- Email: [email protected]
P-B1-23 DNA strands complementary in parallel orientation, form an antiparallel duplex at neutral pH with A-C, G-T and T-C mismatched base pairs
Bl Structure and dynamics P-Bl-21 DNA HYDRATION
P-Bl-22 DYNAMICS
BY NhlRD
HALLE B. DENISOV VD. VENU K. CARLSTROM Department of Chemistry. Lund University, Sweden
A SPECTROSCOPIC APPROACH OF ETHIDIUM BROMIDE IN LIVING
G
CELLS
VIGNY P, FAVARD C, PAGER J, LOCKER D
Water is essential for maintaining the double helix conformation of DNA and for its specific interactions with proteins and drugs. We report here a study of the dynamics of the highly ordered water molecules previously identified in AT-rich regions of the minor groove. Methods: Synthetic d-(CGCGAATTCGCG)2 in aqueous solution was studied by NMRD (Nuclear Magnetic Relaxation Dispersion), using all water isotopes (IH, ?H, 170). Nuclear Overhausereffects between water and DNA were also studied. Results: The NMRD technique yields direct dynamic information in the frequency domain. The mean residence time of 5 highly ordered water molecules could thus be determined as 1.5 f 0.5 ns at 4°C. Further, a comparison of the 2H and 170 NMRD profiles showed that these waters undergo a subnanosecond180” flip motion. Conclusions: The internal motion and mean residence time of 5 highly ordered water molecules in a DNA duplex have been quantified. On the basis of (i) previous X-ray crystal and NMR solution studies and (ii) previous NMRD studies of small proteins, the long-lived water molecules seen here can be confidently assigned to the narrow AT-rich part of the minor groove.
Centre
P-Bl-23
P-Bl-24 NMR CHARACTERISATION OF A DNA TRIPLEX FORMED BY HOMO-PURINE AND HOMO-PYRIMEDINIf STRANDS AT 1: 1 MOLAR RATIO AND ACIDIC pH
Purpose:
Mokculaire
CNRS. Orl&ns
(F),
: The molecular interactions of Ethidium Bromide (EtB) have been checked in situ as a model for vital intercalating drugs. Methods : An Argon laser confocal microspectrofluorometer allows ponctual spectral analysis and imaging. Epithelial cells of excised Drosophila salivary glands, stained with 10” M EtB, have been excited at 514 nm with a = 20 pW power in situ. Cell viability has been checked at the end of experiments. Results : After 10 min staining, EtB has been spectrally detected throughout the living cells. The spectralparametersand kinetics have been measured and analyzed in the cytoplasm, the nucleusand the nucleolus. The emission spectra (550 to 800 nm) have been also deconvoluted in terms of f?ee and bound Et and used in linear imaging of the cells. Bound Et only could be detected in the cytoplasm ; in the nucleus, free Et was estimated to reach up to more than half of the total nuclear Et concentration. Conclusions : In a living system, EtB doesn’t behave as a specific dye for nucleic acids, but as a sensitiveindicator of the cell functional state. Pmpose
DNA STRANDS COMPLtMENTARY IN PARALLEL ORIENTATION, FQRM AN ANTIPARALLEL DUPLEX AT NEUTRAL pH WITH A-C, G-T AND T-C MISMATCHED BASE PAIRS.
BHAUMIK SR,’ CHARY KVR,’ GOVIL G,’ LIU K,* MILES HT.’
CHARY KVR,’ BHAUMIK SR,’ GOVIL G,’ LIU K,ZMILES HT. * ‘Tata Institute of Fundamental Research, Mumbai, *National Institutes of Health, Bethesda (USA)
de Biophysique
‘Tata Institute of Fundamental Research, Murnbai, 2National Institutes of Health, Bethesda (USA)
(IN )
(IN)
Purpose: Characterisationof the complex
Homo-purine (d-TGAGGAAAGAAG GT) and homo-pyrimidine (d-CTCCTTTCTTC C) oligomers have been designed to look for the structural feasibility of parallel stranded duplex at neutral pH.
Methods:
2D NMR experiments have been carried out on the complex in aqueoussolution.
Results & Conclusions:
Purpose:
formed at 1: 1 molar ratio of homo-purine (dTGAGGAAAGAAGGT) and homo-pyrimidine (6CTCCTTTCTTCC) oligomers at acidic pH. 2D NMR experimentshave been carried out on the complex in aqueous solution.
Methods:
NMR results provide the first structural evidence for the pH driven disproportionation of the oligonucleotide sample, into an intermolecular DNA triplex and a single stranded oligopurine. This study provides some insight into the interactions stabilizing the basetriplets, which include C+.G-Tand T.A-C. This mode of recognition offers new possibilities for the targeting of mismatched sequencesby oligonucleotidedirectedtriple-helix formation.
Results and Conclusions: NMR resultsprovide the evidencefor the formation of antiparallel duplex with three mismatched base pairs, namely G-T, A-C and T-C. All the three mismatced basepairs form two hydrogen bonds each. The adenosinein A-C mismatch is protonated at Nl, even at neutral pH. The study shows that the parallel duplex is lessstablethan the antiparallel duplex in spite of its perfect complementarity. 74
P-Bl-22 DYNAMICS
BY NhlRD
HALLE B. DENISOV VD. VENU K. CARLSTROM Department of Chemistry. Lund University, Sweden
A SPECTROSCOPIC APPROACH OF ETHIDIUM BROMIDE IN LIVING
G
CELLS
VIGNY P, FAVARD C, PAGER J, LOCKER D
Water is essential for maintaining the double helix conformation of DNA and for its specific interactions with proteins and drugs. We report here a study of the dynamics of the highly ordered water molecules previously identified in AT-rich regions of the minor groove. Methods: Synthetic d-(CGCGAATTCGCG)2 in aqueous solution was studied by NMRD (Nuclear Magnetic Relaxation Dispersion), using all water isotopes (IH, ?H, 170). Nuclear Overhausereffects between water and DNA were also studied. Results: The NMRD technique yields direct dynamic information in the frequency domain. The mean residence time of 5 highly ordered water molecules could thus be determined as 1.5 f 0.5 ns at 4°C. Further, a comparison of the 2H and 170 NMRD profiles showed that these waters undergo a subnanosecond180” flip motion. Conclusions: The internal motion and mean residence time of 5 highly ordered water molecules in a DNA duplex have been quantified. On the basis of (i) previous X-ray crystal and NMR solution studies and (ii) previous NMRD studies of small proteins, the long-lived water molecules seen here can be confidently assigned to the narrow AT-rich part of the minor groove.
Centre
P-Bl-23
P-Bl-24 NMR CHARACTERISATION OF A DNA TRIPLEX FORMED BY HOMO-PURINE AND HOMO-PYRIMEDINIf STRANDS AT 1: 1 MOLAR RATIO AND ACIDIC pH
Purpose:
Mokculaire
CNRS. Orl&ns
(F),
: The molecular interactions of Ethidium Bromide (EtB) have been checked in situ as a model for vital intercalating drugs. Methods : An Argon laser confocal microspectrofluorometer allows ponctual spectral analysis and imaging. Epithelial cells of excised Drosophila salivary glands, stained with 10” M EtB, have been excited at 514 nm with a = 20 pW power in situ. Cell viability has been checked at the end of experiments. Results : After 10 min staining, EtB has been spectrally detected throughout the living cells. The spectralparametersand kinetics have been measured and analyzed in the cytoplasm, the nucleusand the nucleolus. The emission spectra (550 to 800 nm) have been also deconvoluted in terms of f?ee and bound Et and used in linear imaging of the cells. Bound Et only could be detected in the cytoplasm ; in the nucleus, free Et was estimated to reach up to more than half of the total nuclear Et concentration. Conclusions : In a living system, EtB doesn’t behave as a specific dye for nucleic acids, but as a sensitiveindicator of the cell functional state. Pmpose
DNA STRANDS COMPLtMENTARY IN PARALLEL ORIENTATION, FQRM AN ANTIPARALLEL DUPLEX AT NEUTRAL pH WITH A-C, G-T AND T-C MISMATCHED BASE PAIRS.
BHAUMIK SR,’ CHARY KVR,’ GOVIL G,’ LIU K,* MILES HT.’
CHARY KVR,’ BHAUMIK SR,’ GOVIL G,’ LIU K,ZMILES HT. * ‘Tata Institute of Fundamental Research, Mumbai, *National Institutes of Health, Bethesda (USA)
de Biophysique
‘Tata Institute of Fundamental Research, Murnbai, 2National Institutes of Health, Bethesda (USA)
(IN )
(IN)
Purpose: Characterisationof the complex
Homo-purine (d-TGAGGAAAGAAG GT) and homo-pyrimidine (d-CTCCTTTCTTC C) oligomers have been designed to look for the structural feasibility of parallel stranded duplex at neutral pH.
Methods:
2D NMR experiments have been carried out on the complex in aqueoussolution.
Results & Conclusions:
Purpose:
formed at 1: 1 molar ratio of homo-purine (dTGAGGAAAGAAGGT) and homo-pyrimidine (6CTCCTTTCTTCC) oligomers at acidic pH. 2D NMR experimentshave been carried out on the complex in aqueous solution.
Methods:
NMR results provide the first structural evidence for the pH driven disproportionation of the oligonucleotide sample, into an intermolecular DNA triplex and a single stranded oligopurine. This study provides some insight into the interactions stabilizing the basetriplets, which include C+.G-Tand T.A-C. This mode of recognition offers new possibilities for the targeting of mismatched sequencesby oligonucleotidedirectedtriple-helix formation.
Results and Conclusions: NMR resultsprovide the evidencefor the formation of antiparallel duplex with three mismatched base pairs, namely G-T, A-C and T-C. All the three mismatced basepairs form two hydrogen bonds each. The adenosinein A-C mismatch is protonated at Nl, even at neutral pH. The study shows that the parallel duplex is lessstablethan the antiparallel duplex in spite of its perfect complementarity. 74