Abstract
G-quadruplex (GQ) stabilizing small molecule (SM) ligands have been used to stabilize human telomeric GQ (hGQ) to inhibit telomerase activity, or non-telomeric GQs to manipulate gene ...expression at transcription or translation level. GQs are known to inhibit DNA replication unless destabilized by helicases, such as Bloom helicase (BLM). Even though the impact of SM ligands on thermal stability of GQs is commonly used to characterize their efficacy, how these ligands influence helicase-mediated GQ unfolding is not well understood. Three prominent SM ligands (an oxazole telomestatin derivative, pyridostatin, and PhenDC3), which thermally stabilize hGQ at different levels, were utilized in this study. How these ligands influence BLM-mediated hGQ unfolding was investigated using two independent single-molecule approaches. While the frequency of dynamic hGQ unfolding events was used as the metric in the first approach, the second approach was based on quantifying the cumulative unfolding activity as a function of time. All three SM ligands inhibited BLM activity at similar levels, 2–3 fold, in both approaches. Our observations suggest that the impact of SM ligands on GQ thermal stability is not an ideal predictor for their inhibition of helicase-mediated unfolding, which is physiologically more relevant.
We report dynamic light scattering measurements of the orientational (Frank) elastic constants and associated viscosities among a homologous series of a liquid crystalline dimer, trimer, and tetramer ...exhibiting a uniaxial nematic (N) to twist-bend nematic (N
) phase transition. The elastic constants for director splay (K
), twist (K
) and bend (K
) exhibit the relations K
> K
> K
and K
/K
> 2 over the bulk of the N phase. Their behavior near the N-N
transition shows dependency on the parity of the number (n) of the rigid mesomorphic units in the flexible n-mers. Namely, the bend constant K
in the dimer and tetramer turns upward and starts increasing close to the transition, following a monotonic decrease through most of the N phases. In contrast, K
for the trimer flattens off just above the transition and shows no pretransitional enhancement. The twist constant K
increases pretransitionally in both even and odd n-mers, but more weakly so in the trimer, while K
increases steadily on cooling without evidence of pretransitional behavior in any n-mer. The viscosities associated with pure splay, twist-dominated twist-bend, and pure bend fluctuations in the N phase are comparable in magnitude to those of rod-like monomers. All three viscosities increase with decreasing temperature, but the bend viscosity in particular grows sharply near the N-N
transition. The N-N
pretransitional behavior is shown to be in qualitative agreement with the predictions of a coarse-grained theory, which models the N
phase as a "pseudo-layered" structure with the symmetry (but not the mass density wave) of a smectic-A* phase.
We performed single molecule studies to investigate the impact of several prominent small molecules (the oxazole telomestatin derivative L2H2-6OTD, pyridostatin, and Phen-DC
) on intermolecular ...G-quadruplex (i-GQ) formation between two guanine-rich DNA strands that had 3-GGG repeats in one strand and 1-GGG repeat in the other (3+1 GGG), or 2-GGG repeats in each strand (2+2 GGG). Such structures are not only physiologically significant but have recently found use in various biotechnology applications, ranging from DNA-based wires to chemical sensors. Understanding the extent of stability imparted by small molecules on i-GQ structures, has implications for these applications. The small molecules resulted in different levels of enhancement in i-GQ formation, depending on the small molecule and arrangement of GGG repeats. The largest enhancement we observed was in the 3+1 GGG arrangement, where i-GQ formation increased by an order of magnitude, in the presence of L2H2-6OTD. On the other hand, the enhancement was limited to three-fold with Pyridostatin (PDS) or less for the other small molecules in the 2+2 GGG repeat case. By demonstrating detection of i-GQ formation at the single molecule level, our studies illustrate the feasibility to develop more sensitive sensors that could operate with limited quantities of materials.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
We present a collection of single molecule work on the i-motif structure formed by the human telomeric sequence. Even though it was largely ignored in earlier years of its discovery due to its modest ...stability and requirement for low pH levels (pH < 6.5), the i-motif has been attracting more attention recently as both a physiologically relevant structure and as a potent pH sensor. In this manuscript, we establish single molecule Förster resonance energy transfer (smFRET) as a tool to study the i-motif over a broad pH and ionic conditions. We demonstrate pH and salt dependence of i-motif formation under steady state conditions and illustrate the intermediate states visited during i-motif folding in real time at the single molecule level. We also show the prominence of intermediate folding states and reversible folding/unfolding transitions. We present an example of using the i-motif as an
in-situ
pH sensor and use this sensor to establish the time scale for the pH drop in a commonly used oxygen scavenging system.
The occurrence of a smectic-B (Sm-B) phase is demonstrated in concentrated aqueous solutions of “gapped” DNA constructs consisting of fully paired duplexes bridged by a flexible, unpaired strand of ...nucleotides. The Sm-B phase, identified by small and wide angle x-ray scattering measurements and optical microscopy, develops from a smectic-A (Sm-A) phase with increasing DNA concentration at room temperature. It transitions (reversibly) to the Sm-A when the temperature is raised above ∼50^{∘}C.
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CMK, CTK, FMFMET, IJS, NUK, PNG, UL, UM, UPUK
Positionally ordered bilayer liquid crystalline nanostructures formed by gapped DNA (GDNA) constructs provide a practical window into DNA–DNA interactions at physiologically relevant DNA ...concentrations; concentrations several orders of magnitude greater than those in commonly used biophysical assays. The bilayer structure of these states of matter is stabilized by end-to-end base stacking interactions; moreover, such interactions also promote in-plane positional ordering of duplexes that are separated from each other by less than twice the duplex diameter. The end-to-end stacked as well as in-plane ordered duplexes exhibit distinct signatures when studied via small-angle X-ray scattering (SAXS). This enables analysis of the thermal stability of both the end-to-end and side-by-side interactions. We performed synchrotron SAXS experiments over a temperature range of 5–65 °C on GDNA constructs that differ only by the terminal base-pairs at the blunt duplex ends, resulting in identical side-by-side interactions, while end-to-end base stacking interactions are varied. Our key finding is that bilayers formed by constructs with GC termination transition into the monolayer state at temperatures as much as 30 °C higher than for those with AT termination, while mixed (AT/GC) terminations have intermediate stability. By modeling the bilayer melting in terms of a temperature-dependent reduction in the average fraction of end-to-end paired duplexes, we estimate the stacking free energies in DNA solutions of physiologically relevant concentrations. The free-energies thereby determined are generally smaller than those reported in single-molecule studies, which might reflect the elevated DNA concentrations in our studies.
Full text
Available for:
IJS, KILJ, NUK, PNG, UL, UM
We performed single-molecule studies to investigate the impact of several prominent small molecules (the oxazole telomestatin derivative L2H2-6OTD, pyridostatin, and Phen-DC 3) on intermolecular ...G-quadruplex (i-GQ) formation between two guanine-rich DNA strands that have 3-GGG repeats in one strand and 1-GGG repeat in the other (3+1 GGG), or 2-GGG repeats in each strand (2+2 GGG). Such structures are not only physiologically significant but have recently found use in various biotechnology applications, ranging from DNA-based wires to chemical sensors. Understanding the extent of stability imparted by small molecules on i-GQ structures has implications for these applications. The small molecules resulted in different levels of enhancement in i-GQ formation, depending on the small molecule and arrangement of GGG repeats. The largest enhancement we observed was in the 3+1 GGG arrangement, where i-GQ formation increased by an order of magnitude, in the presence of L2H2-6OTD. On the other hand, the enhancement was limited to three-fold with Pyridostatin (PDS) or less for the other small molecules in the 2+2 GGG case. By demonstrating detection of i-GQ formation at the single-molecule level, our studies illustrate the feasibility to develop more sensitive sensors that could operate with limited quantities of materials.In another study, although its mesomorphic properties have been studied for many years, only recently has the molecule of life begun to reveal the true range of its rich liquid crystalline (LC) behavior. End-to-end interactions between concentrated, ultra-short DNA duplexes' self-assembling to form longer aggregates that then organize into LC phases — and the incorporation of flexible single-stranded "gap" regions in otherwise fully-paired duplexes —leading to the first convincing evidence of an elementary lamellar (smectic-A) phase in DNA solutions — are two exciting developments that have opened new avenues for discovery. In this dissertation, we used a combination of optical microscopy and synchrotron small-angle x-ray scattering to characterize the nature and temperature dependence of elementary lamellar ordering in concentrated solutions of various "gapped" DNA (GDNA) constructs. We examine symmetric GDNA constructs consisting of two 48 base-pair duplex segments bridged by an unpaired, single-stranded sequence ("gap") of 2 — 20 thymine bases. Two distinct, elementary smectic layer structures are observed for DNA concentration in the range 220 - 270 mg/ml. One exhibits an interlayer periodicity comparable to two duplex lengths ("bilayer" structure), and the other has a period similar to a single duplex length ("monolayer" structure). The GDNA with a 20T gap exhibits a "bilayer" structure, with four observable diffractions, and, when heated to a temperature between 30 ºC to 35 ºC melts into the cholesteric phase. At a concentration of about 260 mg/ml, the GDNA constructs with a gap length of 10T or shorter (<10T) exhibit the "monolayer" structure with two observable diffraction peaks, which predominate upon heating to 40 ºC. We discuss models for the two-layer structures and mechanisms for their stability. We also report results for novel asymmetric "gapped" constructs and for constructs with terminal overhangs. These results further test and support the model layer structures and illustrate the rich liquid crystalline phases formed by gapped DNA structures.
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