Isothermal folding of G-quadruplexes

Thermodynamic studies of G-quadruplex stability are an essential complement to structures obtained by NMR or x-ray crystallography. An understanding of the energetics of quadruplex folding provides a necessary foundation for the physical interpretation of quadruplex formation and reactivity. While thermal denaturation methods are most commonly used to evaluate quadruplex stability, it is also possible to study folding using isothermal titration methods. G-quadruplex folding is tightly coupled to specific cation binding. We describe here protocols for monitoring the cation-driven quadruplex folding transition using circular dichroism or absorbance, and for determination of the distribution of free and bound cation using a fluorescence indicator. Together these approaches provide insight into quadruplex folding at constant temperature, and characterize the linkage between cation binding and folding

Characterization of a K+-induced conformational switch in a human telomeric DNA oligonucleotide using 2-aminopurine fluorescence

Human telomeric DNA consists of tandem repeats of the DNA sequence d(GGGTTA). Oligodeoxynucletotide telomere models such as d[A(GGGTTA)(3)GGG] (Tel22) fold in a cation-dependent manner into quadruplex structures consisting of stacked G-quartets linked by d(TTA) loops. NMR has shown that in Na(+) solutions Tel22 forms a ‘basket’ topology of four antiparallel strands; in contrast, Tel22 in K(+) solutions consists of a mixture of unknown topologies. Our previous studies on the mechanism of folding of Tel22 and similar telomere analogs utilized changes in UV absorption between 270 and 325 nm that report primarily on G-quartet formation and stacking showed that quadruplex formation occurs within milliseconds upon mixing with an appropriate cation. In the current study, we assessed the dynamics and equilibria of folding of specific loops by using Tel22 derivatives in which the dA residues were serially substituted with the fluorescent reporter base, 2-aminopurine (2-AP). Tel22 folding induced by Na(+) or K(+) assessed by changes in 2-AP fluorescence consists of at least three kinetic steps with time constants spanning a range of ms to several hundred seconds. Na(+)-dependent equilibrium titrations of Tel22 folding could be approximated as a cooperative two-state process. In contrast, K(+)-dependent folding curves were biphasic, revealing that different conformational ensembles are present in 1 mM and 30 mM K(+). This conclusion was confirmed by (1)H NMR. Molecular dynamics simulations revealed a K(+) binding pocket in Tel22 located near dA1 that is specific for the so-called hybrid-1 conformation in which strand 1 is in a parallel arrangement. The possible presence of this topologically specific binding site suggests that K(+) may play an allosteric role in regulating telomere conformation and function by modulating quadruplex tertiary structure

Experimental and theoretical optical properties of two emerging nanoparticles

As nanoparticles and materials are introduced into commercial applications, new emerging materials continue to be discovered. Many of these new materials have interesting properties that make them well-suited for use in a variety of fields, including electronics, energy production/storage, sensing, and biomedical. However, before these emerging particles can be used, it is critical that the properties of these materials are well-characterized. Two particular nanoparticles, carbon nanodots (CNDs) and copper sulfide nanoparticles (CuS NPs), are of interest, owing to their unique properties. Specifically, CNDs are of interest in biomedical applications due to their small size and strong fluorescence, though the mechanism of this fluorescence remains unknown. CuS is of interest in many optical and electronic applications due to its near-metallic nature and asymmetrical crystal structure. The nature of both nanomaterials is such that purely experimental characterization is insufficient in exploring the properties of interest. Thus, experimental and theoretical studies of both materials have been explored to better elucidate the properties of these materials. Specifically, both particles have been synthesized, characterized, and modeled using density functional theory. For the study of CNDs, the major findings were in regard to how structural modifications and deformations affect the optical properties and these relations were compared to experimental findings. A novel synthesis of CuS NPs was developed and the charge carrier effective mass and density were studied from a theoretical perspective for the first time. Additionally, photothermal therapy has been explored as a potential application for the CuS NPs

English secondary students’ thinking about the status of scientific theories: consistent, comprehensive, coherent and extensively evidenced explanations of aspects of the natural world – or just ‘an idea someone has’

Teaching about the nature of science (NOS) is seen as a priority for science education in many national contexts. The present paper focuses on one central issue in learning about NOS: understanding the nature and status of scientific theories. A key challenge in teaching about NOS is to persuade students that scientific knowledge is generally robust and reliable, yet also in principle always open to challenge and modification. Theories play a central role, as they are a form of conjectural knowledge that over time may be abandoned, replaced, modified, yet sometimes become well established as current best scientific understanding. The present paper reports on findings from interviews with 13–14 year olds in England where target knowledge presents theories as ‘consistent, comprehensive, coherent and extensively evidenced explanations of aspects of the natural world’. Student thinking reflected a two-tier typology of scientific knowledge in which largely unsupported imaginative ideas (‘theories’) became transformed into fairly definitive knowledge (such as laws) through relatively straightforward testing. These results are considered in relation to research into intellectual development which indicates that effective teaching in this area requires careful scaffolding of student learning, but has potential to contribute to supporting intellectual development across the curriculum.This is the author accepted manuscript. The final version is available from Taylor & Francis via http://dx.doi.org/10.1080/09585176.2015.104392

Preliminary spectroscopic characterization of a synthetic DNA oligomer containing a B-Z junction at high salt.

It is well known that the local conformation of a segment of DNA is dependent upon both the sequence of the segment and the conditions under which the DNA is prepared. In extreme cases, the DNA may contain regions of both right and left-handed conformations, mandating the existence of a conformational junction between the two. These B-Z junctions have been observed in plasmids but, to date, no model systems have been characterized to determine the molecular nature of these junctions. Preliminary CD, UV, and NMR studies on such a model are presented here. A 16 base pair oligonucleotide, containing a potential B-Z junction, has been synthesized and characterized by the above techniques. The results suggest that this molecule contains both right and left-handed conformations under condition of high salt, and thus a B-Z junction

Conformational properties of Z-forming DNA oligomers bearing terminal unpaired bases

Three sets of semi-self-complementary deoxyribonucleotide decamers with the sequence XX-(5meCG)4, (5meCG)4-XX, or Y-(5meCG)4-Y, where XX = AA, CC, GG, or TT and Y = A, C, G, or T, were synthesized along with the self-complementary octamer (5meCG)4. The 8-mer duplex readily undergoes a B-to-Z conformational conversion upon increasing the NaCl concentration with a transitional midpoint of approximately 1.1 M NaCl. The 10-mers should form 8-bp duplexes a with core sequence of [(5meCG)4]2 with 5'-XX overhangs, 3'-XX overhangs, or 5',3'-Y/Y mismatches. Circular dichroism was employed to determine the conformations of all oligomers. Salt titrations were performed to measure the effect of overhangs and terminal mismatches on the B-to-Z conversion. In general, the presence of 5'-XX overhangs results in a transition midpoint equal to or slightly higher than the control, whereas the presence of 3'-XX overhangs results in a transition midpoint slightly lower than the control. The 3'-CC and 5'-GG overhangs are exceptions, with transition midpoints much higher than the control. These oligomers apparently form duplexes with 5',3'-C/C or 5',3'-G/G mismatches abutting a [(G5meC)4]2 duplex core. The presence of terminal mismatches in the third set of oligomers results in transition midpoints higher than the control. Ultraviolet absorbance methods were used to evaluate the effect of the various stacking motifs of the 10-mers on the thermodynamics of melting relative to the 8-mer for both B and Z conformations. We found that in both the B and Z conformations, the presence of an overhang stabilizes the [(5meCG)4]2 duplex, with the 5' overhangs having a greater stabilizing effect relative to the 3' overhangs. The presence of 5',3'-Y/Y mismatches also imparts a stabilizing effect on the control 8-mer in both the B and Z conformations. These results are discussed in terms of stacking interactions of the terminal unpaired bases