RESUMEN
The layered liquid crystalline 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 (helix-to-helix) lateral interactions, forming a model system to study such interactions at physiologically relevant DNA and ion concentrations. The observed layer structure exhibits long-range interlayer and in-layer positional correlations. In particular, the in-layer order has implications for DNA condensation, as it reflects whether these normally repulsive interactions become attractive under certain ionic conditions. Using synchrotron small-angle X-ray scattering measurements, we investigate the impact of divalent Mg2+ cations (in addition to a constant 150 mM Na+) on the stability of the inter- and in-layer DNA ordering as a function of temperature between 5 and 65 °C. DNA constructs with different terminal base pairings were created to mediate the strength of the attractive end-to-end stacking interactions between the blunt ends of the gapped DNA constructs. We demonstrate that the stabilities at a fixed DNA concentration of both interlayer and in-layer order are significantly enhanced even at a few mM Mg2+ concentration. The stabilities are even higher at 30 mM Mg2+; however, a marked decrease is observed at 100 mM Mg2+, suggesting a change in the nature of side-by-side interactions within this Mg2+ concentration range. We discuss the implications of these results in terms of counterion-mediated DNA-DNA attraction and DNA condensation.
Asunto(s)
Cristales Líquidos , Cationes Bivalentes , ADN/química , Cationes , TemperaturaRESUMEN
Telomeres terminate with a 50-300 bases long single-stranded G-rich overhang, which can be misrecognized as a DNA damage repair site. Shelterin plays critical roles in maintaining and protecting telomere ends by regulating access of various physiological agents to telomeric DNA, but the underlying mechanism is not well understood. Here, we measure how shelterin affects the accessibility of long telomeric overhangs by monitoring transient binding events of a short complementary peptide nucleic acid (PNA) probe using FRET-PAINT in vitro. We observed that the POT1 subunit of shelterin reduces the accessibility of the PNA probe by â¼2.5-fold, indicating that POT1 effectively binds to and protects otherwise exposed telomeric sequences. In comparison, a four-component shelterin stabilizes POT1 binding to the overhang by tethering POT1 to the double-stranded telomeric DNA and reduces the accessibility of telomeric overhangs by â¼5-fold. This enhanced protection suggests shelterin restructures the junction between single and double-stranded telomere, which is otherwise the most accessible part of the telomeric overhang.
Asunto(s)
Complejo Shelterina , Telómero , ADN/metabolismo , Complejo Shelterina/metabolismo , Telómero/genética , Telómero/metabolismo , Proteínas de Unión a Telómeros/metabolismoRESUMEN
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 [Formula: see text] 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 â³10 bases and melts into the cholesteric phase at a temperature between 30 °C and 35 °C. The monolayer structure predominates for gap length â²10 bases and persists to [Formula: see text]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.
Asunto(s)
ADN/química , Cristales Líquidos/química , Estructura Molecular , SolucionesRESUMEN
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.
Asunto(s)
ADN , Dispersión del Ángulo Pequeño , Difracción de Rayos X , ADN/química , Emparejamiento Base , Temperatura , Termodinámica , Conformación de Ácido NucleicoRESUMEN
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.
Asunto(s)
G-Cuádruplex , RecQ Helicasas/metabolismo , Humanos , Ligandos , Telómero/metabolismoRESUMEN
Ribosomes are ribonucleoprotein particles that are essential for protein biosynthesis in all forms of life. During ribosome biogenesis, transcription, folding, modification, and processing of rRNA are coupled to the assembly of proteins. Various assembly factors are required to synchronize all different processes that occur during ribosome biogenesis. Herein, the RNA chaperone and RNA strand annealing activity of rRNA modification enzyme ribosome small subunit methyltransferaseâ C (RsmC), which modifies guanine to 2-methylguanosine (m2 G) at position 1207 of 16S rRNA (Escherichia coli nucleotide numbering) located at helixâ 34 (h34), are reported. A 25-fold increase in the h34 RNA strand annealing rates is observed in the presence of RsmC. Single-molecule FRET experiments confirmed the ability of protein RsmC to denature a non-native structure formed by one of the two h34 strands and to form a native-like duplex. This observed RNA chaperone activity of protein RsmC might play a vital role in the rapid generation of functional ribosomes.
Asunto(s)
Metiltransferasas/metabolismo , ARN Ribosómico 16S/metabolismo , Escherichia coli/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Cinética , Metilación , Conformación de Ácido Nucleico , ARN Ribosómico 16S/químicaRESUMEN
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 (NTB) phase transition. The elastic constants for director splay (K11), twist (K22) and bend (K33) exhibit the relations K11 > K22 > K33 and K11/K22 > 2 over the bulk of the N phase. Their behavior near the N-NTB transition shows dependency on the parity of the number (n) of the rigid mesomorphic units in the flexible n-mers. Namely, the bend constant K33 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, K33 for the trimer flattens off just above the transition and shows no pretransitional enhancement. The twist constant K22 increases pretransitionally in both even and odd n-mers, but more weakly so in the trimer, while K11 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-NTB transition. The N-NTB pretransitional behavior is shown to be in qualitative agreement with the predictions of a coarse-grained theory, which models the NTB phase as a "pseudo-layered" structure with the symmetry (but not the mass density wave) of a smectic-A* phase.
RESUMEN
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.
Asunto(s)
G-Cuádruplex/efectos de los fármacos , Descubrimiento de Drogas , Estructura Molecular , Conformación de Ácido Nucleico/efectos de los fármacos , Oxazoles/química , Oxazoles/farmacología , Repeticiones de TrinucleótidosRESUMEN
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.
RESUMEN
We report a case of a 42-year-old man with a submitral aneurysm who presented to the emergency room in circulatory collapse, with left ventricular obstruction and severe mitral valve stenosis and regurgitation secondary to the aneurysm. Resection of the aneurysm and mitral valve replacement were performed through a median sternotomy without any complication and with good results.