ass
T
ass
[Delta]T
[Delta]T/C
+
pH 7.0
pH 5.5
(5.5-7.0)
pH 7.0
pH 5.5
(5.5-7.0)
2
36.2
52.2
16.0
4.0
37.8
52.2
14.4
3.6
4
57.3
68.3
11.0
2.2
64.8
69.3
4.5
0.9
5
49.8
67.3
17.5
2.5
59.2
68.2
9.0
1.3
11
57.8
56.8
-1.0
-0.02
75.3
73.3
-2.0
-0.04
a
[Delta]T is the difference between T
ass
measured at pH 5.5 and 7.0; [Delta]T/C
+
is [Delta]T per cytosine residues in Hoogsteen PNA strand
K
50
values for pyrimidine PNA binding to single stranded targets
K
50
was measured for each PNA listed in Table
1
. K
50
values varied from 30 pM up to 300 nM demonstrating a wide range of PNA
affinity to the targets (Table
3
). It should be noted that at high PNA concentration, the major shifted band in
the polyacrylamide gel was sometimes accompanied by one or a few minor bands of
slower mobility suggesting formation of more than one type of complex.
Comparison of dissociation constants (K
50
) and T
ass
and T
dis
All T
dis
and T
ass
values derived from melting-cooling curves and K
50
values derived from gel shift mobility experiments are compiled in Table
3
. For all PNAs (except case 9'), T
ass
was lower than T
dis
and no other correlation between T
dis
and T
ass
was observed. For some PNAs, a small difference in T
ass
corresponded to a big difference in T
dis
(e.g. PNAs 1 and 2 versus parallel targets or PNAs 6+5 and PNA 6 versus antiparallel target). For some others, a small difference in T
dis
corresponded to a big difference in T
ass
(e.g. PNA 6 versus parallel target and bis PNA 9 versus DNA target). The most
important observation was that similar T
dis
values corresponded to K
50
values differing from each other as much as 10-20-fold. Thus two parameters routinely used to characterize the
affinity of the oligonucleotides and their analogs to DNA/RNA targets did not
correlate with each other. On the other hand, decrease in K
50
correlated well with increase in T
ass
values. Realizing this, we plotted T
ass
versus log K
50
for all PNA-DNA complexes studied (Fig.
4
a) to find out whether the T
ass
quantitatively correlated with K
50
's and could be used for the PNA affinity evaluation. Comparison of the two plots in Figure
4
demonstrated there was a reasonable correlation between T
ass
and K
50
(Fig.
4
a) while no correlation between T
dis
and K
50
values was observed (Fig.
4
b). All T
ass
values for single PNAs fell on a single line. Points corresponding to bis PNAs
in four of five cases fell higher than the correlation predicted and we discuss
this below.
DISCUSSION
Relationship between T
ass
and K
50
Developed for measurement of slow protein-nucleic acid binding (
15
,
16
), electrophoretic shift mobility assays are also successfully used now for
evaluation of slow complex formation between structured DNA, RNA and
oligonucleotides or analogs (
9
,
11
,
12
). However, gel mobility shift assay is time and labor intensive and
quantitative reproducibility is limited (
9
). Measurement of dissociation and reassociation curves by UV absorption
spectroscopy is a rapid, easy, and reproducible technique for determination of
complex formation if it occurs under equilibrium conditions. Under
nonequilibrium conditions, temperatures of dissociation and reassociation
depend on the heating rate (
17
).
.
Temperatures of Triplex Dissociation (T
dis
) and Reassociation (T
ass
) and K
50
values for PNAs binding DNA targets in 10 mM phosphate buffer (pH 7.0), 100 mM
Na
+
, 0.1 mM EDTA
PNA
a
Both PNA strands parallel to target
Both PNA strands antiparallel to target
PNA strands parallel and antiparallel to target
T
dis
T
ass
[Delta]
b
K
50
(nM)
T
dis
T
ass
D
K
50
(nM)
T
dis
T
ass
[Delta]
a
K
50
(nM)
1
47.8
36.8
-11.0
300
40.0
39.8
-0.2
-
2
72.2
36.2
-36.0
125
71.2
37.8
-33.4
150
3
83.2
47.7
-35.5
12.0
82.8
50.3
-28.5
20.0
4
81.0
57.3
-23.7
0.4
83.7
64.8
-18.9
0.7
5
84.1
49.8
-34.3
9.3
83.7
59.2
-24.5
1.2
6
85.7
49.8
-35.9
12.0
69.7
61.2
-8.5
0.8
5+6*
93.6
63.2
-30.4
0.4
6+5*
96.0
61.2
-34.8
0.6
7*
74.7
47.9
-26.8
9.0
8
80.8
69.8
-11.0
0.2
9
85.2
76.3
-8.9
0.3
9'
82.8
82.8
0
0.3
9
*
92.8
87.8
4.0
0.03
10
85.2
84.3
-0.9
0.16
a
PNA numbers in Table 1.
b
[Delta] =
T
ass
-T
dis
is hysteresis between dissociation and reassociation curves.
5+6*, PNA 5 bound DNA target in parallel direction and PNA (6) bound target in
antiparallel direction;
6+5*, PNA 6 bound DNA target in parallel direction and PNA (5) bound target in
antiparallel direction;
7*, PNA 7 has symmetrical base sequence and can bind the same target in both
orientation
9', T
dis
and T
ass
were obtained in melting-cooling experiment with heating rate 0.1oC/min.
9*, PNA 9 bound RNA target.
Hysteresis of 25-30oC has been reported for PNA triplexes
heated and cooled at rates of 0.1 and 1oC/min
(
5
-
7
). At a rate 0.7oC/min, we observed hysteresis of 8-36oC
for triplexes with single PNAs
and 1-10oC for triplexes with bis PNAs depending mainly on cytosine content
in the PNA strands and linker chemistry. Decrease of the heating/cooling rate
down to 0.2oC/min resulted in an increase of T
ass
by 1-3oC and in a decrease of T
dis
by 3-7oC (measured for four triplexes). A temperature rate of 0.1oC/min reduced hysteresis even more but did not eliminate it for
triplexes with single PNAs (data not shown ) indicating the characteristic time
of experiments was still much lower than the relaxation time of triplex
dissociation and reassociation at equilibrium (
18
). Therefore, we cannot reach equilibrium in routine UV experiments and thus,
cannot evaluate true PNA affinity directly from melting curves (
7
). In contrast to T
dis
and T
ass
, K
50
values obtained under equilibrium conditions reflect genuine PNA affinities.
The observation of a correlation between K
50
and T
ass
values enabled us to use simple and rapid UV spectroscopy method for evaluation
of PNA affinity to single stranded DNA/RNA targets.
Effect of PNA orientation on PNA binding
Using the correlation obtained we tried to evaluate the effect of PNA
orientation and cytosine protonation on PNA binding. The data in Table
3
indicated there was no preferential orientation of PNA 2 and 3 to DNA targets
while
for PNAs 4, 5, and 6, antiparallel binding to complementary DNA was preferred.
Simultaneous parallel and antiparallel PNA binding to the same target (5+6 and
6+5 triplexes in Table
3
) resulted in a very small decrease in K
50
and increase in T
ass
compared to antiparallel binding of both PNA strands. This observation
correlates with molecular mechanics calculations (
20
) and conformational analysis data (
21
) predicting a small gain in triplex stabilization when the Watson-Crick (W-C)
and Hoogsteen (H) PNA strands are antiparallel to each other. PNA 1 with 70%
cytosines apparently formed a triplex with the parallel DNA target as indicated
by the bi-phasic dissociation and 11oC hysteresis (
1
, Table
3
). A monophasic dissociation curve (data not shown) and lack of hysteresis for
the complex of PNA 1 with antiparallel DNA target (Table
3
) suggests only a PNA-DNA duplex formed at pH 7.0. The gel shift assay indicated that K
50
for PNA 1 binding antiparallel DNA target at 37oC was weaker than 10 [mu]M.
The reported X-ray structure of a pyrimidine bis PNA-DNA triplex (
22
) suggests a strong Van der Waals interaction and hydrogen bonding between DNA
and H-PNA backbones. These data explain enhanced affinity of H-PNA strand with moderate cytosine content compared to affinity of
Hoogsteen oligonucleotide strands in DNA triplexes at pH 7.0 (
23
,
24
). Moreover, the X-ray data indicate that W-C PNA strand of bis PNA is antiparallel to DNA
target suggesting this is preferable orientation for W-C PNA strand in both
duplex and triplex PNA-DNA structures (
22
). Based on this suggestion, we can speculate that extra minor peaks in
dissociation curves reflected melting out of complexes with alternative
mismatched or shifted structures including complexes with reversed binding of
W-C PNA strands to parallel DNA targets as shown in Figure
5
.
Effect of cytosine protonation
At pH 5.5, protonation of cytosines results in additional electrostatic
attraction and Hoogsteen G-C
+
hydrogen bond formation between H-PNA and DNA target. The change of pH from 7.0 to 5.5 resulted in a small
decrease of T
ass
for mixed purine-pyrimidine PNA 11 (Table
2
) which forms a PNA-DNA duplex with only W-C hydrogen bonds (
4
). Therefore protonation of the Hoogsteen PNA strand is responsible for the significant increase in T
ass
at pH 5.5 for homopyrimidine PNAs. The [Delta]T
ass
/C
+
values (Table
2
) enabled us to evaluate the effect of protonation per one cytosine in Hoogsteen
PNA strand. The data showed the effect of protonation was much higher for PNA 2
without positively charged
lysine
than for PNA 4 and 5 with
lysines
. At pH 7.0, antiparallel orientation of PNA 4 and 5 to DNA targets was
preferable compared to parallel one. Protonation of cytosines affected
triplexes with parallel PNAs twice as much as triplexes with antiparallel PNAs
judging by [Delta]T
ass
/C
+
values. As a result, at pH 5.5, T
ass
became similar suggesting there was no preferable binding orientation for
positively charged H-PNA strands toward DNA target. Since it is not clear which PNA strand
binds tighter to the DNA target under these conditions, it is difficult to
assign the peaks in melting curves (Fig.
3
a, b) to Hoogsteen or Watson-Crick PNA strand dissociation.
In conclusion, we have shown that T
ass
values for PNA
2
-DNA triplex formation derived from nonequilibrium reassociation curves correlated well with log K
50
derived from equilibrium mobility shift experiments. The correlation obtained
enabled evaluation of affinity of single and bis pyrimidine PNAs which bind
slowly single stranded DNA/RNA targets using fast and convenient UV absorption
method.
ACKNOWLEDGEMENTS
We thank Dr Richard Friedman for very useful discussion, Dr Alexander Vologodsky
for critical reading of the manuscript, and Dr Rolf Berg for synthesis of some
of the PNAs.
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*
To whom correspondence should be addressed. Tel: +1 619 603 2377; Fax: +1 619
931 0209; Email: elesnik@isisph.com
Present addresses:
+
Chiron Viagene, 11055 Roselle Street, San Diego, CA 92121, USA and
[Dagger]
Houghten Pharmaceuticals, 3550 General Atomic Court, San Diego, CA 92121, USA
