Biomedical subjects

Erin Shammel Baker

Publications and source records attributed to Erin Shammel Baker.

7 recordsLinked to original sources

PubMed2006

PNA/dsDNA complexes: site specific binding and dsDNA biosensor applications.

The ability of peptide nucleic acids (PNA) to form specific higher-order (i.e., three- and four-stranded) complexes with DNA makes it an ideal structural probe for designing strand-specific dsDNA biosensors. Higher-order complexes are formed between a dye-labeled charge-neutral PNA probe and complementary dsDNA. Addition of a light-harvesting cationic conjugated polymer (CCP) yields supramolecular structures held together by electrostatic forces that incorporate the CCP and the dye-labeled PNA/DNA complexes. Optimization of optical properties allows for excitation of the CCP and subsequent fluorescence resonance energy transfer (FRET) to the PNA-bound dye. In the case of noncomplementary dsDNA, complexation between the probe and target does not occur, and dye emission is weak. The binding between PNA and noncomplementary and complementary dsDNA was examined by several methods. Gel electrophoresis confirms specificity of binding and the formation of higher-order complexes. Nano-electrospray mass spectrometry gives insight into the stoichiometric composition, including PNA/DNA, PNA(2)/DNA, PNA/DNA(2), and PNA(2)/DNA(2) complexes. Finally, structural characteristics and binding-site specificity were examined using ion mobility mass spectrometry in conjunction with molecular dynamics. These results give possible conformations for each of the higher-order complexes formed and show exclusive binding of PNA to the complementary stretch of DNA for all PNA/DNA complexes. Overall, the capability and specificity of binding indicates that the CCP/PNA assay is a feasible detection method for dsDNA and eliminates the need for thermal denaturing steps typically required for DNA hybridization probe assays.

Biosensing Techniques↗

PubMed2006

Cyclo[n]pyrroles: size and site-specific binding to G-quadruplexes.

Inhibiting the enzyme telomerase by stabilizing the G-quadruplex has potential in anticancer drug design. Diprotonated cyclo[n]pyrroles represent a set of expanded porphyrin analogues with structures similar to that of telomestatin, a natural product known to bind to and stabilize G-quadruplexes. As a first step toward testing whether cyclo[n]pyrroles display a similar function, a series of diprotonated cyclo[n]pyrroles (where n = 6, 7, and 8) was each added to the human telomere repeat sequence d(T(2)AG(3))(4) and examined with mass spectrometry, ion mobility, and molecular dynamics calculations. Nano-ESI-MS indicated that the smaller the cyclo[n]pyrrole, the more strongly it binds to the telomeric sequence. It was also found that cyclo[6]pyrrole bound to d(T(2)AG(3))(4) better than octaethylporphyrin, a finding rationalized by cyclo[6]pyrrole having a 2+ charge, while octaethylporphyrin bears no charge. Ion mobility measurements were used to measure the collision cross section of each d(T(2)AG(3))(4)/cyclo[n]pyrrole complex. Only one peak was observed in the arrival time distributions for all complexes, and the experimental cross sections indicated that only structures with d(T(2)AG(3))(4) in an antiparallel G-quadruplex arrangement and each cyclo[n]pyrrole externally stacked below the G-quartets occur under these experimental conditions. When the cyclo[n]pyrroles were intercalated or nonspecifically bound to the quadruplex, or if conformations different than antiparallel were considered for d(T(2)AG(3))(4), the theoretical cross sections did not match experiment. On this basis, it is inferred that (1) external stacking represents the dominant binding mode for the interaction of cyclo[n]pyrroles with d(T(2)AG(3))(4) and (2) the overall size and charge of the cyclo[n]pyrroles play important roles in defining the binding strength.

Binding Sites↗

PubMed2005

Probing shapes of bichromophoric metal-organic complexes using ion mobility mass spectrometry.

Ion mobility mass spectrometry (IM-MS) was used to probe the structures of several metal complexes carrying pendant chromophores. The three complexes investigated were the copper(II) complex Cu(DAC)2+ (DAC = 1,8-bis(9-methylanthracyl)cyclam, cyclam = 1,4,8,11-tetraazacyclotetradecane), the N-nitrosylated ligand DAC-NO, and the Roussin's red salt ester (mu-S,mu-S')-protoporphyrin-IX-bis(2-thioethyl ester)tetranitrosyldiiron (PPIX-RSE). From the IM-MS data coupled with theoretical calculations, it was found that [Cu(II)(DAC - H)]+ exists as a single conformer, with one anthracenyl group above the cyclam and the other below, similar to the crystal structure of Cu(II)(DAC)2+. The metal-free N-nitrosylated ligand (DAC-NO + H)+ has two conformations: one family of structures has one anthracenyl group above the cyclam and one below, while the other has both anthracenyl groups on the same side of the cyclam. These observations are consistent with 1H NMR data for the neutral DAC-NO complex that indicate the presence of two geometric isomers in solution. The third species, PPIX-RSE, has a porphyrin chromophore covalently linked to an Fe2S2(NO)4 cluster for use as a precursor for the photochemical delivery of nitric oxide in single- and two-photon excitation processes. Ion mobility indicates the presence of two (PPIX-RSE + H)+ conformations, consistent with the previous interpretation of the bimodal fluorescence lifetime decay seen for PPIX-RSE. DFT structures, in good agreement with the IM-MS cross sections, indicate two "bent" conformations with the planes of the porphyrin and Fe2S2 rings at different angles with respect to each other.

Journal Article↗

PubMed2005

Structural analysis of metal interactions with the dinucleotide duplex, dCG x dCG, using ion mobility mass spectrometry.

The metal binding properties of the dinucleotide duplex, dCG x dCG, were analyzed in the gas phase with ion mobility mass spectrometry. Both MALDI and ESI were used to generate [M(dCG x dCG)]+ complexes. The collision cross section of each complex was measured in helium using ion mobility based methods and compared to calculated cross sections of theoretical structures. When metal cations classified as hard acids were combined with dCG x dCG, the [M(dCG x dCG)]+ complex organized into a globular structure. However, when soft acid metal cations were examined, a structure was observed where the two C-G base pairs were Watson-Crick bound.

Mass Spectrometry↗

PubMed2005

Structural characterization of G-quadruplexes in deoxyguanosine clusters using ion mobility mass spectrometry.

The aggregation and conformation of deoxyguanosine (dG) in an ammonium acetate buffer solution were examined using mass spectrometry, ion mobility, and molecular mechanics/dynamics calculations. The nano-ESI mass spectrum indicated that 4 and 6 dGs cluster with 1 NH4+; 11 dGs with 2 NH4+; 14, 16, and 17 dGs with 3 NH4+; and 23 dGs with 4 NH4+. The collision cross sections with helium were measured and compared with calculated cross sections of theoretical structures generated by molecular mechanics/dynamics calculations. Three distinct arrival time distribution (ATD) peaks were observed for (4dG + NH4)+. One peak was assigned to the quadruplex structure of (4dG + NH4)+, while the other two peaks corresponded to the quadruplex structures of (8dG + 2NH4)2+ and (12dG + 3NH4)3+, all with the same m/z. Four ATD peaks were observed for (6dG + NH4)+ and assigned to the globular structure of (6dG + NH4)+, and the quadruplex structures of (12dG + 2NH4)2+, (18dG + 3NH4)3+, and (24dG + 4NH4)4+. Two ATD peaks were observed for (11dG + 2NH4)2+ and assigned to the quadruplex structures of (11dG + 2NH4)2+ and (22dG + 4NH4)4+. All of the other clusters in the mass spectrum (14, 16, and 17 dGs with 3 NH4+ and 23 dGs with 4 NH4+) only had one peak in their ATDs and in all cases the theoretical structures in a quadruplex arrangement agreed with the experimental cross sections. These results provide compelling evidence that quadruplexes are present in solution and retain their structure during the spray process, dehydration, and detection.

Deoxyguanosine↗

PubMed2004

Duplex formation and the onset of helicity in poly d(CG)n oligonucleotides in a solvent-free environment.

The gas-phase conformations of a series of cytosine/guanine DNA duplexes were examined by ion mobility and molecular dynamics methods. Deprotonated duplex ions were formed by electrospray ionization, and their collision cross sections measured in helium were compared to calculated cross sections of theoretical models generated by molecular dynamics. The 4-mer (dCGCG) and 6-mer (dCGCGCG) duplexes were found to have globular conformations. Globular and helical structures were observed for the 8-mer (dCGCGCGCG) duplex, with the globular form being the more favored conformer. For the 10-mer (dCGCGCGCGCG), 14-mer (dCGCGCGCGCGCGCG), and 18-mer (dCGCGCGCGCGCGCGCGCG) duplexes, only helical structures were observed in the ion mobility measurements. Theory predicts that the helical structures are less stable than the globular forms in the gas phase and should collapse into the globular form given enough time. However, molecular dynamics simulations at 300 K indicate the helical structures are stable in aqueous solution and will retain their conformations for a limited time in the gas phase. The presence of helical structures in the ion mobility experiments indicates that the duplexes retain "solution structures" in the gas phase on the millisecond time scale.

Circular Dichroism↗

PubMed2004

Diastereomer assignment of an olefin-linked bis-paracyclophane by ion mobility mass spectrometry.

trans-1,2-Bis([2.2]paracyclophanyl)ethene (1) exists as a pair of diastereomers whose conformations, and thus effective collision cross sections, are quite different. The two forms can be obtained by different transition metal-catalyzed reactions. To assign meso and racemic structures, a novel method is reported in which experimental gas-phase ion mobility data are compared with theoretical structures obtained from molecular mechanics calculations.

Journal Article↗