TG mismatched base pairs in DNA are responsible for most of the common mutations leading to formation of tumors in humans. TG mismatches are particularly abundant in cells lacking mismatch repair mechanism (MMR). MMR deficiency increases 50-1000-fold spontaneous mutation rates (microsatellite instability MSI). An increase of MSI is observed in hereditary nonpolyposis colon cancer (HNPCC) and in a series of sporadic tumor types. In addition, MMR deficiency can lead to resistance to several chemotherapeutic agents (DNA damaging agent). The aim of the present work is the development and validation of an ESI-MS screening method for the identification of new molecules able to recognize TG mismatched base pairs in DNA. This would help in the synthesis of new chemotherapeutic agents particularly effective on MMR deficient cell lines. ESI MS spectra were recorded on a Q-Tof Ultima mass spectrometer (Waters, Manchester UK) with negative ion detection by continuous infusion at 5 L/min. Experiments performed on mixture of ligands, whereas resolving power above 10,000 are required, were carried out on a LTQ-Orbitrap (Thermo Fisher Scientific, Bremen, Germany). Different self complementary DNA sequence were synthesized. The duplex fully matched sequence (HFM) was formed from the single strand d(GAACCGGTTC) and was used as control. A duplex DNA structure incorporating two T:G mismatched base pairs (HDM) were obtained from the sequence d(GAACTGGTTC). Since a tandem T:G/G:T mismatch is seldom found in vivo, an hairpin DNA sequence (HSM) d(GAACTGGTCCTCTGACTGGTTC) bearing a single T:G mismatch was prepared. A poliAT DNA duplex (A5TG) d(ACCTTTTTGATGT) was also tested. Solutions of oligonucleotides were heated to 90°C for 5 min and cooled to room temperature overnight to form duplexes. Different papers confirm that the duplex DNA structure is maintained in the gas phase and the Watson-Crick base pairing is preserved [1,2]. In our study the formation of duplexes was confirmed by ESI-MS and dissociation curves were obtained for the different oligonucleotides confirming a decreasing stability of the duplexes when TG mismatches are present. This hairpin DNA was used to set up the method for studying the complexes formed with minor groove binders and intercalators (doxorubicin). The association constants (Kas) were directly determined from the MS spectrum following a procedure developed by Rosu et all. [3]. Different standard compounds were tested against the three DNA targets at 5 and 25 M (HFM; HSM; A5TG). The amount of bound ligand was used to determine the selectivity of a binder among the different DNA sequences [4]. As expected the minor groove binders (Distamycin A, H33258, H33342, DAPI) show an evident selectivity for the poliAT sequence A5TG and a poor affinity for the DNA sequence bearing a single mismatch (HSM). Intercalator doxorubicin did not show any selectivity between the Hairpin Single Mismatch (HSM) and A5TG. Different Lexitropsin derivatives, able to recognize single and double mismatches, were designed, synthesized and tested with this procedure. NMS-077 reveals a significant selectivity for the Hairpin Single mismatch and the ligand binds with positive cooperativity Ka1<4Ka2. [1] Schnier, PD; Klassen, JS; Strittmatter, EF; Williams, ER; J. Am. Chem. Soc. 120 (1998) 9605 [2] Gabelica, V; De Pauw, E; Int. J. Mass Spectrom 219 (2002) 151 [3] Rosu, F; Gabelica, V; Hossier, C; De Pauw, E; Nucleic Acid Res 30/16 (2002) e82 [4] Rosu, F; De Pauw , E; Gabelica, V; Biochimie 90(7) (2008) 1074

MOLECULAR RECOGNITION OF T:G MISMATCHED BASE PAIRS IN DNA STUDIED BY ELECTROSPRAY IONIZATION MASS SPECTROMETRY / F. Riccardi Sirtori, R. D’Alessio, J. Malyszko, G. Aldini, M. Colombo. ((Intervento presentato al 5. convegno MS-Pharmaday tenutosi a Verona nel 2008.

MOLECULAR RECOGNITION OF T:G MISMATCHED BASE PAIRS IN DNA STUDIED BY ELECTROSPRAY IONIZATION MASS SPECTROMETRY

F. Riccardi Sirtori
Primo
;
G. Aldini
Penultimo
;
2008

Abstract

TG mismatched base pairs in DNA are responsible for most of the common mutations leading to formation of tumors in humans. TG mismatches are particularly abundant in cells lacking mismatch repair mechanism (MMR). MMR deficiency increases 50-1000-fold spontaneous mutation rates (microsatellite instability MSI). An increase of MSI is observed in hereditary nonpolyposis colon cancer (HNPCC) and in a series of sporadic tumor types. In addition, MMR deficiency can lead to resistance to several chemotherapeutic agents (DNA damaging agent). The aim of the present work is the development and validation of an ESI-MS screening method for the identification of new molecules able to recognize TG mismatched base pairs in DNA. This would help in the synthesis of new chemotherapeutic agents particularly effective on MMR deficient cell lines. ESI MS spectra were recorded on a Q-Tof Ultima mass spectrometer (Waters, Manchester UK) with negative ion detection by continuous infusion at 5 L/min. Experiments performed on mixture of ligands, whereas resolving power above 10,000 are required, were carried out on a LTQ-Orbitrap (Thermo Fisher Scientific, Bremen, Germany). Different self complementary DNA sequence were synthesized. The duplex fully matched sequence (HFM) was formed from the single strand d(GAACCGGTTC) and was used as control. A duplex DNA structure incorporating two T:G mismatched base pairs (HDM) were obtained from the sequence d(GAACTGGTTC). Since a tandem T:G/G:T mismatch is seldom found in vivo, an hairpin DNA sequence (HSM) d(GAACTGGTCCTCTGACTGGTTC) bearing a single T:G mismatch was prepared. A poliAT DNA duplex (A5TG) d(ACCTTTTTGATGT) was also tested. Solutions of oligonucleotides were heated to 90°C for 5 min and cooled to room temperature overnight to form duplexes. Different papers confirm that the duplex DNA structure is maintained in the gas phase and the Watson-Crick base pairing is preserved [1,2]. In our study the formation of duplexes was confirmed by ESI-MS and dissociation curves were obtained for the different oligonucleotides confirming a decreasing stability of the duplexes when TG mismatches are present. This hairpin DNA was used to set up the method for studying the complexes formed with minor groove binders and intercalators (doxorubicin). The association constants (Kas) were directly determined from the MS spectrum following a procedure developed by Rosu et all. [3]. Different standard compounds were tested against the three DNA targets at 5 and 25 M (HFM; HSM; A5TG). The amount of bound ligand was used to determine the selectivity of a binder among the different DNA sequences [4]. As expected the minor groove binders (Distamycin A, H33258, H33342, DAPI) show an evident selectivity for the poliAT sequence A5TG and a poor affinity for the DNA sequence bearing a single mismatch (HSM). Intercalator doxorubicin did not show any selectivity between the Hairpin Single Mismatch (HSM) and A5TG. Different Lexitropsin derivatives, able to recognize single and double mismatches, were designed, synthesized and tested with this procedure. NMS-077 reveals a significant selectivity for the Hairpin Single mismatch and the ligand binds with positive cooperativity Ka1<4Ka2. [1] Schnier, PD; Klassen, JS; Strittmatter, EF; Williams, ER; J. Am. Chem. Soc. 120 (1998) 9605 [2] Gabelica, V; De Pauw, E; Int. J. Mass Spectrom 219 (2002) 151 [3] Rosu, F; Gabelica, V; Hossier, C; De Pauw, E; Nucleic Acid Res 30/16 (2002) e82 [4] Rosu, F; De Pauw , E; Gabelica, V; Biochimie 90(7) (2008) 1074
28-ott-2008
mass spectrometry ; non covalent adduct; ESI
Settore CHIM/08 - Chimica Farmaceutica
MOLECULAR RECOGNITION OF T:G MISMATCHED BASE PAIRS IN DNA STUDIED BY ELECTROSPRAY IONIZATION MASS SPECTROMETRY / F. Riccardi Sirtori, R. D’Alessio, J. Malyszko, G. Aldini, M. Colombo. ((Intervento presentato al 5. convegno MS-Pharmaday tenutosi a Verona nel 2008.
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