Conformational Dynamics of Guanine Residues Within the Human Telomeric G-quadruplex
Item
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Title
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Conformational Dynamics of Guanine Residues Within the Human Telomeric G-quadruplex
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Identifier
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d_2009_2013:9c0669f259a2:11443
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identifier
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11884
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Creator
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Liu, Susan Y.,
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Contributor
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Lesley Davenport
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Date
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2012
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biophysics | Biochemistry | conformation | DNA | fluorescence | guanine | human telomere | quadruplex
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Abstract
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In our studies, we have focused on the conformational dynamics of the human telomeric HT4 intramolecular G-quadruplex in solution, specifically the role each guanine residue plays in the quadruplex folding process, through investigating their contributions to the global quadruplex stabilities and the local environmental changes around single guanine residue. To accomplish this aim, we have substituted single guanine residues with the fluorescence analog---6MI at guanine positions (G1, G2, G4, G5, G7, G9, G11 and G12) along the HT4 oligonucleotide. Serving as either both a base mutation and a sensitive probe for local environment changes, the 6MI-substituted oligonucleotides have provided insights into the quadruplex dynamics under physiological conditions.;We have confirmed the formation of G-quadruplex conformations in the presence of 100mM KCl or NaCl by all 6MI-substituted sequences using thermal difference spectroscopy. In general, the global stabilities of the 6MI-labeled sequences (as judged by mid-point of thermal UV-melting profiles and DeltaG folding) are weaker than for the parent HT4, but to varying extents, suggesting that different guanine positions of the quadruplex do indeed play distinct roles in the formation and stabilization of the G-quadruplex.;Studies of local environmental effects around individual guanine positions have been explored using the sensitivity of 6MI fluorescence. With quadruplex folding promoted through either decreasing temperatures or addition of K +, a decrease in fluorescence intensity generally corresponds to formation of quadruplex. The degree of fluorescence quenching however, varied for different 6MI-labeled sequences, suggesting different extents of base stacking interactions associated with quadruplex folding for each guanine residue. Analyses of fluorescence data from thermal folding or K+ titration studies suggested a cooperative quadruplex folding pathway, with base nucleation starting at relatively low K+-ion concentrations (<20mM).;Interestingly, the G1 labeled position (close to the 5'-end) shows abnormal behavior compared with other substituted positions on folding, with a fluorescence intensity enhancement and spectral shifts to longer wavelength. Our data suggests the flexibility of G1 on the 5'-end of the sequence in the folded quadruplex conformation with possible strand fraying and H-bonding interactions, over base stacking interactions, as predicted for all other guanine positions.;Time-resolved fluorescence studies were performed to address possible mechanisms for the observed steady-state fluorescence quenching with quadruplex folding. Fluorescence intensity decay profiles were best fit using three decay components for all 6MI-labeled sequences. Although static quenching is evident, presumably arising from base stacking interactions, the observed intensity quenching is dominated by an ultrafast quasi-static self-quenching deactivation route (QSSQ). This effect is greatest for 6MI positions G5 and G11 sandwiched by two guanines, and lowest for G1 near the 5'-end. On K+-initiated folding, an increase in QSSQ is detected, suggesting that the additional fluorescence intensity quenching may arise from additional (sub-picosecond) electron transfer events or base stacking interactions around the 6MI probe.;Fluorescence decay-associated spectra (DAS), which associate lifetimes (or decay rates) with fluorescence spectral envelopes, can provide insights into the origins of the heterogeneous fluorescence decay observed for the 6MI-labeled oligonucleotides. DAS revealed that the longer wavelength spectral shifts observed for G1 with quadruplex folding are associated with the longest fluorescence decay time tau1, and appear to originate from enhanced solvent interactions around the G1 fluorophore.;Environmental heterogeneity has been further examined using single value decomposition (SVD) analyses for the absorbance and fluorescence thermal folding profiles. We have extracted at least four components for the unlabeled HT 4 sequence and at least three for the fluorescence melting profiles of the 6MI-labeled sequences. Our data suggest that the folding pathway for the quadruplex formation process involves intermediate states. (Abstract shortened by UMI.).
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Type
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dissertation
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Source
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2009_2013.csv
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degree
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Ph.D.
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Program
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Biochemistry
