Interstrand cross-link of DNA by covalently linking a pair of abasic sites

Ichikawa, Kohei; Kojima, Naoshi; Hirano, Yu; Takebayashi, Toshie; Kowata, Keiko; Komatsu, Yasuo

doi:10.1039/c2cc16785a

Cited by 20 publications

(22 citation statements)

References 35 publications

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“…This feature makes the assay much more adaptable for large-scale screening than any existing ICLR assay methods. Fourth, the ICL is an aromatic ring unit (aoNao, (7)) that is chemically stable and easily produced in a site-specific manner on a large scale, enabling production of enough ICLR reporter plasmids for chemical library screening. Fifth, the screening is performed in a non–SV40-transformed HT1080 fibrosarcoma cell line.…”

Section: Resultsmentioning

confidence: 99%

“…N, N -bis(aminooxyacetyl)-1,5-diaminonaphthalene (aoNao) was synthesized as described previously (7). All chemical compounds were prepared as 10 mM solutions in DMSO, except aoNao (100 mM in DMSO), cisplatin (5 mM in 0.9% aqueous sodium chloride), and gemcitabine (10 mM in water).…”

Section: Methodsmentioning

confidence: 99%

“…B ) The control plasmid’s backbone is identical to that of the ICLR reporter plasmid ( A ) but lacks the ICLs and encodes Renilla luciferase as the reporter. C ) The chemical structure of the ICL (aoNao (7) in bold) in the reporter plasmid ( A ). The ICL is replacing two nucleotide bases at the interstrand counter positions and, thus, is not projected from the helix.…”

Section: Figurementioning

confidence: 99%

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A novel assay revealed that ribonucleotide reductase is functionally important for interstrand DNA crosslink repair

Fujii

Evison

Actis

et al. 2015

Bioorganic & Medicinal Chemistry

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Cells have evolved complex biochemical pathways for DNA interstrand crosslink (ICL) removal. Despite the chemotherapeutic importance of ICL repair, there have been few attempts to identify which mechanistic DNA repair inhibitor actually inhibits ICL repair. To identify such compounds, a new and robust ICL repair assay was developed using a novel plasmid that contains synthetic ICLs between a CMV promoter region that drives transcription and a luciferase reporter gene, and an SV40 origin of replication and the large T antigen (LgT) gene that enables self-replication in mammalian cells. In a screen against compounds that are classified as inhibitors of DNA repair or synthesis, the reporter generation was exquisitely sensitive to ribonucleotide reductase (RNR) inhibitors such as gemcitabine and clofarabine, but not to inhibitors of PARP, ATR, ATM, Chk1, and others. The effect was observed also by siRNA downregulation of RNR. Moreover, the reporter generation was also particularly sensitive to 3-AP, a non-nucleoside RNR inhibitor, but not significantly sensitive to DNA replication stressors, suggesting that the involvement of RNR in ICL repair is independent of incorporation of a nucleotide RNR inhibitor into DNA to induce replication stress. The reporter generation from a modified version of the plasmid that lacks the LgT-SV40ori motif was also adversely affected by RNR inhibitors, further indicating a role for RNR in ICL repair that is independent of DNA replication. Intriguingly, unhooking of cisplatin-ICL from nuclear DNA was significantly inhibited by low doses of gemcitabine, suggesting an unidentified functional role for RNR in the process of ICL unhooking. The assay approach could identify other molecules essential for ICLR in quantitative and flexible manner.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Figurementioning

confidence: 99%

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A novel assay revealed that ribonucleotide reductase is functionally important for interstrand DNA crosslink repair

Fujii

Evison

Actis

et al. 2015

Bioorganic & Medicinal Chemistry

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“…Several articles have reported that AMOs flanked with double‐stranded duplexes improve the inhibitory effects (Haraguchi et al., ; Haraguchi, Ozaki, & Iba, ; Robertson et al., ; Vermeulen et al., ); however, the stability of a double‐stranded structure depends on the sequences or the physicochemical conditions such as the temperature or ionic strength of the solution. We developed a site‐specific CL reaction that links a pair of AP sites in a complementary DNA duplex, as described in Basic Protocol 2 (Ichikawa et al., ). Recently, we constructed an AMO flanked by an interstrand cross‐linked 2′‐O‐methylated RNA duplex (CL) and found that the CL affects the anti‐miRNA activity (Mie et al., ).…”

Section: Commentarymentioning

confidence: 99%

“…In addition, the AP site becomes a recognition site for a number of small ligands that interact with the nucleobases surrounding the AP site. In contrast to the naturally occurring cross-linked AP sites, we previously developed a sequence-specific cross-link reaction that conjugates a pair of AP sites on complementary oligodeoxynucleotides (ODNs) using a bifunctional cross-linker containing the bisaminooxy groups (Ichikawa et al, 2012). The cross-link structure makes the duplex rigid (Mie, Kowata, Kojima, & Komatsu, 2012) and provides a stable DNA scaffold wherein enzymes are immobilized for use in a glucose microbiosensor (Hirano, Ikegami, Kowata, & Komatsu, 2017).…”

Section: Interstrand Cross-linked Duplexmentioning

confidence: 99%

Synthesis and Application of Interstrand Cross‐Linked Duplexes by Covalently Linking a Pair of Abasic Sites

Hirano

Kojima

Komatsu

2018

CP Nucleic Acid Chemistry

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Interstrand cross‐linking of DNA or RNA inhibits the double strands from dissociating into single strands. This article contains detailed procedures for the synthesis of a novel interstrand cross‐linker that comprises a bis‐aminooxy naphthalene derivative and a description of its use in the preparation of sequence‐specific interstrand cross‐linked oligonucleotide duplexes. The interstrand cross‐linker covalently connects a pair of apurinic/apyrimidinic sites in DNA/RNA duplexes with bis(aminooxy) groups. The resulting oxime linkages are stable under physiological conditions and greatly improve the thermal stability of the duplex. In addition, we construct a novel anti‐miRNA oligonucleotide (AMO) flanked by interstrand cross‐linked 2′‐O‐methylated RNA duplexes (CLs). AMO flanked by CLs at the 5′‐ and 3′‐termini exhibited high inhibition activity toward miRNA function in cells. The novel interstrand cross‐linker indicates potent activity and is applicable in biophysical studies, oligonucleotide therapeutics, and materials science. © 2018 by John Wiley & Sons, Inc.

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A Simple, High‐Yield Synthesis of DNA Duplexes Containing a Covalent, Thermally Cleavable Interstrand Cross‐Link at a Defined Location

Varela

Gates

2015

Angewandte Chemie

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Interstrand DNA-DNAc ross-links are highly toxic to cells because these lesions block the extraction of information from the genetic material. The pathwaysb yw hich cells repair cross-links are important, but not well understood. The preparation of chemically well-defined cross-linked DNA substrates represents as ignificant challenge in the study of cross-link repair.H ere as imple method is reported that employs "post-synthetic" modifications of commercially available 2'-deoxyoligonucleotides to install as ingle cross-link in high yield at as pecified location within aD NA duplex. The cross-linking process exploits the formation of ah ydrazone between an on-natural N 4 -amino-2'-deoxycytidine nucleobase and the aldehyde residue of an abasic site in duplex DNA. The resulting cross-link is stable under physiological conditions, but can be readily dissociated and re-formed through heatingcooling cycles.

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Interstrand cross-link of DNA by covalently linking a pair of abasic sites

Cited by 20 publications

References 35 publications

A novel assay revealed that ribonucleotide reductase is functionally important for interstrand DNA crosslink repair

A novel assay revealed that ribonucleotide reductase is functionally important for interstrand DNA crosslink repair

Synthesis and Application of Interstrand Cross‐Linked Duplexes by Covalently Linking a Pair of Abasic Sites

A Simple, High‐Yield Synthesis of DNA Duplexes Containing a Covalent, Thermally Cleavable Interstrand Cross‐Link at a Defined Location

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