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ELECTRONIC SUPPLEMENTARY INFORMATION (ESI)
Unusual RNA and DNA binding properties of
a novel pyrrolidine–amide oligonucleotide mimic (POM)
David T. Hickman,a Paul M. King,b Matthew A. Cooper,c Jonathan M. Slater,b
and Jason Micklefield*a
a
Department of Chemistry, UMIST, Faraday building, PO Box 88, Manchester M60
1QD, UK.
b
Department of Chemistry, Birkbeck College, University of London, Gordon House,
29 Gordon Square, London WC1H 0PP, UK.
c
University Chemical Laboratory, Lensfield Road, Cambridge, CB2 1EW, UK.
Dr M. A. Cooper
UV thermal denaturation curves, Job plots and SPR sensograms for T 5–
POM 3 binding to DNA and RNA.
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2
1.15
Abs (260nm)
1.1
1.05
fast heating
1
slow cooling
slow heating
0.95
0.9
0.85
0.8
15
25
35
45
55
65
75
85
95
temperature / deg C
Figure 1. Variation of Absorption (A260) with temperature curves for an equimolar
mixture of T5-POM 3 and Poly(rA) (42 M each in bases) in buffer A (10 mM K2HPO4
adjusted to 120 mM K+, pH 7.0) for a typical cycle of fast heating (5 ˚C/min), slow
cooling (0.2 ˚C/min) and slow heating (0.2 ˚C/min). The large hysteresis between the
slow heating and cooling curves indicates that the rate of association or dissociation is
less than the rate of heating/cooling such that equilibrium between 3 and Poly(rA) is not
attained. Typically at a heating/cooling rate of 0.2 ˚C/min little or no hysteresis is
observed for native duplex forming oligonucleotides, suggesting that 3 and Poly(rA)
associate and dissociate slowly compared with the hybridisation of native nucleic acids.
This cycle of heating/cooling was used for determining Tms except where stated
otherwise.
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3
1
Abs (260nm)
0.95
5 degC/min
2 degC/min
1 degC/min
0.5 degC/min
0.1 degC/min
0.9
0.85
0.8
0.75
0.7
15
25
35
45
55
65
75
85
temperature / degC
Figure 2. UV melting curves for T5-POM 3 and Poly(rA) (42 M each in bases) in
buffer A following fast heating (5 ˚C/min) then cooling and heating at different rates (5,
2, 1, 0.5 and 0.1 ˚C/min). The larger hyperchromic shifts at slower rates of heating are
presumably a consequence of the greater extent of duplex association and dissociation
during the cooling and heating cycles respectively.
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50
49
Tm / degC
48
47
46
45
44
43
0
1
2
3
4
5
Rate of heating (degC/min)
Figure 3. Plot of Tm vs rate of heating/cooling (from figure 2). This shows that the
observed Tm for T5-POM 3 and Poly(rA) is higher at lower rates of heating/cooling. By
extrapolating to an infinitely slow rate of heating/cooling the true equilibrium Tm was
determined to be 49˚C.
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normalised Abs (260nm)
1.3
1.25
Poly (rU)
Poly (rC)
Poly (rG)
Poly (rA)
1.2
1.15
1.1
1.05
1
0.95
15
25
35
45
55
65
75
85
95
temperature / deg C
Figure 4. UV thermal denaturation curves for T5-POM 3 and an equimolar amount
(42M each strand in bases) of Poly(rA), (rU), (rC) or (rG) in buffer A. The absence of
significant hyperchromicity with non-complementary RNAs suggests that T5-POM
binds sequence specifically to Poly(rA).
n o r m a lis e d A b s ( 2 6 0 n m )
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1 .3
1 .2 5
1 .2
120m M
220m M
620m M
1200m M
1 .1 5
1 .1
K+
K+
K+
K+
1 .0 5
1
0 .9 5
0 .9
15
25
35
45
55
65
75
85
95
te m p e r a tu r e / d e g C
Figure 5. UV thermal denaturation curves of T5- POM 3 and Poly(rA) (42 M each in
bases, 10 mM K2HPO4, pH 7.0) at different ionic strengths. An increase in ionic
strength is accompanied by a moderate increase in Tm. The lower hyperchromic shifts
observed at higher ionic strength are probably due to slower rates of
association/dissociation of T5-POM with Poly(rA).
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normalised Abs (260nm)
1.5
1.4
pH 6.0
pH 6.5
pH 7.0
pH 7.5
pH 8.0
1.3
1.2
1.1
1
0.9
25
35
45
55
65
75
temperature / degC
Figure 6. UV thermal denaturation curves of T5- POM 3 and Poly(rA) at different pHs
(42 M each in bases, 10 mM K2HPO4, 120 mM K+). Considerably higher Tm values
and larger hyperchromic shifts are observed at lower pH. The later is a reflection of
faster association at lower pH such that during the annealing cycle a higher proportion
of strands have hybridised.
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60
58
Tm / degC
56
54
52
50
48
46
44
42
40
6
6.5
7
7.5
8
pH
Figure 7. Plot of Tm vs pH for T5-POM 3 and Poly(rA) derived from figure 6. The most
marked change in Tm occurs between pH 6.5 - 7.0.
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0.6
0% A
10% A
20% A
30% A
40% A
50% A
60% A
70% A
80% A
90% A
100% A
0.5
Abs
0.4
0.3
0.2
0.1
0
220
230
240
250
260
270
280
290
300
Wavelength / nm
Figure 8. UV spectra of T5-POM 3 and Poly(rA) at different molar ratios. Mixtures of 3
and Poly(rA) 50 M total base concentration in buffer A were allowed to anneal for 24
hr at 25 ˚C before UV spectra were recorded. The lowest absorbance maximum in the
range 255–275 nm is obtained at a 1:1 (T:A) molar ratio indicative of duplex formation.
0.6
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Figure 9. A Job plot of T5-POM 3 and Poly(rA) showing UV maximum absorbance
(Amax) in the 255–275 nm region vs molar ratio derived from figure 8. Job plots of A260
vs molar ratio also show a minima at a 1:1 (T:A) molar ratio, but the minima is less
well defined due to the large difference in max for poly(rA) (257 nm) and 3 (272 nm).
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1.2
normalised Abs (260nm)
1.18
1.16
1.14
1.12
1.1
1.08
1.06
1.04
1.02
1
15
25
35
45
55
65
75
85
95
temperature / degC
Figure 10. Melting curve for T5-POM 3 and Poly(dA), 120 mM K+, pH 7 at an heating
rate of 0.2 ˚C/min. The samples were incubated for 48 h at a fivefold increased
concentration (210 M each in bases) prior to dilution to 42 M as described in the
main text. Even after such a long incubation period it is likely that only a fraction of the
complex has formed.
Supplementary Material (ESI) for Chemical Communications
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Figure 11. Melting curve for T5-POM 3 and Poly(dA), 1200 mM K+, pH 7.0.
12
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normalised Abs (260nm)
1.026
1.022
1.018
1.014
1.01
1.006
1.002
0.998
15
25
35
45
55
65
75
85
95
temperature / degC
Figure 12. Melting curve for T5-POM (3) and Poly(dA) 620 mM K+, pH 7.0, after 96 h
incubation . Although the absorbance change is small, two hyperchromic shifts are
observed consistent with melting of a triplex to a duplex and then melting of a duplex to
single strands.
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n o rm alised Ab s (260n m )
1.1
1.08
1.06
1.04
1.02
1
0.98
15
25
35
45
55
65
75
85
95
tem perature / degC
Figure 13. Melting curve for T5-POM (3) and Poly(dA) at 0.2 ˚C/min, 120 mM K+, pH
6.0 after 96 h incubation. Again two hyperchromic shifts are observed indicative of
triplex denaturation.
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0.7
0.6
0% A
10% A
Abs
0.5
30% A
40% A
0.4
50% A
60% A
0.3
80% A
0.2
90% A
100% A
0.1
0
220
230
240
250
260
270
280
290
300
wavelength / nm
Figure 14. UV spectra T5-POM 3 and Poly(dA) at different molar ratios. Mixtures of 3
and Poly(rA) 210 M total base concentration in buffer A were allowed to anneal for 48
hr at 25 ˚C, then diluted with buffer A to a total base concentration of 42 M, before UV
spectra were recorded. The lowest absorbance maximum in the range 257–272 nm is
obtained at 30% Poly(dA), a 2:1 (T:A) molar ratio, indicative of triplex formation.
(Scans of 20 % and 70 % Poly(dA) have been omitted to improve clarity).
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0.58
0.56
0.54
Abs max
0.52
0.5
0.48
0.46
0.44
0.42
0.4
0
10
20
30
40
50
60
70
80
90
100
mol % Polyd(A)
Figure 15. A Job plot of T5-POM 3 and Poly(dA) showing UV maximum absorbance
(Amax) in the 257-272 nm region vs molar ratio derived from figure 14.
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Figure 16. SPR response (RU) vs. time for T5-POM 3 injected across r(A)20 (), d(A)20
(), d(AGC TTC AGA GAT CGA TCG GAG AGA GTA GTG–3´) () derivatised
surfaces and an underivatised control surface (). Response profiles were determined
using a BIAcore 2000 instrument (Biacore AB, UK) under standard conditions
following injection of T5-POM 3 (40M strand concentration, 10 mM K2HPO4, pH 7
adjusted to 0.12 M K+) across each surface at a flow rate of 20 L/min for 300 sec
followed by buffer to allow dissociation. All oligonucleotides were biotinylated at the
5´–end (Cruachem, UK) and immobilised on a streptavidin derivatised carboxymethyl
dextran sensor chip (SA) (Biacore).
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700
600
500
400
RU
300
200
100
0
-100
-100
100
300
500
700
t / sec
Figure 17. SPR response (RU) vs. time for T5-POM 3 injected across r(A)20 (), d(A)20
(), d(AGC TTC AGA GAT CGA TCG GAG AGA GTA GTG–3´) () derivatised
surfaces and an underivatised control surface (). Response profiles were determined as
described in figure 16 except 80M strand concentration of T5-POM 3 was injected
across each surface.