Synthesis of cross-linked DNA containing oxidized abasic site analogues (original) (raw)
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The Journal of Organic Chemistry, 2014
Duplex DNA with terminal and internal sugar cross-links were synthesized by the CuAAC reaction from oligonucleotides containing 2′-O-propargyl-2-aminoadenosine as a clickable site and a bifunctional azide (4). Stepwise click chemistry was employed to introduce cross-links at internal and terminal positions. Copper turnings were used as catalyst, reducing the copper load of the reaction mixture and avoiding complexing agents. For oligonucleotide building block synthesis, a protecting group strategy was developed for 2′-O-propargyl-2-aminoadenosine owing to the rather different reactivities of the two amino groups. Phosphoramidites were synthesized bearing clickable 2′-O-propargyl residues (14 and 18) as well as a 2′-deoxyribofuranosyl residue (10). Hybridization experiments of non-cross-linked oligonucleotides with 2,6-diaminopurine as nucleobase showed no significant thermal stability changes over those containing adenine. Surprisingly, an isobutyryl group protecting the 2-amino function has no negative impact on the stability of DNA−DNA and DNA−RNA duplexes. Oligonucleotide duplexes with cross-linked 2′-O-propargylated 2-aminoadenosine (1) and 2′-O-propargylated adenosine (3) at terminal positions are significantly stabilized (ΔT m = +29°C). The stability results from a molecularity change from duplex to hairpin melting and is influenced by the ligation position. Terminal ligation led to higher melting duplexes than corresponding hairpins, while duplexes with central ligation sites were less stable.
Efficient Synthesis of DNA Duplexes Containing Reduced Acetaldehyde Interstrand Cross-Links
Journal of the American Chemical Society, 2022
DNA interstrand cross-links (ICLs) prevent DNA replication and transcription and can lead to potentially lethal events, such as cancer or bone marrow failure. ICLs are typically repaired by proteins within the Fanconi Anemia (FA) pathway, although the details of the pathway are not fully established. Methods to generate DNA containing ICLs are key to furthering the understanding of DNA cross-link repair. A major route to ICL formation in vivo involves reaction of DNA with acetaldehyde, derived from ethanol metabolism. This reaction forms a threecarbon bridged ICL involving the amino groups of adjacent guanines in opposite strands of a duplex resulting in amino and imino functionalities. A stable reduced form of the ICL has applications in understanding the recognition and repair of these types of adducts. Previous routes to creating DNA duplexes containing these adducts have involved lengthy post-DNA synthesis chemistry followed by reduction of the imine. Here, an efficient and high-yielding approach to the reduced ICL using a novel N 2-((R)-4-trifluoroacetamidobutan-2-yl)-2′-deoxyguanosine phosphoramidite is described. Following standard automated DNA synthesis and deprotection, the ICL is formed overnight in over 90% yield upon incubation at room temperature with a complementary oligodeoxyribonucleotide containing 2-fluoro-2′deoxyinosine. The cross-linked duplex displayed a melting transition 25°C higher than control sequences. Importantly, we show using the Xenopus egg extract system that an ICL synthesized by this method is repaired by the FA pathway. The simplicity and efficiency of this methodology for preparing reduced acetaldehyde ICLs will facilitate access to these DNA architectures for future studies on cross-link repair.
Probing DNA interstrand cross-link formation by an oxidized abasic site using nonnative nucleotides
Bioorganic & Medicinal Chemistry, 2011
The C4'-oxidized abasic site (C4-AP) forms two types of interstrand cross-links with the adjacent nucleotides in DNA. Previous experiments revealed that dG does not react with the lesion and that formation of one type of cross-link is catalyzed by the opposing dA. Iso-guanosine•dC and 2aminopurine•dT base pairs were used to determine why dG does not cross-link with C4-AP despite its well known reactivity with other bis-electrophiles. 7-Deaza-2'-deoxyadenosine was used to probe the role of the nucleotide opposite C4-AP in the catalysis of interstrand cross-link formation.
Site-specific inter-strand cross-links of DNA duplexes
Chemical science (Royal Society of Chemistry : 2010), 2013
We report the development of technology that allows inter-strand coupling across various positions within one turn of DNA. Four 2'-modified nucleotides were synthesized as protected phosphoramidites and incorporated into DNA oligonucleotides. The modified nucleotides contain either 5-atom or 16-atom linker components, with either amine or carboxylic acid functional groups at their termini, forming 10 or 32 atom (11 or 33 bond) linkages. Chemical coupling of the amine and carboxylate groups in designed strands resulted in the formation of an amide bond. Coupling efficiency as a function of trajectory distance between the individual linker components was examined. For those nucleotides capable of forming inter-strand cross-links (ICLs), coupling yields were found to depend on temperature, distance, and linker length, enabling several approaches that can control regioselective linkage. In the most favorable cases, the coupling yields are quantitative. Spectroscopic measurements of ...
Repair and replication of oxidized DNA bases using modified oligodeoxyribonucleotides
Biochimie, 2000
Modified oligodeoxyribonucleotides (ODNs) are powerful tools to assess the biological significance of oxidized lesions to DNA. For this purpose, we developed original synthetic pathways for the site-specific insertion of several oxidized bases into DNA fragments. Thus, the chemical solid-phase synthesis of ODNs using original strategies of protection and mild conditions of deprotection, as well as a specific post-oxidation approach of an unique nucleoside residue within the sequence have been applied. These two approaches of preparation allowed us to have access to a set of modified ODNs that contain a single modified nucleoside, i.e., N-(2-deoxy--D-erythro-pentofuranosyl)formylamine (dF), 5-hydroxy-2'-deoxycytidine (5-OHdCyd), thymidine glycol , 5,6-dihydrothymidine (DHdThd), 2,2-diamino-4-[(2-deoxy--D-erythro-pentofuranosyl)-amino]-5(2H)-oxazolone (dZ), N-(2-deoxy--D-erythro-pentofuranosyl)cyanuric acid (dY), 5',8-cyclo-2'-deoxyguanosine (cyclodGuo) and 5',8-cyclo-2'-deoxyadenosine (cyclodAdo). The substrates were used to investigate recognition and removal of the lesions by bacterial DNA N-glycosylases, including endonuclease III (endo III) and Fapy glycosylase (Fpg). In addition, the DNA polymerase-mediated nucleotide incorporation opposite the damage was determined using modified ODNs as templates. © 2000 Société française de biochimie et biologie moléculaire/Éditions scientifiques et médicales Elsevier SAS oxidized DNA bases / synthetic oligonucleotides / DNA N-glycosylases / DNA polymerases * Correspondence and reprints Abbreviations: ODN, oligodeoxyribonucleotide; BER, base excision repair; Fpg, formamidopyrimidine-DNA N-glycosylase; endo III, endonuclease III; Kf, Klenow fragment of E. coli polymerase I; Taq pol, Taq DNA polymerase; MALDI-TOF MS, matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry.
Targeted Generation of DNA Strand Breaks Using Pyrene-Conjugated Triplex-Forming Oligonucleotides †
Biochemistry, 2008
Gene targeting by triplex-forming oligonucleotides (TFOs) 1 has proven useful for gene modulation in vivo. Photoreactive molecules have been conjugated to TFOs to direct sequence-specific damage in double-stranded DNA. However, the photoproducts are often repaired efficiently in cells. This limitation has led to the search for sequence-specific photoreactive reagents that can produce more genotoxic lesions. Here we demonstrate that photoactivated pyrene-conjugated TFOs (pyr-TFOs) induce DNA strand breaks near the pyrene moiety with remarkably high efficiency, and also produce covalent pyrene-DNA adducts. Free radical scavenging experiments demonstrated a role for singlet oxygen activated by the singlet-excited state of pyrene in the mechanism of pyr-TFO-induced DNA damage. In cultured mammalian cells, the effect of photoactivated pyr-TFO-directed DNA damage was to induce mutations, in the form of deletions, ~7-fold over background levels, at the targeted site. Thus, pyr-TFOs represent a potentially powerful new tool for directing DNA strand breaks to specific chromosomal locations for biotechnological and potential clinical applications. Triplex-forming oligonucleotides (TFOs) ‡ have been employed to modulate gene structure and function, both in vitro and in vivo. When conjugated to DNA damaging agents, TFOs can induce site-directed DNA damage resulting in enhanced mutagenesis and recombination [reviewed in (1-8)]. Reactive molecules that have been conjugated to TFOs include, but are not limited to, 2-amino-6-vinylpurine, haloacetyl amide, aryl nitrogen mustard, N 4 ,N 4etheno-5-methyldeoxycytidine, and various psoralen derivatives. All of these induce sitespecific cross-linking and/or alkylation in double-stranded DNA (dsDNA) (9-15). Psoralenlinked TFOs (pso-TFOs), which can induce covalent monoadducts on one strand or cross-links with both strands of duplex DNA with high efficiency and sequence specificity, are the most studied of the photoreactive TFOs. Pso-TFOs can yield site-specific DNA damage with high efficiency and have been used to introduce mutations and inactivate target genes. However, the existence of cellular mechanisms for repairing the pso-TFO photoproducts without introduction of sequence errors has limited their utility.
Tetrahedron, 1999
Protected forms of 1,2,3-propanetriol and cis, cis-l,3,5-cyclohexanetriol were incorporated onto solid supports which were exploited in the solid phase synthesis of 3'-3' linked oligodeoxyribonucleotides (ODNs), involving only nucleoside 3'-phosphoramidites as building blocks. UV thermal denaturation analysis showed the ability of ODNs with this inversion of polarity motif to cooperatively hybridize with duplexes of the type 5'-(Pu)m(PY)n-3' in an alternate strand recognition approach.
Journal of the American Chemical Society, 2013
We recently reported that the aldehyde residue of an abasic (Ap) site in duplex DNA can generate an interstrand cross-link via reaction with a guanine residue on the opposing strand. This finding is intriguing because the highly deleterious nature of interstrand cross-links suggests that even small amounts of Ap-derived cross-links could make a significant contribution to the biological consequences stemming from the generation of Ap sites in cellular DNA. Incubation of 21-bp duplexes containing a central 5′-CAp sequence under conditions of reductive amination (NaCNBH 3 , pH 5.2) generated much higher yields of cross-linked DNA than reported previously. At pH 7, in the absence of reducing agents, these Ap-containing duplexes also produced cross-linked duplexes that were readily detected on denaturing polyacrylamide gels. Cross-link formation was not highly sensitive to reaction conditions, and the cross-link, once formed, was stable to a variety of workup conditions. Results of multiple experiments including MALDI-TOF mass spectrometry, gel mobility, methoxyamine capping of the Ap aldehyde, inosine-for-guanine replacement, hydroxyl radical footprinting, and LC−MS/MS were consistent with a cross-linking mechanism involving reversible reaction of the Ap aldehyde residue with the N 2-amino group of the opposing guanine residue in 5′-CAp sequences to generate hemiaminal, imine, or cyclic hemiaminal cross-links (7−10) that were irreversibly converted under conditions of reductive amination (NaCNBH 3 /pH 5.2) to a stable amine linkage. Further support for the importance of the exocyclic N 2-amino group in this reaction was provided by an experiment showing that installation of a 2-aminopurine-thymine base pair at the cross-linking site produced high yields (15−30%) of a cross-linked duplex at neutral pH, in the absence of NaCNBH 3 .