250 300 350 400 4500

0.1

0.2

0.3

0.4

Wavelength (nm)

Abs

orba

nce

(AU

)

250 300 350-2

0

2

4

6

8

Wavelength (nm)

Elli

ptic

ity (

mD

)

250 300 350 400 4500

5

10

15x 10

4

Wavelength (nm)

Abs

ortiv

ity (

l/mol

·cm

)

220 240 260 280 300 320-1

0

1

2

3x 10

6

Wavelength (nm)

Mol

ar E

llipt

icity

(l/m

ol·c

m)

20 40 60 800

0.5

1

1.5

2

2.5x 10

-6

Temperature (ºC)

Con

cent

ratio

n (M

)

a b

c d e

Study of the interaction between the porphyrin TmPyP4 and the Thrombin Binding Aptamer (TBA) G-quadruplex

Miquel del Toro1, Ramon Eritja2, Raimundo Gargallo1, Joaquim Jaumot1

1. Department of Analytical Chemistry, University of Barcelona, Diagonal 647, E-08028 Barcelona, Spain

2. Institut de Biologia Molecular de Barcelona, CSIC, Jordi Girona 18-26, 08034 Barcelona, Spain 

EXPERIMENTAL Spectrophotometric techniques:

UV-VIS molecular absorption and circular dichroism

Melting experiments: spectra between 220 and 700 nm have been recorded at 1 ºC step between 20 and 80 ºC.

Acid base titration: Spectra between 220 and 700 nm have been recorded in the pH range 2 - 11.

Mole-ratio studies: Spectra between 220 and 700 nm have been recorded at increasing CTmPyP4:Coligo values.

Spectroscopic data analysis:

Experimental melting data have been analyzed using the multivariate resolution method MCR-ALS3. The data from acid base titration and mole-ratio studies have been analyzed using hard-modelling Equispec4.

RP-HPLC-DAD:

Experimental conditions:

Temperature: 30ºC

Oligonucleotide concentration: 40M

TmPyP4 concentrations: 0, 20, 40, 80, 120M

Buffer A: TEAA 0,1M + KCl 0,1M

Buffer B: Acetonitrile

Flow: 1ml/min

Injection volume: 20l

Column: Clarity 3u Oligo-RP 50 x 4,6 mm

CONCLUSIONS

REFERENCES

The acid base and melting behavior of TBA have been studied. Upon addition of the porphyrin TmPyP4, 1:1 interaction complex is formed with weak stability. The results seem to point out to an interaction near the loops.

INTRODUCTION

The G-quadruplex structure has been found in telomers, entity related with the cycle of life in the cell, and in some oncogens and aptamers. These parts of the genome have been suggested as potentials targets for anti-cancer therapies. For this reason, nowadays, there is an increasing interest in the development of drugs which could stabilize this structure1.

In this work, the interaction between the TBA G-quadruplex and the TmPyP4 porphyrin has been studied using RP-HPLC-DAD and two different spectrophotometric techniques (UV-VIS molecular absorption and circular dichroism).

G-QUADRUPLEX AND TmPyP4

Oligonucleotides with a guanine rich sequence are able to form a secondary structure known as G-quadruplex. This structure is characterized by the association of four guanines in a cyclic hydrogen-bonding arrangement.

An intramolecular and anti-parallel G-quadruplex structure results from the Thrombin-Binding Aptamer sequence, TBA (5’-GGT TGG TGT GGT TGG-3’).

Several interaction mechanisms between TmPyP4 and TBA have been proposed. One of them shows the porphyrin inserted between the two planes defined by the G-tetrads. Another mechanism shows the drug interacting through the loop2.

guanine1 guanine2

guanine3guanine4

1- Han, H.; Hurley, L. H. G-quadruplex DNA : a potential target for anti - cancer drug design. Trends in Pharmacological Sciences (2000), 21(4), 136-142

2- Haq, Ihtshamul; Trent, John O.; Chowdhry, Babur Z.; Jenkins, Terence C. Intercalative G-Tetraplex Stabilization of Telomeric DNA by a Cationic Porphyrin. Journal of the American Chemical Society (1999), 121(9), 1768-1779

3- Jaumot, Joaquim; Gargallo, Raimundo; de Juan, Anna; Tauler, Roma. A graphical user-friendly interface for MCR - ALS : a new tool for multivariate curve resolution in MATLAB. Chemometrics and Intelligent Laboratory Systems (2005), 76(1), 101-110

4- Dyson, Raylene M.; Kaderli, Susan; Lawrance, Geoffrey A.; Maeder, Marcel; Zuberbuhler, Andreas D. Second order global analysis: the evaluation of series of spectrophotometric titrations for improved determination of equilibrium constants. Analytica Chimica Acta (1997), 353(2-3), 381-393.

ACKNOWLEDGMENT

This research was supported be the Spanish Ministerio de Educación y Ciencia (CTQ2006-15052-C02-01 and BFU2004-02048/BMC) .

gradient

MOLECULAR ABSORPTION AND CIRCULAR DICHROISM RESULTS

TmPyP4Melting

(a) Absorbance data. (b) Pure spectra considering two species. (c) Solved concentration profiles. (d) Residuals.

A little variation in the absorbance is observed upon temperature increase

Acid base titration

pKa = 6,2 0,2 pKa = 1,8 0,7

(a) Absorbance data. (b) Pure spectra considering three species. (c) solved concentration profiles (d) Residuals.

pKa = 10,5 0,1

ComplexMelting

(a) Absorbance data. (b) Pure spectra considering four species. (c) Solved concentration profile. (f) Residuals.

Mole- ratio experiment

(a) Ellipticity data (b) Absorbance data. (c) Molar ellipticity pure spectra considering three species. (d) Absorptivity pure spectra considering three species. (e) Solved concentration profiles. (f) Residuals. ( TMPyP4 TBA interaction).

TBA:TmPyP4 complex is formed with a log = 5,7 0,1.

TBA

(a) Absorbance data. (b) Ellipticity data. (c) Pure molecular absorption spectra considering two species. (d) Pure circular dichroism soectra considerin two species. (e) Solved concentration profiles. ( G-Quadruplex, single strand).

Melting temperature: 48 1ºC

Melting Acid base titration

(a) Absorbance data. (b) Pure spectra considering two species. (c) Solved concentration profiles. (d) Residuals. ( bases protonated, bases deprotonated).

RP-HPLC-DAD RESULTS

Mole-ratio studies between TBA and TmPyP4

The oligonucleotide doesn’t show signal at 422nm

A new peak, related with the complex, appears upon addition of TmPyP4

Free TmPyP4 sample shows two peaks.

Chromatograms of the mixtures at 422nm Overlay chromatograms of the mixtures at 422nm

N

NH

N

NH

N +

N +N +

N +CH

3

CH3

CH3

CH3

0

10

20

30

40

50

60

70

0 2,5 5 7,5 8,5 9,5 10,5 13

Analysis time (min)

% A

CN

300 400 500 600 7000

0.2

0.4

0.6

0.8

Wavelength (nm)

Abs

orba

nce

(AU

)

300 400 500 600 7000

0.5

1

1.5

2

2.5x 10

5

Wavelength (nm)

Abs

orpt

ivity

(l/m

ol·c

m)

4 6 8 100

1

2

3

x 10-6

pH

Con

cent

ratio

n (M

)

4 6 8 10-0.02

-0.01

0

0.01

0.02

pH

Res

idua

ls

a b

c d

300 400 500 600 7000

0.2

0.4

0.6

0.8

Wavelength (nm)

Abs

orba

nce

(AU

)

300 400 500 600 7000

5

10

x 104

Wavelength (nm)

Abs

orpt

ivity

(l/m

ol·c

m)

20 40 60 800

2

4

6

8x 10

-6

Temperature (ºC)

Con

cent

ratio

n (M

)

20 40 60 80-0.01

-0.005

0

0.005

0.01

0.015

Temperature (ºC)R

esid

uals

a b

c d

-2 0 2 4 6 8 10 12 14 16 18 20-50

0

50

100

150

200

250

300

350

Retention time (min)

Abs

orba

nce

(mA

U)

200 300 400 500 6000

50

100

150

200

Wavelength (nm)

Abs

orba

nce

(mA

U)

420 425 430

120

140

160

TBA spectrumFree TmPyP4 spectrum, peak 3,8minComplex spectrum, peak 4,4minTBA

1:21:12:13:1TmPyP4

0 2 4 6 8 10 120

200

400

0 2 4 6 8 10 120

200

400

0 2 4 6 8 10 120

200

400

0 2 4 6 8 10 120

200

400

0 2 4 6 8 10 120

200

400

0 2 4 6 8 10 120

200

400

Retention time (min)

Abs

orba

nce

(mA

U)

oligo 4e-5M

1TmPyP4:2 TBA

1:1

2:1

3:1

tmpyp 8e-5M

250 300 350 400 450 500 5500

0.5

1

220 240 260 280 300 320

0

5

10

0 0.5 1 1.5 20

1

2

3

4x 10

-6

0 0.5 1 1.5 2-1

-0.5

0

0.5

1

220 240 260 280 300 320-1

0

1

2

3x 10

6

250 300 350 400 450 500 5500

1

2

3

x 106

a

b

c

d

e f

Wavelength (nm) Wavelength (nm)

Elli

ptic

ity (

mD

)A

bsor

banc

e (A

U) M

olar

elli

ptic

ity

(l/m

ol·c

m)

Abs

orpt

ivity

(l/

mol

·cm

)

Mole-ratio Mole-ratio

Con

cent

ratio

n (M

)

Res

idua

ls

250 300 3500

0.1

0.2

0.3

0.4

Wavelength (nm)

Abs

orba

nce

(AU

)

250 300 3500

5

10

15x 10

4

Wavelength (nm)

Abs

orpt

ivity

(l/m

ol·c

m)

7 8 9 10 110

1

2

3x 10

-6

pH

Con

cent

ratio

n (M

)

7 8 9 10 11-6

-4

-2

0

2

4

x 10-3

pH

Res

idua

ls

a

c

b

d