Probing non-selective cation binding in the hairpin ribozyme with Tb(III)

Walter, Nils G.; Yang, Ning; Burke, John M.

doi:10.1006/jmbi.2000.3691

Cited by 92 publications

(114 citation statements)

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“…(The subsequent measurements of particle biodistribution took into account the differing Gd loadings per particle.). In a complementary approach, we took advantage of a natural affinity of lanthanide metals for nucleic acids [6,57], and found that Gd and Tb ions interact stably with the viral genomic RNA (and probably also the associated protein) inside the particles ( Figure 1C) [45]. Wild-type CPMV was soaked with either GdCl 3 or TbCl 3 in the presence of EDTA, the excess metal was dialyzed away, and the number of bound Gd +3 or Tb +3 ions per virus particle was determined using ICP-OES following virus purification.…”

Section: Resultsmentioning

confidence: 99%

Bio-distribution, toxicity and pathology of cowpea mosaic virus nanoparticles in vivo

Singh

Prasuhn

Yeh

et al. 2007

Journal of Controlled Release

227

224

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Virus-based nanoparticles (VNPs) from a variety of sources are being developed for biomedical and nanotechnology applications that include tissue targeting and drug delivery. However, the fate of most of those particles in vivo has not been investigated. Cowpea mosaic virus (CPMV), a plant comovirus, has been found to be amenable to the attachment of a variety of molecules to its coat protein, as well as to modification of the coat protein sequence by genetic means. We report here the results of studies of the bio-distribution, toxicology, and pathology of CPMV in mice. Plasma clearance and tissue biodistribution were measured using CPMV particles derivatized with lanthanide metal complexes. CPMV particles were cleared rapidly from plasma, falling to undetectable levels within 20 minutes. By 30 minutes the majority of the injected VNPs were trapped in the liver and to a lesser extent the spleen with undetectable amounts in other tissues. At doses of 1 mg, 10 mg and 100 mg per kg body weight, no toxicity was noted and the mice appeared to be normal. Hematology was essentially normal, although with the highest dose examined, the mice were somewhat leukopenic with relative decreases in both neutrophils and lymphocytes. Histological examination of spleen showed cellular infiltration, which upon flow cytometry analyses revealed elevated B lymphocytes on the first day following virus administration that subsequently subsided. Microscopic evaluation of various other tissues revealed a lack of apparent tissue degeneration or necrosis. Overall, CPMV appears to be a safe and non-toxic platform for in vivo biomedical applications.

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Section: Resultsmentioning

confidence: 99%

Bio-distribution, toxicity and pathology of cowpea mosaic virus nanoparticles in vivo

Singh

Prasuhn

Yeh

et al. 2007

Journal of Controlled Release

227

224

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“…This unexpectedly high reactivity was attributed to local effects such as potential general base catalysis by RNA functional groups and specific orientations of the flanking 3 ′ phosphate group (McGinnis et al 2012), which may occur in gene 60 mRNA as well. Third, Tb 3+ ions bind preferentially to and can displace metals from cation binding sites (Hargittai and Musier-Forsyth 2000;Walter et al 2000). Nucleotides proximal to specific metal ion binding sites may thus be expected to be more reactive to Tb 3+ , while potentially being protected from SHAPE reagents.…”

Section: Discussionmentioning

confidence: 99%

“…Briefly, in vitro transcribed, gel-purified mRNA was refolded in the same S30 buffer where we observed bypassing, and probed with either 1 mM Tb 3+ ions as described (Walter et al 2000;Walter 2003, 2005;Jeong et al 2003;Harris et al 2004;Sefcikova et al 2007a,b) or 2 mM of the SHAPE reagent 1M7 (Mortimer and Weeks 2007;Gherghe et al 2008Gherghe et al , 2010Watts et al 2009). These two complementary reagents target accessible 2 ′ -OH groups, either to deprotonate them and cleave the adjacent phosphodiester bond (Tb 3+ ) or to acylate them (1M7).…”

Section: Conditions For Translation and Bypassing Of Gene 60 Mrna In mentioning

confidence: 99%

“…On this normalized scale, 1M7 reacted overall slightly more strongly than Tb 3+ , giving an average reactivity along the mRNA of ∼0.43 ± 0.12 compared with ∼0.35 ± 0.06 for Tb 3+ . Although addition of 1 mM Tb 3+ to probing reactions altered the ionic strength by <3%, ions of greater charge density, such as Tb 3+ , can preferentially stabilize structural elements (including tertiary structures) in an RNA by shielding its negatively charged backbone (Walter et al 2000;Walter 2003, 2005;Jeong et al 2003;Harris et al 2004;Newby Lambert et al 2006;Sefcikova et al 2007b). This feature is a plausible cause for the on average lower overall reactivity of gene 60 mRNA to Tb 3+ ions than to the neutral SHAPE reagent.…”

Section: Conditions For Translation and Bypassing Of Gene 60 Mrna In mentioning

confidence: 99%

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Secondary structure of bacteriophage T4 gene 60 mRNA: Implications for translational bypassing

Todd

Walter

2013

RNA

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Translational bypassing is a unique phenomenon of bacteriophage T4 gene 60 mRNA wherein the bacterial ribosome produces a single polypeptide chain from a discontinuous open reading frame (ORF). Upon reaching the 50-nucleotide untranslated region, or coding gap, the ribosome either dissociates or bypasses the interruption to continue translating the remainder of the ORF, generating a subunit of a type II DNA topoisomerase. Mutational and computational analyses have suggested that a compact structure, including a stable hairpin, forms in the coding gap to induce bypassing, yet direct evidence is lacking. Here we have probed the secondary structure of gene 60 mRNA with both Tb 3+ ions and the selective 2 ′ -hydroxyl acylation analyzed by primer extension (SHAPE) reagent 1M7 under conditions where bypassing is observed. The resulting experimentally informed secondary structure models strongly support the presence of the predicted coding gap hairpin and highlight the benefits of using Tb 3+ as a second, complementary probing reagent. Contrary to several previously proposed models, however, the rest of the coding gap is highly reactive with both probing reagents, suggesting that it forms only a short stem-loop. Mutational analyses coupled with functional assays reveal that two possible base-pairings of the coding gap with other regions of the mRNA are not required for bypassing. Such structural autonomy of the coding gap is consistent with its recently discovered role as a mobile genetic element inserted into gene 60 mRNA to inhibit cleavage by homing endonuclease MobA.

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“…These applications take advantage of a few of their unique properties. First, lanthanides and their complexes can efficiently cleave nucleic acids, 3 and thus are used as RNA structural probes 4 and DNA cleaving agents. 5 In addition, a number of in vitro selection experiments were carried out using lanthanides as metal cofactors to obtain DNA-based catalysts (so called DNAzymes [6][7][8][9][10][11][12] for RNA or DNA cleavage.…”

mentioning

confidence: 99%

The group trend of lanthanides binding to DNA and DNAzymes with a complex but symmetric pattern

et al. 2014

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Using sensitized Tb 3+ luminescence spectroscopy as a tool, binding of 14 lanthanides to a lanthanide-dependent DNAzyme is studied, where the binding affinity is symmetric cross the series and the tightest binding occurs with Nd 3+ and Ho 3+ . This trend does not correlate with DNAzyme activity, suggesting that metal binding may not be the rate-limiting step of the DNAzyme catalysis.Lanthanides refer to the elements in the periodic table from La to Lu. Beyond their critical roles in modern technologies, lanthanides also emerge as important probes for biology and medicine. 1,2 In particular, they have been extensively used to study the structure and function of nucleic acids. These applications take advantage of a few of their unique properties. First, lanthanides and their complexes can efficiently cleave nucleic acids, 3 and thus are used as RNA structural probes 4 and DNA cleaving agents. 5 In addition, a number of in vitro selection experiments were carried out using lanthanides as metal cofactors to obtain DNA-based catalysts (so called DNAzymes 6-12 ) for RNA or DNA cleavage. 13,14 Second, a few lanthanides (especially Tb 3+ ) are luminescent and DNA can act as an antenna to increase their light absorption and thus emission intensity. This is useful for probing metal binding sites, 15 and for developing biosensors. 16 Third, lanthanides are hard Lewis acids and some have a similar size as Ca 2+ . Lanthanides can compete with other metal ions in enzymes and act as enzyme inhibitors. For example, both the 17E DNAzyme and the hammerhead ribozyme are inhibited by lanthanides. 15,17 On the other hand, the Leadzyme and a DNA-based ligase are accelerated by lanthanides. [18][19][20] All these examples suggest strong interactions between lanthanides and nucleic acids. Finally, nucleotides and lanthanides can form coordination complexes with useful luminescence and DNA binding properties. [21][22][23][24] Given these progresses, few studies explored the binding of the whole lanthanide series with DNA or correlated metal binding with enzyme activity. Such studies are important for revealing the coordination chemistry of lanthanides, and for DNA bioinorganic chemistry in general. 25,26 We recently performed an in vitro selection experiment using Ce 4+ as the intended metal cofactor. The selected DNAzyme (named Ce13d) was found to have similar activity with all trivalent lanthanides. 14 Therefore, Ce13d must bind all the lanthanides and this DNAzyme might provide a good scaffold for studying lanthanide binding. Herein, we employed Tb 3+ luminescence as a tool to study lanthanide binding to DNAzymes and its relation to catalytic activity. The secondary structure of the Ce13d DNAzyme complex is shown in Figure 1A. It consists of a substrate strand named Sub-rA with a single RNA linkage (rA, ribo-adenosine), and an enzyme strand named Ce13d. In the presence of a trivalent lanthanide, the substrate is cleaved into two fragments at the position pointed by the arrowhead. Lanthanides alone can catalyze the reaction and no div...

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Probing non-selective cation binding in the hairpin ribozyme with Tb(III)

Cited by 92 publications

References 68 publications

Bio-distribution, toxicity and pathology of cowpea mosaic virus nanoparticles in vivo

Bio-distribution, toxicity and pathology of cowpea mosaic virus nanoparticles in vivo

Secondary structure of bacteriophage T4 gene 60 mRNA: Implications for translational bypassing

The group trend of lanthanides binding to DNA and DNAzymes with a complex but symmetric pattern

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