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Interstrand cross-link and bioconjugate formation in RNA from a modified nucleotide.

ABSTRACT: RNA oligonucleotides containing a phenyl selenide derivative of 5-methyluridine were chemically synthesized by solid-phase synthesis. The phenyl selenide is rapidly converted to an electrophilic, allylic phenyl seleneate under mild oxidative conditions. The phenyl seleneate yields interstrand cross-links when part of a duplex and is useful for synthesizing oligonucleotide conjugates. Formation of the latter is illustrated by reaction of an oligonucleotide containing the phenyl selenide with amino acids in the presence of mild oxidant. The products formed are analogous to those observed in tRNA that are believed to be formed posttranslationally via a biosynthetic intermediate that is chemically homologous to the phenyl seleneate.

SUBMITTER: Sloane JL

PROVIDER: S-EPMC4201359 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Interstrand cross-link and bioconjugate formation in RNA from a modified nucleotide.

Sloane Jack L JL Greenberg Marc M MM

The Journal of organic chemistry 20141008 20

RNA oligonucleotides containing a phenyl selenide derivative of 5-methyluridine were chemically synthesized by solid-phase synthesis. The phenyl selenide is rapidly converted to an electrophilic, allylic phenyl seleneate under mild oxidative conditions. The phenyl seleneate yields interstrand cross-links when part of a duplex and is useful for synthesizing oligonucleotide conjugates. Formation of the latter is illustrated by reaction of an oligonucleotide containing the phenyl selenide with amin ...[more]

PMID: 25295850

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Project description:DNA interstrand cross-links have important biological consequences and are useful biotechnology tools. Phenylselenyl substituted derivatives of thymidine (1) and 5-methyl-2'-deoxycytidine (5) produce interstrand cross-links in duplex DNA when oxidized by NaIO4. The mechanism involves a [2,3]-sigmatropic rearrangement of the respective selenoxides to the corresponding methide type intermediates, which ultimately produce the interstrand cross-links. Determination of the rate constants for the selenoxide rearrangements indicates that the rate-determining step for cross-linking is after methide formation. Cross-linking by the thymidine derivative in duplex DNA shows a modest kinetic preference when flanked by pyrimidines as opposed to purines. In contrast, the rate constant for cross-link formation from 5 opposite dG in duplex DNA is strongly dependent upon the flanking sequence and, in general, is at least an order of magnitude slower than that for 1 in an otherwise identical sequence. Introduction of mispairs at the base pairs flanking 5 or substitution of the opposing dG by dI significantly increases the rate constant and yield for cross-linking, indicating that stronger hydrogen bonding between the methide derived from it and dG compared to dA and the respective electrophile derived from 1 limits reaction by increasing the barrier to rotation into the required syn-conformation. Incorporation of 1 or 5 in triplex forming oligonucleotides (TFOs) that utilize Hoogsteen base pairing also yields interstrand cross-links. The dC derivative produces ICLs approximately 10x faster than the thymidine derivative when incorporated at the 5'-termini of the TFOs and higher yields when incorporated at internal sites. The slower, less efficient ICL formation emanating from 1 is attributed to reaction at N1-dA, which requires local melting of the duplex. In contrast, 5 produces cross-links by reacting with N7-dG. The cross-linking reactions of 1 and 5 illustrate the versatility and utility of these molecules as mechanistic probes and tools for biotechnology.

Project description:DNA interstrand cross-links (ICLs) are products of chemotherapeutic agents and cellular metabolic processes that block both replication and transcription. If left unrepaired, ICLs are extremely toxic to cells, and ICL repair mechanisms contribute to the survival of certain chemotherapeutic resistance tumors. A critical step in ICL repair involves unhooking the cross-link. In the absence of a homologous donor sequence, the resulting gap can be filled in by a repair synthesis step involving bypass of the cross-link remnant. Here, we examine the effect of cross-link structure on the ability of unhooked DNA substrates to undergo repair synthesis in mammalian whole cell extracts. Using 32P incorporation assays, we found that repair synthesis occurs efficiently past the site of damage when a DNA substrate containing a single N4C-ethyl-N4C cross-link is incubated in HeLa or Chinese hamster ovary cell extracts. This lesion, which can base pair with deoxyguanosine, is readily bypassed by both Escherichia coli DNA polymerase I and T7 DNA polymerase in a primer extension assay. In contrast, bypass was not observed in the primer extension assay or in mammalian cell extracts when DNA substrates containing a N3T-ethyl-N3T or N1I-ethyl-N3T cross-link, whose linkers obstruct the hydrogen bond face of the bases, were used. A modified phosphorothioate sequencing method was used to analyze the ICL repair patches created in the mammalian cell extracts. In the case of the N4C-ethyl-N4C substrate, the repair patch spanned the site of the cross-link, and the lesion was bypassed in an error-free manner. However, although the N3T-ethyl-N3T and N1I-ethyl-N3T substrates were unhooked in the extracts, bypass was not detected. These and our previous results suggest that although the chemical structure of an ICL may not affect initial cross-link unhooking, it can play a significant role in subsequent processing of the cross-link. Understanding how the physical and chemical differences of ICLs affect repair may provide a better understanding of the cytotoxic and mutagenic potential of specific ICLs.

Dataset Information

Interstrand cross-link and bioconjugate formation in RNA from a modified nucleotide.

Publications

Interstrand cross-link and bioconjugate formation in RNA from a modified nucleotide.

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