Guanine Specific DNA Cleavage by Photoirradiation of Dibenzoyldiazomethane−Oligonucleotide Conjugates

Abstract

Photoirradiation of dibenzoyldiazomethane (DBDM) produced highly electrophilic benzoylketene via Wolff rearrangement. DBDM derivative possessing an aminoalkyl side chain induced a DNA cleavage selectively at guanine (G) residues upon photoirradiation and subsequent piperidine treatment. In order to devise photochemical DNA cleavers that can specifically alkylate a guanine residue proximal to the target sequence of long DNA fragments, a new reagent, DBDM-OSu, which facilitates the connection of DBDM unit to various DNA binders, was developed. DBDM-oligonucleotide (ODN) conjugates 5 and 6 were obtained by the coupling of 5‘-aminohexyl 8-mer [H2N-(CH2)6-d(ACGTCAGG)-3‘] and 15-mer [H2N-(CH2)6-d(ACGTCAGGTGGCACT)-3‘], respectively, with DBDM-OSu in aqueous acetonitrile in the presence of sodium bicarbonate. Photoirradiation of 5 and 6 in the presence of 25-mer 5‘-d(AGTGCCACCTGACGTCTG18CTCTCTC)-3‘ having a complementary sequence induced cross-linking of both oligomers. A distinct cleavage band at guanine residue (G18) was observed upon heating the cross-linked oligomers with piperidine. A similar DNA cleavage reaction of 5‘-d(AGTGCCACCTGACG14TG16CG18TG20CG22TCT)-3‘ having multiple guanine sites in the presence of DBDM-ODN conjugate 6 indicated that the most effectively cleaved site is G16. These results demonstrated that DBDM-oligonucleotide conjugates can serve as a new class of photonucleases that can cleave single-stranded DNA at predetermined guanine sites. Furthermore, the reagent DBDM-OSu can be used as a convenient and effective photoinducible electrophile for the cross-linking or the modification of biopolymers.

Mechanistic Studies on Photoinduced Cross-Linking and Specific Cleavage at Guanine by Dibenzoyldiazomethane-Oligodeoxynucleotide Conjugate

“…G-selective DNA cleavage may also occur by singlet oxygen mediated mechanism (24)(25)(26)(27) or by one-electron oxidation of DNA (28)(29)(30)(31)(32)(33)(34)(35). The experiment on the DNA cleavage by 1b in the presence of singlet oxygen quencher indicated that the G cleavage mediated by singlet oxygen, if it occurs, is only a minor process in the present case (22). The observed equal G cleavage of 5ЈGG3Ј sequence is also inconsistent with the one-electron oxidation mechanism, since 5Ј-G of 5ЈGG3Ј sequence is known to be the most readily oxidizable site in DNA one-electron oxidation (29)(30)(31)(32)(33)(34)(35).…”

“…Thus, the postactivation of an electrophile precursor by a phototriggered process would be highly attractive for sequencespecific DNA alkylation by designed ODN 2 conjugates. Toward this end, we have designed a novel photoinducible DNA-alkylating agent that can generate a strong electrophile after complexation with the target sequence triggered by photoirradiation and alkylate guanine base at the close vicinity (21)(22)(23). Our attention has been focused on the compounds having dibenzoyldiazomethane (DBDM) 1a as a chromophore, which produces highly electrophilic benzoyl ketene (BK) 2 via photochemical Wolff rearrangement (21)(22)(23).…”

“…Toward this end, we have designed a novel photoinducible DNA-alkylating agent that can generate a strong electrophile after complexation with the target sequence triggered by photoirradiation and alkylate guanine base at the close vicinity (21)(22)(23). Our attention has been focused on the compounds having dibenzoyldiazomethane (DBDM) 1a as a chromophore, which produces highly electrophilic benzoyl ketene (BK) 2 via photochemical Wolff rearrangement (21)(22)(23). We previously observed that DNA cleavage occurred selectively at guanine (G) residues when DNA was photoirradiated in the presence of water-soluble monomer DBDM 1b followed by hot piperidine treatment (21,22).…”

“…Our attention has been focused on the compounds having dibenzoyldiazomethane (DBDM) 1a as a chromophore, which produces highly electrophilic benzoyl ketene (BK) 2 via photochemical Wolff rearrangement (21)(22)(23). We previously observed that DNA cleavage occurred selectively at guanine (G) residues when DNA was photoirradiated in the presence of water-soluble monomer DBDM 1b followed by hot piperidine treatment (21,22). G-selective DNA cleavage may also occur by singlet oxygen mediated mechanism (24)(25)(26)(27) or by one-electron oxidation of DNA (28)(29)(30)(31)(32)(33)(34)(35).…”

Chemical Stability of Nucleic Acid–Derived Drugs

“…[106][107][108][109][110] Such electron transfer (hole migration) ultimately ends up at guanine, known as the most readily oxidizable site among the nucleobases. [111][112][113] The guanine moiety is also a preferred target of many other oxidizing agents, such as 1 O 2 , 114 or different electron-accepting photosensitizers.…”