Chapter 3 Direct Quantitation of Mg2+‐RNA Interactions by Use of a Fluorescent Dye

Grilley, Dan; Soto, Ana M.; Draper, David E.

doi:10.1016/s0076-6879(08)04203-1

Cited by 51 publications

(74 citation statements)

References 25 publications

(28 reference statements)

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“…Although ion counting has been demonstrated using the Donnan effect in equilibrium dialysis experiments (26,27) or with ion-binding fluorescent dyes (28), ASAXS is the only method that provides both the number and distribution of ions around nucleic acids, which is essential for the most accurate comparison to theory. However, the achievable signal/noise ratio in ASAXS measurements is limited by the small difference signals (typically <10%) and sensitivity of macromolecule solutions to radiation damage (29).…”

Section: Introductionmentioning

confidence: 99%

Determining the Locations of Ions and Water around DNA from X-Ray Scattering Measurements

Meisburger

Pabit

Pollack

2015

Biophysical Journal

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Nucleic acids carry a negative charge, attracting salt ions and water. Interactions with these components of the solvent drive DNA to condense, RNA to fold, and proteins to bind. To understand these biological processes, knowledge of solvent structure around the nucleic acids is critical. Yet, because they are often disordered, ions and water evade detection by x-ray crystallography and other high-resolution methods. Small-angle x-ray scattering (SAXS) is uniquely sensitive to the spatial correlations between solutes and the surrounding solvent. Thus, SAXS provides an experimental constraint to guide or test emerging solvation theories. However, the interpretation of SAXS profiles is nontrivial because of the difficulty in separating the scattering signals of each component: the macromolecule, ions, and hydration water. Here, we demonstrate methods for robustly deconvoluting these signals, facilitating a more straightforward comparison with theory. Using SAXS data collected on an absolute intensity scale for short DNA duplexes in solution with Na(+), K(+), Rb(+), or Cs(+) counterions, we mathematically decompose the scattering profiles into components (DNA, water, and ions) and validate the decomposition using anomalous scattering measurements. In addition, we generate a library of physically motivated ion atmosphere models and rank them by agreement with the scattering data. The best-fit models have relatively compact ion atmospheres when compared to predictions from the mean-field Poisson-Boltzmann theory of electrostatics. Thus, the x-ray scattering methods presented here provide a valuable measurement of the global structure of the ion atmosphere that can be used to test electrostatics theories that go beyond the mean-field approximation.

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Section: Introductionmentioning

confidence: 99%

Determining the Locations of Ions and Water around DNA from X-Ray Scattering Measurements

Meisburger

Pabit

Pollack

2015

Biophysical Journal

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“…This can be useful to establish the number of strong divalent metal ion binding sites in a high-salt background [143], but also to evaluate the affinities of different metal ions with respect to each other. Here, fluorescent indicators have been widely used to determine free metal ion concentrations [278][279][280]. However, such dyes are usually restricted to the detection of one specific kind of metal ion.…”

Section: Stoichiometric Methods -"Ion Counting"mentioning

confidence: 99%

“…Irrespective of the way of detection, those methods are based on the equilibration of the nucleic acid-containing solution (by dialysis or ultrafiltration spin columns) against a buffer solution with well defined metal content. After the equilibrium is reached, metal ion concentrations in both solutions are measured and the number of metal ions retained per nucleic acid molecule can be determined [278,282].…”

Section: Stoichiometric Methods -"Ion Counting"mentioning

confidence: 99%

Characterization of Metal Ion-Nucleic Acid Interactions in Solution

Pechlaner

Sigel

2011

Metal Ions in Life Sciences

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Metal ions are inextricably involved with nucleic acids due to their polyanionic nature. In order to understand the structure and function of RNAs and DNAs, one needs to have detailed pictures on the structural, thermodynamic, and kinetic properties of metal ion interactions with these biomacromolecules. In this review we first compile the physicochemical properties of metal ions found and used in combination with nucleic acids in solution. The main part then describes the various methods developed over the past decades to investigate metal ion binding by nucleic acids in solution. This includes for example hydrolytic and radical cleavage experiments, mutational approaches, as well as kinetic isotope effects. In addition, spectroscopic techniques like EPR, lanthanide(III) luminescence, IR and Raman as well as various NMR methods are summarized. Aside from gaining knowledge about the thermodynamic properties on the metal ion-nucleic acid interactions, especially NMR can be used to extract information on the kinetics of ligand exchange rates of the metal ions applied. The final section deals with the influence of anions, buffers, and the solvent permittivity on the binding equilibria between metal ions and nucleic acids. Little is known on some of these aspects, but it is clear that these three factors have a large influence on the interaction between metal ions and nucleic acids.

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“…The first method uses the dye 8-hydroxyquinoline-5-sulfonic acid (HQS), whose 1:1 Mg 2+ -HQS complex is fluorescent (Romer and Hach 1975;Grilley et al 2009 (Table 2).…”

Section: Counting Bound Mg 2+ Ionsmentioning

confidence: 99%

“…Measurements with HQS were carried out at 298 K on a Varian Carry Eclipse Fluorescence Spectrometer at pH 7, with excitation wavelength 400 nm and with emission wavelength 550 nm (as previously described by Romer and Hach 1975;Grilley et al 2009). Two samples (0.1 mM HQS, 20 mM MOPS buffer, 50 mM, 100 mM, or 2 M KCl), one of which contained 24 µM of CPEB3 RNA, were titrated with Mg 2+ in exactly the same way (2-260 µM Mg 2+ ) in 29-42 steps.…”

Section: Mgmentioning

confidence: 99%

Secondary structure confirmation and localization of Mg²⁺ ions in the mammalian CPEB3 ribozyme

Skilandat

Rowińska‐Żyrek

Sigel

2016

RNA

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Most of today's knowledge of the CPEB3 ribozyme, one of the few small self-cleaving ribozymes known to occur in humans, is based on comparative studies with the hepatitis delta virus (HDV) ribozyme, which is highly similar in cleavage mechanism and probably also in structure. Here we present detailed NMR studies of the CPEB3 ribozyme in order to verify the formation of the predicted nested double pseudoknot in solution. In particular, the influence of Mg 2+ , the ribozyme's crucial cofactor, on the CPEB3 structure is investigated. NMR titrations, Tb 3+ -induced cleavage, as well as stoichiometry determination by hydroxyquinoline sulfonic acid fluorescence and equilibrium dialysis, are used to evaluate the number, location, and binding mode of Mg 2+ ions. Up to eight Mg 2+ ions interact site-specifically with the ribozyme, four of which are bound with high affinity. The global fold of the CPEB3 ribozyme, encompassing 80%-90% of the predicted base pairs, is formed in the presence of monovalent ions alone. Low millimolar concentrations of Mg 2+ promote a more compact fold and lead to the formation of additional structures in the core of the ribozyme, which contains the inner small pseudoknot and the active site. Several Mg 2+ binding sites, which are important for the functional fold, appear to be located in corresponding locations in the HDV and CPEB3 ribozyme, demonstrating the particular relevance of Mg 2+ for the nested double pseudoknot structure.

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Chapter 3 Direct Quantitation of Mg2+‐RNA Interactions by Use of a Fluorescent Dye

Cited by 51 publications

References 25 publications

Determining the Locations of Ions and Water around DNA from X-Ray Scattering Measurements

Determining the Locations of Ions and Water around DNA from X-Ray Scattering Measurements

Characterization of Metal Ion-Nucleic Acid Interactions in Solution

Secondary structure confirmation and localization of Mg²⁺ ions in the mammalian CPEB3 ribozyme

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Chapter 3 Direct Quantitation of Mg2+‐RNA Interactions by Use of a Fluorescent Dye

Cited by 51 publications

References 25 publications

Determining the Locations of Ions and Water around DNA from X-Ray Scattering Measurements

Determining the Locations of Ions and Water around DNA from X-Ray Scattering Measurements

Characterization of Metal Ion-Nucleic Acid Interactions in Solution

Secondary structure confirmation and localization of Mg2+ ions in the mammalian CPEB3 ribozyme

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Product

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Secondary structure confirmation and localization of Mg²⁺ ions in the mammalian CPEB3 ribozyme