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We performed single molecule studies to investigate the impact of several prominent small molecules (the oxazole telomestatin derivative L2H2-6OTD, pyridostatin, and Phen-DC3) 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.

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.

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 behavior. End-to-end interactions between concentrated, ultrashort DNA duplexes—driving the self-assembly of aggregates that organize into liquid crystal phases—and the incorporation of flexible single-stranded “gaps” in otherwise fully paired duplexes—producing clear evidence of an elementary lamellar (smectic-A) phase in DNA solutions—are two exciting developments that have opened avenues for discovery. Here, we report on a wider investigation of the nature and temperature dependence of smectic 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 a single-stranded sequence of 2 to 20 thymine bases. Two distinct smectic layer structures are observed for DNA concentration in the range ∼230 to ∼280 mg/mL. One exhibits an interlayer periodicity comparable with two-duplex lengths (“bilayer” structure), and the other has a period similar to a single-duplex length (“monolayer” structure). The bilayer structure is observed for gap length ≳10 bases and melts into the cholesteric phase at a temperature between 30 °C and 35 °C. The monolayer structure predominates for gap length ≲10 bases and persists to >40 °C. We discuss models for the two layer structures and mechanisms for their stability. We also report results for asymmetric gapped constructs and for constructs with terminal overhangs, which further support the model layer structures.

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 behavior. End-to-end interactions between concentrated, ultrashort DNA duplexes—driving the self-assembly of aggregates that organize into liquid crystal phases—and the incorporation of flexible single-stranded “gaps” in otherwise fully paired duplexes—producing clear evidence of an elementary lamellar (smectic-A) phase in DNA solutions—are two exciting developments that have opened avenues for discovery. Here, we report on a wider investigation of the nature and temperature dependence of smectic 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 a single-stranded sequence of 2 to 20 thymine bases. Two distinct smectic layer structures are observed for DNA concentration in the range ∼ 230 to ∼ 280 mg/mL. One exhibits an interlayer periodicity comparable with two-duplex lengths (“bilayer” structure), and the other has a period similar to a single-duplex length (“monolayer” structure). The bilayer structure is observed for gap length ≳10 bases and melts into the cholesteric phase at a temperature between 30 °C and 35 °C. The monolayer structure predominates for gap length ≲10 bases and persists to > 40 ° C. We discuss models for the two layer structures and mechanisms for their stability. We also report results for asymmetric gapped constructs and for constructs with terminal overhangs, which further support the model layer structures.

In-phase adenine-tracts (A-tracts) introduce intrinsic bending to double stranded DNA resulting in banana-shaped macromolecules. In this study, we investigate how such sequence-dependent bending influences DNA-based liquid crystalline (LC) phases formed by gapped DNA (GDNA) constructs. By incorporating three in-phase A-tracts into each duplex arm, we created a GDNA construct with bent duplexes and examined the LC phases they form using temperature-resolved synchrotron small-angle X-ray scattering and polarizing optical microscopy. Like the analogous constructs containing straight (rod-like) duplexes, the bent constructs exhibit a transition from bi-layer smectic-B phase to a monolayer smectic-A phase, although at ~30 °C lower temperatures. By comparing the monolayer spacing between the bent and straight constructs, we estimate a bending angle of ~11° per A-tract at room temperature and at physiologically relevant salt and cDNA. The bending angle decreases with increasing DNA concentration and temperature. Moreover, we demonstrate that divalent cations enhance the stability of the smectic-B phase up to ~30 mM Mg2+ but reduce it beyond that. The reduced thermal stabilities of the bilayer and in-layer ordering of bent duplexes imply reduced propensity for DNA condensation and heterochromatin formation under physiological conditions.

The layered liquid crystalline (LC) phases formed by DNA molecules which include rigid and flexible segments (‘gapped DNA’) enable the study of both end-to-end stacking and side-to-side lateral interactions that drive the condensation of DNA molecules. The resulting layer structure exhibits long-range inter-layer and intra-layer positional correlations. Using synchrotron small-angle x-ray scattering (SAXS) measurements, we investigate the impact of divalent Mg2+ cations on the stability of the inter- and intra-layer DNA ordering as a function of temperature between 5-65 °C and for different terminal base pairings at the blunt ends of the gapped DNA constructs, which mediate the strength of the attractive end-to-end interaction. We demonstrate that the stabilities at a fixed DNA concentration of both inter-layer and intra-layer order are significantly enhanced even at a few mM Mg2+ concentration. The stability continues to increase up to ∼30 mM Mg2+ concentration, but at higher (∼100 mM) Mg2+ content repulsion between positive ions counteracts and reverses the increase. On the other hand, sufficiently strong base-stacking interactions promote intra-layer order even in the absence of multivalent cations, which demonstrates the impact of liquid crystal layering on the DNA condensation process. We discuss the implications of these results in terms cation-mediated DNA-DNA attraction.

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.

At sufficiently high concentrations, gapped DNA (GDNA) constructs - two duplex arms connected by a flexible single stranded linker - form layered (smectic) liquid crystalline (LC) phases, whose nature and stability depend on both end-to-end stacking and side-by-side electrostatic interactions between the duplex arms. We performed synchrotron small angle x-ray scattering (SAXS) experiments over 5-65 C on GDNA constructs that differ only by the terminal base-pairs at the blunt ends of the duplex arms. Two smectic phases are generally observed at the DNA concentrations studied: a bilayer phase at lower temperature, where the duplexes are stacked end-to-end and also positionally ordered within the layers (smectic-B phase), and a monolayer phase at higher temperature with single duplex layer spacing and short-range order within layers (smectic-A phase). Our key finding is that the stability of the bilayer phase varies remarkably among constructs that differ only in their terminal base-pairs. Bilayers formed by constructs with GC termination at both blunt duplex ends melt into the monolayer phase at temperatures up to 30 C higher than for those with AT termination, while mixed (AT/GC) terminations have intermediate stability. Correlating this compositional variation with macroscopic effects on the thermal stability of the bilayer smectic phase enables us to determine the relative strength of base pair specific, end-to-end stacking interactions. Modeling the bilayer melting in terms of a temperature-dependent reduction in the average fraction of end-to-end paired duplexes enables us to estimate stacking free energies in solutions of duplexes at physiologically relevant DNA concentrations.Competing Interest StatementThe authors have declared no competing interest.

Shelterin plays critical roles in maintaining and protecting telomeres by regulating access of various physiological agents to telomeric DNA. We present single molecule measurements investigating the impact of the POT1 and a four-component shelterin complex on the accessibility of human telomeric DNA overhangs with physiologically relevant lengths (28-150 nt), which to our knowledge is the first direct approach to measure this effect on such telomeric constructs. To quantify telomere accessibility, we monitored transient binding events of a short peptide nucleic acid (PNA) probe that is complementary to telomeric overhangs using FRET-PAINT. Although POT1 has a mild G-quadruplex unfolding activity, it reduced accessibility of the PNA probe by ~2.5 fold, indicating that POT1 effectively binds to and protects otherwise exposed telomeric sequences. In comparison, a four-component shelterin reduced the accessibility of telomeric overhangs by ~5-fold. This enhanced protection suggests shelterin restructures the region between single and double stranded telomere, which is otherwise the most accessible part of the overhang, by a synergistic cooperation of shelterin components located on single and double stranded telomere. Competing Interest Statement The authors have declared no competing interest.

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 physiologically 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 kinetics of 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 establish the time scale for the pH drop in a commonly used oxygen scavenging system. Competing Interest Statement The authors have declared no competing interest.