1. Study of the interaction between the porphyrin TmPyP4 and the Thrombin Binding Aptamer (TBA) G-quadruplex Miquel del Toro 1, Ramon Eritja 2, Raimundo Gargallo 1, Joaquim Jaumot 1 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 C TmPyP4 :C oligo values.  Spectroscopic data analysis: Experimental melting data have been analyzed using the multivariate resolution method MCR-ALS 3. The data from acid base titration and mole-ratio studies have been analyzed using hard-modelling Equispec 4.  RP-HPLC-DAD:  Experimental conditions:  Temperature: 30ºC  Oligonucleotide concentration: 40  M  TmPyP4 concentrations: 0, 20, 40, 80, 120  M  Buffer A: TEAA 0,1M + KCl 0,1M  Buffer B: Acetonitrile  Flow: 1ml/min  Injection volume: 20  l  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 structure 1. 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 loop 2. guanine1 guanine2 guanine3 guanine4 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 TmPyP4 Melting (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 Complex Melting ( 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 MeltingAcid 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 422nmOverlay chromatograms of the mixtures at 422nm Wavelength (nm) Ellipticity (mD) Absorbance (AU) Molar ellipticity (l/mol·cm) Absorptivity (l/mol·cm) Mole-ratio Concentration (M) Residuals