Copper(II)quenched oligonucleotide probes for fluorescent DNA sensing Jens Brunner and Roland Kraemer* Supplementary material Figure Captions Scheme 1: Solid-phase syntheses of oligo 1, 2, 3 Scheme 2: Synthesis of oligo 4 Figures 1, 2, 3, 4: MALDI-TOF spectra of DNA 1, 2, 3 and 4 are given General remarks: The solution of 50% saturated 6-aza-2-thiothymine in acetonitrile and 50% 0.1 M diammoniumcitrate in water was used as a matrix for MALDI-TOF analysis of the DNA conjugates. MALDI-TOF mass spectra were recorded on a Bruker BIFLEX III spectrometer. Figure 5: Fluorescence recovery of Cu(II) quenched 1 by competing ligand 1,2-diaminoethane General remarks: The fluorescence experiments were done on a Varian Eclipse Fluorescence Spectrophotometer with a Hellma 50 µl fluorescence cuvette. The exitation wavelength was λ = 494 nm, spectra were obtained from λ = 500 to 600 nm. Figure 6: Fluorescence of Cu(II) quenched oligo 2 on addition of complementary DNA 1 Figures 7, 8, 9, 10: Fluorimetric titrations of oligo 1, oligo 2, oligo 3 and oligo 4 with Cu(II) General remarks: The fluorescence experiments were done on a Varian Eclipse Fluorescence Spectrophotometer with a Helma 50 µl fluorescence cuvette. The exitation wavelength was λ = 494 nm, spectra were obtained from λ=500 to 600 nm. The pyrene modified oligo 4 was excited at λ = 340 nm and spectra were obtained from λ = 350 to 550 nm. Figures 11, 12: Comparison of optical and fluorescence spectra of oligo 1 and of calcein on titration with Cu(II) General remarks: The UV-Vis absorption measurements were done on a Varian Cary 100 Bio Spectrophotometer. The fluorescence experiments were done on a Varian Eclipse Fluorescence Spectrophotometer. The exitation wavelength was λ = 494 nm, spectra were obtained from λ = 500 to 600 nm. Titration experiments were performed with a 50 µl fluorescence cuvette for oligo 1 and a 1000 µl fluorescence cuvette for Calcein.
Solid-phase syntheses of oligo 1, oligo 2 and oligo 3
O
O
O
O
O
O NH
S
NH(MMT)
NH OC7H13
O
O O
O
N
S
NH
NH OC7H13
PG:
N
HO
N O O
O
Ligand
HO2C
HN
NH3 aq
HO2C N R:
O
N
O NH
NH HOC7H13
C6H12
T APG APG T TPG A PG A T T PG APG PG C G PG PG G A PG T A PG PG A G T T PG PG PG C PGPG G A C A
OH
O
S
R
C6H12
T PG APG A TPG T A A PG T TPG APG PG PG C G PG G A PG T A PG PG A G T T PG PG PG C PG PGG A C A
CCl3COOH
R: HN N
O
NH2
NH OC7H13
PG
A T PG A T PG A G PGPG A PG T A PG A G PG T PG PG T PG C PG PG G A C A
NH
S
C6H12
T APG T A PG TPG CPG G
HOBT/HBTU
O
O
O
O
O
O
O
O
O
T A T A T C G
R HN C6H12 A T A T A G
T A T
C
C AG
A A G T A
O oligo 3 N HN N O oligo 1
Cytidine - benzoyl protected Adenosine - benzoyl protected Guanosine - isobutyryl protected
Scheme 1: Synthesis of oligo 1, 2, 3; the 3´-fluorescein-5´- MMT protected aminolink hairpin-DNA on a CPG solid support was purchased from Metabion, Planegg-Martinsried (Germany). 1-Hydroxybenzotriazole (HOBT) was purchased from Acros Organics and O(Benzotriazol-1-yl)-N,N,N´,N´-tetramethyluronium
hexafluorphosphate
(HBTU)
was
purchased from Fluka. The Ligand 2,2´-Bipyridine-4,4´-dicarboxylic acid was purchased from Sigma-Aldrich and Pyridyl-pyrazolyl carboxylic acid preparation was described earlier[11]. HPLC was performed at room temperature on a Shimadzu liquid chromatograph
equipped with UV-Vis detector and column oven. HPLC was done on a EC 250 x 4.6 mm Nucleosil 300-5 C4. Gradients of CH3CN (solvent B) and 0.1M triethylammoniumacetate in water (solvent A) were used: 1min at 0% B; in 20 min from 0%-30% B; in 6min from 30%90% B; 10min at 90% B.
Synthesis of oligo 4 N HN N O TFA NH OC7H13
TFA
NH(MMT)
NH OC7H13
C6H12
T A A PG T PG TPG A CCl3COOH A T T PG APG G PGPG PG C G A PG T A PG PG A G T T PG PG PG C PGPGG A CA PG
NH2
TFA NH OC7H13
C6H12
T APG PG A T TPG APG HOBT/HBTU A TPG TPG A Ligand PG C G PGPG G A T APGPG PG A G T T PG PG PG C PG PG G A C A
N HN N
O
NH3 aq
NH2 HOC7H13
HPLC Purification
T A T A T C G
HN
A T A T A G
T A T
C
O
C6H12
C AG
O
HN
HN
1) Pyrene NHS Ester HOC7H13 2) HPLC-Purification
A A G T A
C6H12
T A T A T C G
A T A T A G
T A T
C
C AG
oligo 4
PG:
Cytidine - benzoyl protected Adenosine - benzoyl protected
A A G T A
C6H12
T PG APG A T TPG APG A TPG TPG A PG G PG PG C G A PG T A PG PG A G PG T T PG PG C PG PG G A CA
N HN N
HN
Guanosine - isobutyryl protected Scheme 2: Synthesis of oligo 4; the 3´-TFA(Trifluoracetic acid) protected C7-aminolink and 5´- MMT protected C6-aminolink hairpin-DNA on a CPG solid support was purchased from Metabion, Planegg-Martinsried (Germany). 1-Pyrenebutyric acid N-hydroxysuccinimide ester was purchased from Sigma-Aldrich. 1-Hydroxybenzotriazole (HOBT) was purchased from Acros
Organics
and
O-(Benzotriazol-1-yl)-N,N,N´,N´-tetramethyluronium
hexafluorphosphate (HBTU) was purchased from Fluka. The preparation of the ligand Pyridyl-pyrazolyl carboxylic acid was described earlier[11]. HPLC was performed at room temperature on a Shimadzu liquid chromatograph equipped with UV-Vis detector and column oven. HPLC was done on a EC 250 x 4.6 mm Nucleosil 300-5 C4. Gradients of CH3CN (solvent B) and 0.1M triethylammoniumacetate in water (solvent A) were used: 1min at 0% B; in 20 min from 0%-30% B; in 6min from 30%-90% B; 10min at 90% B.
MALDI-mass spectra of oligo 1, oligo2, oligo 3 and oligo 4 r.i. 1.10
1.00
0.90
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
7500
8000
8500
9000
9500
10000 m/z
Figue 1: MALDI Spectrum of oligo 1 Mcalc=8638 (M-H+) ; Mfound=8640
r.i. 1.2
1.0
0.8
0.6
7500
8000
8500
9000
9500
m/z
Figure 2: MALDI Spectrum of oligo 2 Mcalc=8467 (M-H+) ; Mfound=8468
0.100
0.050
0.010 8000
9000
10000
m/z
Figure 3: MALDI Spectrum of oligo 3 Mcalc=8691 (M-H+) ; Mfound=8692
r.i.
1.00
0.90
0.80
0.70
0.60
7600
8100
8600
9100
9600
m/z
Figure 4: MALDI Spectrum of oligo 4 Mcalc=8516 (M-H+); Mfound=8520
Fluorescence recovery of Cu(II) quenched 1 by competing ligand 1,2diaminoethane 1000 1
Fluorescence intensity
800
3
600
400
2
200
0
500
520
540
560
580
600
Wavelength / nm
Figure 5: Fluorescence intensities: 0.1 µM oligo 1 (1), addition of 5 µM Cu(II)SO4 (2), addition of 5 µM 1,2-diaminoethane (3), 10 mM MOPS pH 7, 1 M NaCl, T = 25° We suggest that the quenching process is triggered by intramolecular coordination of a phenolate donor of fluorescein, which is more basic and therefore a better donor than carboxylate [1], to a free coordination site of the (pypz) Cu2+ moiety of 1. This is indicated by 58% recovery of fluorescence when 1,2-diaminoethane (en, 5 µM) is added to the solution containing 5 µM Cu2+ and 0.1 µM 1. Formation of a ternary complex [Cu2+(1)(en)] is expected which compares well to the complex [Cu2+(2,2´-bipyridine)(en)] for which crystallographic [2] data are available: the four in-plane sites of copper are blocked by the two bidentate chelators and not available for coligand coordination. 58 % quenching-inactive ternary complex [Cu2+(1)(en)] and 42 % quenching active [Cu2+(1)] fit a calculated species distribution using published stability data of the en/Cu2+ system [3], a Cu2+(1) formation constant logK = 6.3 (see above), and assuming for the association of 1 and Cu2+(en) a logK value of 5.9, somewhat smaller than the Cu2+ binding constant of 1 [4]. O
O
O
CO2
non-fluorescent
-
N
H2N
NH2
H2 N
Cu N HN
O
N H2
O
N Cu
O-
O
+ N HN
O
CO2
-
fluorescent
Fluorescence quenching by intramolecular fluorescein coordination at Cu2+(1) [1] At pH 7 the nonfluorescent carboxylate monoanion (pKa=4.3) of fluorescein is in equilibrium with the strongly fluorescent carboxylate+phenolate dianion, pKa = 6.7 at 21°C and 70 mM NaCl, S. A. Smith, W. A. Pretorius, Water SA, 2002, 28, 395-402. [2] C. C. Su, Y. L. Lin, S. J. Liu, T. H. Chang, S. L. Wang, L. F. Liao, Polyhedron, 1993, 12, 2687-2696. [3] R. Griesser, H. Sigel, Inorg. Chem., 1970, 9, 1238-1243. The program „Species Ver. 1.2” by L.D. Pettit, Academic Software 1999, was used for calculation of species distribution. [4] This is a reasonable assumption since the association constant for 2,2’-bipyridine and Cu2+(en) is 1.2 logK values smaller than for 2,2’-bipyridine and Cu2+ (logK=8) [3].
Fluorescence of Cu(II) quenched oligo 2 on addition of complementary DNA 1
1000
1 3 2
Fluorescence intensity
800
600
400
200
0 500
520
540
560
580
600
Wavelength / nm
Figure 6: Quenching experiments with oligo 2: 0.1 µM oligo 2 (1), addition of 5 µM Cu(II)SO4 (2), addition of 0.5 µM DNA 1 (3); 10 mM MOPS pH7, 1 M NaCl, T=25° Only 5% quenching was observed for oligo 2 after addition of CuSO4 due to unspecific coordination of Cu2+ Ions to nucleobases.
1000
1000
900
900
800
800
Fluorescence intensity
Fluorescence intensity
Fluorimetric titrations of oligo 1, oligo 2, oligo 3 and oligo 4 with Cu(II)
700 600 500 400 300 200
700 600 500 400 300 200
100
100
0,01
0,1
1
10
0
5
10
15
µM Cu
20
25
30
35
40
µM Cu
Figure 7: Fluorimetric titration (excitation at λ = 494 nm, emission at λ = 522 nm) of oligo 1 (0.1 µM) with CuSO4, logarithmic scale (left), linear scale (right). 10 mM MOPS pH 7, 1 M NaCl, T = 25°
1000
800
Fluorescence intensity
Florescence intensity
1000
600
400
200
0
800
600
400
200
0
0,1
1
10
µM Cu
100
20
40
60
80
100
120
140
µM Cu
Figure 8: Fluorimetric titration (excitation at λ = 494 nm, emission at λ = 522 nm) of oligo 2 (0.1 µM) with CuSO4, logarithmic scale (left), linear scale (right). 10 mM MOPS pH 7, 1 M NaCl, T = 25°
800 800
700
Fluorescence intensity
Fluorescence intensity
700 600 500 400 300 200
600 500 400 300 200 100
100
0
0 0,01
0,1
1
0
10
10
20
30
40
50
µM Cu
µM Cu
Figure 9: Fluorimetric titration (excitation at λ = 494 nm, emission at λ = 522 nm) of oligo 3 (0.1 µM) with CuSO4, logarithmic scale (left), linear scale (right). 10 mM MOPS pH 7, 1 M NaCl, T = 25°
140 140
120
Fluorescence intensity
Fluorescence intensity
120 100 80 60 40
100 80 60 40 20
20 0
0 0,1
1
10
µM Cu
0
10
20
30
40
50
60
70
80
µM Cu
Figure 10: Fluorimetric titration (excitation at λ = 340 nm, emission at λ = 381 nm) of oligo 4 (0.1 µM) with CuSO4, logarithmic scale (left), linear scale (right). 10 mM MOPS pH 7, 1 M NaCl, T = 25°
Fluorimetric and photometric titrations of calcein and oligo 1 with Cu(II) Calcein O
O
(HOCCH2)2NCH3 HO
CH2N(CH2COH)2 O
O
OH
C O
0,425
600
700
0,4
600
0,420
Intensity
400 0,2
Intensity 515 nm
Absorption
Absorption 492 nm
0,415
500
500
0,3
0,410 0,1
0,405
450
500
Wavelength
0,400 0,395 0,390
400
300 200 100
300
0 500
520
540
560
580
600
Wavelength
200
100
0,385 0
10
20
30
40
0
2+
µM Cu
0
10
20
30
µM Cu
Figure 11: top:
40
2+
Structure of Calcein ; bottom left: Copper(II) titration absorption
measurement of Calcein; bottom right: Copper(II) titration fluorescence measurement of Calcein; 10 µM Calcein, 10 mM MOPS pH7, 1 M NaCl, T = 25°C
0,084 900
1000
800
800
0,082 0,06
600
700 0,03
0,076 0,00
0,074
450
500
550
W avelength
0,072
Intensity
0,078
Intensity 522 nm
Absorption
Absorption 494 nm
0,080
600
400
200
500 0
400
500
520
540
560
580
600
Wavelength
300
0,070 200
0,068
100
0,066 0
5
10
15 2+
µM Cu
20
25
30
0
5
10
15
µM Cu
20
25
2+
Figure 12: left : Copper(II) titration absorption measurement; right: Copper(II) titration fluorescence measurement; 5 µM oligo1, 10 mM MOPS pH7, 1 M NaCl, T = 25°C
30
Absorption and fluorescence spectra of calcein (Fig. 7) and oligo 1 (Fig. 8) show similar decrease of absorption and fluorescence after adition of CuSO4. In case of strong Cu2+ binder Calcein, a 1:1 complex is apparently formed quantitatively on addition of 1 equivalent metal ion (= 10 µM). In case of oligo 1, decrease of optical optical absorbance and fluorescence is more sluggish due to weaker Cu(II) binding by the pyridyl-pyrazole chelator.
