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The effect of osmolytes and small molecule on Quadruplex-WC duplex equilibrium: a fluorescence resonance energy transfer study.

ABSTRACT: The structural competition between the G-quadruplex and Watson-Crick duplex has been implicated for the repetitive DNA sequences, but the factors influencing this competitive equilibrium in the natural and pharmacological context need to be elucidated. Using a 21mer 5'-Fluorescein-d[(G3TTA)3G3]-TAMRA-3' as a model system, extensive fluorescence resonance energy transfer analysis was carried out to investigate sensitivity of this equilibrium to osmotic stress and quadruplex selective small molecule. The binding affinities and kinetics involved in the hybridization of quadruplex to its complementary strand in the absence and presence of different concentrations of osmolytes (ethylene glycol and glycerol) and a quadruplex selective ligand (cationic porphyrin-TMPyP4) were determined. The presence of osmolytes and cationic porphyrin decreased the binding affinity of quadruplex to its complementary strand and slowed the kinetics of the reaction by delaying the hybridization process. Our binding data analysis indicates that the presence of either osmolytes or porphyrin increase the amount of quadruplex in the equilibrium. In 100 mM KCl solution, when 30 nM of each of the components, i.e. quadruplex and the complementary strand, were mixed together, the amount of quadruplex present in the system under equilibrium were 17.6, 23.4, 23.1 and 19.6 nM in the absence and presence of 10% ethylene glycol, 10% glycerol and 150 nM TMPyP4, respectively. Fluorescence melting profile of quadruplex in the absence and presence of these perturbants confirm the findings that osmolytes and cationic porphyrin stabilize quadruplex, and thus, shift the equilibrium to quadruplex formation.

SUBMITTER: Kumar N

PROVIDER: S-EPMC1301592 | biostudies-literature | 2005

REPOSITORIES: biostudies-literature

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The effect of osmolytes and small molecule on Quadruplex-WC duplex equilibrium: a fluorescence resonance energy transfer study.

Kumar Niti N Maiti Souvik S

Nucleic acids research 20051130 21

The structural competition between the G-quadruplex and Watson-Crick duplex has been implicated for the repetitive DNA sequences, but the factors influencing this competitive equilibrium in the natural and pharmacological context need to be elucidated. Using a 21mer 5'-Fluorescein-d[(G3TTA)3G3]-TAMRA-3' as a model system, extensive fluorescence resonance energy transfer analysis was carried out to investigate sensitivity of this equilibrium to osmotic stress and quadruplex selective small molecu ...[more]

PMID: 16321964

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Project description:Conjugated polydiacetylene (PDA) possessing stimuli-responsive properties has been intensively investigated for developing efficient sensors. We report here fluorescence resonance energy transfer (FRET) in liposomes synthesized using different molar ratios of dansyl-tagged diacetylene and diacetylene-carboxylic acid monomers. Photopolymerization of diacetylene resulted in cross-linked PDA liposomes. We used steady-state electronic absorption, emission, and fluorescence anisotropy (FA) analysis to characterize the thermal-induced FRET between dansyl fluorophores (donor) and PDA (acceptor). We found that the monomer ratio of acceptor to donor ( R ad) and length of linkers (functional part that connects dansyl fluorophores to the diacetylene group in the monomer) strongly affected FRET. For R ad = 10 000, the acceptor emission intensity was amplified by more than 18 times when the liposome solution was heated from 298 to 338 K. A decrease in R ad resulted in diminished acceptor emission amplification. This was primarily attributed to lower FRET efficiency between donors and acceptors and a higher background signal. We also found that the FRET amplification of PDA emissions after heating the solution was much higher when dansyl was linked to diacetylene through longer and flexible linkers than through shorter linkers. We attributed this to insertion of dansyl in the bilayer of the liposomes, which led to an increased dansyl quantum yield and a higher interaction of multiple acceptors with limited available donors. This was not the case for shorter and more rigid linkers where PDA amplification was much smaller. The present studies aim at enhancing our understanding of FRET between fluorophores and PDA-based conjugated liposomes. Furthermore, receptor tagged onto PDA liposomes can interact with ligands present on proteins, enzymes, and cells, which will produce emission sensing signal. Therefore, using the present approach, there exist opportunities for designing FRET-based highly sensitive and selective chemical and biochemical sensors.

Dataset Information

The effect of osmolytes and small molecule on Quadruplex-WC duplex equilibrium: a fluorescence resonance energy transfer study.

Publications

The effect of osmolytes and small molecule on Quadruplex-WC duplex equilibrium: a fluorescence resonance energy transfer study.

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