Cisplatin and siRNA interference with structure and function of Wnt-5a mRNA: design and in vitro evaluation of targeting AU-rich elements in the 3′ UTR

Hägerlöf, Margareta; Papsai, Pal; Hedman, Hanna; Jungwirth, Ute; Jenei, Veronika; Elmroth, Sofi K. C.

doi:10.1007/s00775-007-0327-6

Cited by 12 publications

(17 citation statements)

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“…Factors that may be relevant to structurally diverse RNAs have been encountered in studies of platinated DNA hairpins, platinum cross-linked quadruplex structures, and platinated DNA–protein complexes 47–53. Additionally, preliminary investigations have already begun to address cisplatin’s use as a structural probe and as drug conjugate for targeting RNA 15,17,54,55…”

Section: Discussionmentioning

confidence: 99%

Rapid Cross-Linking of an RNA Internal Loop by the Anticancer Drug Cisplatin

2009

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Cisplatin is the most prominent member of a series of platinum(II) antitumor drugs that demonstrate activity based on binding to adjacent purines on genomic DNA. The interactions between cisplatin and alternate biomolecules, including chemically similar RNA, are less understood than are those for DNA. In order to investigate potential implications of platinum(II) drug binding to a structurally complex RNA, we have characterized the reaction between cisplatin and the internal loop of a 41-nucleotide subdomain derived from the U2:U6 spliceosomal RNAs. This "BBD" RNA subdomain consists of a hairpin structure containing a purine-rich asymmetric internal loop. Aquated cisplatin is found to cross-link G nucleobases on opposing sides of the internal loop, forming an intramolecular internal loop cross-link in BBD and an analogous intermolecular cross-link in a two-piece construct containing the same internal loop sequence. The two opposing guanine residues involved in the crosslink were identified via limited alkaline hydrolysis. The kinetics of aquated cisplatin binding to the BBD RNA, a related RNA hairpin, and its DNA hairpin analogue were investigated in an ionic background of 0.1 M NaNO 3 and 1 mM Mg(NO 3 ) 2 . Both BBD and the RNA hairpin react 5-6-fold faster than the DNA hairpin, with calculated second-order rate constants of 2.0(2), 1.7(3), and 0.33 (3) M −1 s −1 , respectively, at 37 °C, pH 7.8. MALDI-MS data corroborate the biochemical studies and support a model in which kinetically preferred platinum binding sites compete with less reactive sites in these oligonucleotides. Taken together, these data indicate that cisplatin treatment has potential to create internal loop and other unusual cross-links in structurally complex RNAs, on a time scale that is relevant for RNA-dependent biological processes.

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Section: Discussionmentioning

confidence: 99%

Rapid Cross-Linking of an RNA Internal Loop by the Anticancer Drug Cisplatin

2009

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“…Subsequent mechanistic studies have shown that isomerization reactions between these 1,3 G*pNpG* transplatin adducts and complementary oligonucleotides are sequence-specific [91], can result in unusual G-N7* to C-N3* crosslinks [89], and are also observed using 2'-OMe [92] and chemically modified RNAs [93]. Further work has shown that transplatin-modified 2'-OMe RNAs [94] and cisplatinmodified RNAs [95,96] can be used as antisense oligos for attenuating gene expression in cell lysates and in living cells.…”

Section: Transplatin Cross-linking and Pt(ii) Drug Conjugatesmentioning

confidence: 99%

Binding of Kinetically Inert Metal Ions to RNA: The Case of Platinum(II)

Chapman

Hostetter

Osborn

et al. 2011

Structural and Catalytic Roles of Metal Ions in RNA

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In this chapter several aspects of Pt(II) are highlighted that focus on the properties of Pt(II)-RNA adducts and the possibility that they influence RNA-based processes in cells. Cellular distribution of Pt(II) complexes results in significant platination of RNA, and localization studies find Pt(II) in the nucleus, nucleolus, and a distribution of other sites in cells. Treatment with Pt(II) compounds disrupts RNA-based processes including enzymatic processing, splicing, and translation, and this disruption may be indicative of structural changes to RNA or RNA-protein complexes. Several RNA-Pt(II) adducts have been characterized in vitro by biochemical and other methods. Evidence for Pt(II) binding in non-helical regions and for Pt(II) cross-linking of internal loops has been found. Although platinated sites have been identified, there currently exists very little in the way of detailed structural characterization of RNA-Pt(II) adducts. Some insight into the details of Pt(II) coordination to RNA, especially RNA helices, can be gained from DNA model systems. Many RNA structures, however, contain complex tertiary folds and common, purine-rich structural elements that present suitable Pt(II) nucleophiles in unique arrangements which may hold the potential for novel types of platinum-RNA adducts. Future research aimed at structural characterization of platinum-RNA adducts may provide further insights into platinum-nucleic acid binding motifs, and perhaps provide a rationale for the observed inhibition by Pt(II) complexes of splicing, translation, and enzymatic processing.

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“…26 In the initial study, we were able to observe that, first, both cisplatin and siRNAs were independently able to silence translation. 23 Second, the measured silencing ability after initial transfection of siRNA followed by subsequent cisplatin exposure was found to be additive. It was therefore concluded that cisplatin had a non-significant effect on direct siRNA-induced gene silencing under the experimental conditions used, with short-term evaluation on translational levels (48 h).…”

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confidence: 98%

Platinum Interference with siRNA Non-seed Regions Fine-Tunes Silencing Capacity

2011

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Knowledge concerning the molecular mechanisms governing the influence of non-coding RNAs on protein production has emerged rapidly during the past decade. Today, two main research areas can be identified, one oriented toward the use of artificially introduced siRNAs for manipulation of gene expression, and the other one focused on the function of endogenous miRNAs. In both cases, the active molecule consists of a ∼20-nucleotide-long RNA duplex. In the siRNA case, improved systemic stability is of central interest for its further development toward clinical applications. With respect to miRNA processing and function, understanding its influence on mRNA targeting and the silencing ability of individual miRNAs, e.g., under pathological conditions, remains a scientific challenge. In the present study, a model system is presented where the influence of the two clinically used anticancer drugs, cisplatin and oxaliplatin, on siRNA's silencing capacity has been evaluated. More specifically, siRNAs targeting the 3' UTR region of Wnt-5a mRNA (NM_003352) were constructed, and the biologically active antisense RNA strand was pre-platinated. Platinum adducts were detected and characterized by a combination of gel electrophoresis and MALDI-MS techniques, and the silencing capacity was evaluated in cellular luciferase-expressing systems using HB2 cells. Data show that platination of the antisense strand of the siRNAs results in adducts with protection against hydrolytic cleavage in the proximity of the platination sites, i.e., with altered degradation patterns compared to native RNAs. The MALDI-MS method was successfully used to further identify and characterize platinated RNA, with the naturally occurring platinum isotopic patterns serving as sensitive fingerprints for metalated sites. Expression assays all confirm biological activity of antisense-platinated siRNAs, here with platination sites located outside of the seed region. A significant reduction of silencing capacity was observed as a general trend, however. Of the two complexes studied, oxaliplatin exhibits the larger influence, thus indicating subtle differences between the abilities of cis- and oxaliplatin to interfere with si- and miRNA processing.

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Cisplatin and siRNA interference with structure and function of Wnt-5a mRNA: design and in vitro evaluation of targeting AU-rich elements in the 3′ UTR

Cited by 12 publications

References 58 publications

Rapid Cross-Linking of an RNA Internal Loop by the Anticancer Drug Cisplatin

Rapid Cross-Linking of an RNA Internal Loop by the Anticancer Drug Cisplatin

Binding of Kinetically Inert Metal Ions to RNA: The Case of Platinum(II)

Platinum Interference with siRNA Non-seed Regions Fine-Tunes Silencing Capacity

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