Use of terbium fluorescence enhancement as a new probe for assessing the single-strand content of DNA

Ringer, David P.; Howell, Boyd A.; Kizer, Donald E.

doi:10.1016/0003-2697(80)90620-x

Cited by 58 publications

(13 citation statements)

References 15 publications

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“…Thus far there has been no specific staining method for RNA in ultrastructural cytochemistry. Available data suggest that Tb ions in their oxidation form 3 ϩ can interact with single-stranded nucleic acids (Hörer et al 1977) at their guanine sites (Ringer et al 1978), at the phosphate in unpaired residues (Topal and Fresco 1980), and particularly with guanosine monophosphate in RNA (Ringer et al 1980).…”

Section: Discussionmentioning

confidence: 99%

“…Recently a renewed interest has characterized several studies involving the use of elements of the lanthanide family, such as europium and terbium. Data in the literature point out that these ions in their oxidation form 3 ϩ can interact with single-stranded nucleic acids (Hörer et al 1977) at their guanine sites (Ringer et al 1978), at the phosphate in unpaired residues (Topal and Fresco 1980), and especially with guanosine monophosphate in RNA (Ringer et al 1980). At the electron microscopic (EM) level, these ions (particularly lanthanum) have been known to precipitate in the presence of phosphate groups (Hayat 1993), and members of the family, such as cerium, are now routinely used for EM detection of phosphatase activity (Van Noorden and Frederiks 1993).…”

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confidence: 99%

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Fine Structural Specific Visualization of RNA on Ultrathin Sections

Biggiogera

Fakan²

1998

J Histochem Cytochem.

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SUMMARYWe describe a new technique that allows specific visualization of RNA at the electron microscopic level by means of terbium citrate. Under the conditions presented here, terbium binds selectively to RNA and stains nucleoli, interchromatin granules, perichromatin fibrils, perichromatin granules, and coiled bodies in the cell nucleus, whereas ribosomes are the only contrasted structures in the cytoplasm. All the cell components contrasted by terbium are known to contain RNA. When ultrathin sections are pretreated with RNase A or nuclease S1 (specific for single-stranded nucleic acids), staining does not occur. Neither DNase nor pronase influences the reaction. We conclude that terbium staining is selective for RNA and especially for single-stranded RNA. The staining can be performed on thin sections of material embedded both in epoxy and in acrylic resins. The technique is not influenced by the aldehyde fixative used and can also be utilized after immunolabeling. The endproduct is very fine and, although weak in contrast, is suitable for high-resolution observations. (J Histochem Cytochem 46:389-395, 1998)

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Fine Structural Specific Visualization of RNA on Ultrathin Sections

Biggiogera

Fakan²

1998

J Histochem Cytochem.

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“…Because Mn(II) can often replace Mg(II) in RNA crystal structures (26)(27)(28)(29), we propose that a Mg(II) ion also binds at a site close to that observed for Tb(III). Although the general binding site has remained constant, the shift in the coordination mode may reflect the preference of Tb(III) to bind to single-stranded guanosine nucleotides (30).…”

Section: Inhibition Of the Hammerhead Ribozymementioning

confidence: 99%

Inhibition of the Hammerhead Ribozyme Cleavage Reaction by Site-Specific Binding of Tb(III)

Feig

Scott

Uhlenbeck

1998

Science

127

123

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Terbium(III) [ Tb(III)] was shown to inhibit the hammerhead ribozyme by competing with a single magnesium(II) ion. X-ray crystallography revealed that the Tb(III) ion binds to a site adjacent to an essential guanosine in the catalytic core of the ribozyme, approximately 10 angstroms from the cleavage site. Synthetic modifications near this binding site yielded an RNA substrate that was resistant to Tb(III) binding and capable of being cleaved, even in the presence of up to 20 micromolar Tb(III). It is suggested that the magnesium(II) ion thought to bind at this site may act as a switch, affecting the conformational changes required to achieve the transition state.RNA enzymes require divalent metal ions for activity, either to promote folding or for direct participation in catalysis. The hammerhead ribozyme (Fig. 1A), a self-cleaving RNA found naturally in plant viroids and virusoids, is an excellent system in which to study metal ion-RNA interactions because of the extensive structural and mechanistic data available (1-4). Two independent crystal structures of the hammerhead ribozyme have revealed divalent metal ions binding to six different sites on the molecule (5-7). Biochemical methods available to evaluate the role of these metal ions in ribozyme function are limited. The most common approach is to introduce a phosphorothioate modification into the RNA and to examine its effect on the metal specificity of the catalytic reaction (8-13). We present an approach to studying metal binding to ribozymes based on the observation that ions that compete efficiently for critical Mg-binding sites can thereby inhibit catalysis. The powerful enzymatic and spectroscopic tools originally developed for use with protein metalloenzymes can then be applied to RNA systems such as the hammerhead ribozyme.Interactions between lanthanide ions and RNA molecules have been studied (14,15). (Fig. 1B). This signal was absent from control samples lacking either RNA or Tb(III). These data indicate that the Tb ion binds to the RNA, resulting in energy transfer from the RNA to the lanthanide ion. We therefore investigated the effects of Tb(III) binding on the hammerhead-catalyzed reaction, a site-specific cleavage of a phosphodiester bond to form 2Ј,3Ј-cyclic phosphate and 5Ј-hydroxyl termini.Terbium(III) proved an efficient inhibitor of hammerhead cleavage (18). For example, Tb(III) inhibited the HH8 single-turnover cleavage reaction with an apparent inhibition constant (K i,app ) of 2.0 Ϯ 0.3 M at 25 mM Mg(II) (Fig. 2A), and similar values were obtained under multiple-turnover conditions. Two other well-characterized hammerheads, HH16 (19) and HH␣1 (20), showed K i,app values for Tb(III) of 1.1 Ϯ 0.4 and 0.57 Ϯ 0.08 M, respectively, at 10 mM Mg(II) (21). Because all three ribozymes were inhibited similarly by Tb(III) despite sequence differences in the peripheral base-paired regions and loops, the data indicate that the binding event that underlies inhibition results from the interaction of Tb(III) with a site in the conserved catalytic...

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“…22,23 Also, their ability to substitute Zn(II) 24,25 and Mg(II) at their binding sites makes lanthanide luminescence a useful tool to study interaction between DNA and these ions. [26][27][28][29] In addition, polyelectrolyte/lanthanide ion systems are amenable to modeling by Monte Carlo and other techniques, which can help provide a more detailed understanding of the binding behavior. 30 The luminescence of the lanthanide ions arises from f f f electron transitions and this can give information on both the coordination environment 31,32 and degree of hydration of these ions.…”

Section: Introductionmentioning

confidence: 99%

Changes in Hydration of Lanthanide Ions on Binding to DNA in Aqueous Solution

2005

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The interaction of the trivalent lanthanides Ce(III), Eu(III), and Tb(III) with sodium deoxyribonucleic acid (DNA) in aqueous solution has been studied using their luminescence spectra and decays. Complexation with DNA is indicated by changes in luminescence intensity. In the system terbium(III)-DNA, changes in luminescence with pH are suggested to be due to the protonation of phosphate groups. The degree of hydration of Tb(III) on binding to DNA is followed by luminescence lifetime measurements in water and deuterium oxide solutions, and it is found that the lanthanide ion loses at least one hydration water on binding to long double stranded DNA at pH 4.7 and pH 7. Rather different behavior is observed on binding to long or short single stranded DNA, where six water molecules are lost, independent of pH. It is suggested that in this case the lanthanide probably binds to the bases of the DNA backbone. The DNA conformation seems to be an important factor in the binding. In addition, the isotopic effect on terbium luminescence lifetime may provide a useful method to distinguish between single and double stranded DNA. DSC results are consistent with cleavage of the double helix of DNA at pH 9 in the presence of terbium.

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Use of terbium fluorescence enhancement as a new probe for assessing the single-strand content of DNA

Cited by 58 publications

References 15 publications

Fine Structural Specific Visualization of RNA on Ultrathin Sections

Fine Structural Specific Visualization of RNA on Ultrathin Sections

Inhibition of the Hammerhead Ribozyme Cleavage Reaction by Site-Specific Binding of Tb(III)

Changes in Hydration of Lanthanide Ions on Binding to DNA in Aqueous Solution

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