Reduced mitomycin C induces heat-labile sites in DNA at specific sequences.
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We have investigated the sequence specificity of DNA damage induced by mitomycin C reduced with NaBH4, by using 3'- or 5'-end labeled DNA fragments of defined sequence. Mitomycin C reduced with NaBH4 induced heat-labile sites in DNA preferentially at specific sequences. The most preferred trinucleotide sequence for induction of heat-labile sites was GGT, followed by GGG, AGT, GAG, GGC and AGG. Active oxygens such as hydroxyl radical and singlet oxygen, and metal ions were involved in the induction of heat-labile sites. DNA was broken at the 3' side of deoxyguanosines and some of deoxyadenosines by heat-treatment. The produced oligonucleotides contained phosphoryl groups at the 5' termini. The 3' termini seemed not to have simple structures.Keywords:
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Mitomycin C
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We have investigated the sequence specificity of DNA damage induced by mitomycin C reduced with NaBH4, by using 3'- or 5'-end labeled DNA fragments of defined sequence. Mitomycin C reduced with NaBH4 induced heat-labile sites in DNA preferentially at specific sequences. The most preferred trinucleotide sequence for induction of heat-labile sites was GGT, followed by GGG, AGT, GAG, GGC and AGG. Active oxygens such as hydroxyl radical and singlet oxygen, and metal ions were involved in the induction of heat-labile sites. DNA was broken at the 3' side of deoxyguanosines and some of deoxyadenosines by heat-treatment. The produced oligonucleotides contained phosphoryl groups at the 5' termini. The 3' termini seemed not to have simple structures.
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Mitomycin C
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Impact of DNA Sequence and Oligonucleotide Length on a Polythiophene‐Based Fluorescent DNA Biosensor
Abstract DNA hybridization is a universal and specific mechanism for the recognition of biological targets. Some cationic polythiophene transducers sensitive to DNA structure have been previously utilized to detect such biomolecules. Further characterization of these systems indicates that both DNA sequence composition and length modulate the biosensor performance. It appears that different repeated sequence patterns cause different conformational changes of the polythiophene, from a more relaxed form to an extremely rigid one. A length difference between the DNA oligonucleotide probe and target has a detrimental effect on the fluorescent signal, but it can be attenuated by changing the sequence composition of the protruding target sequence. This demonstrates that the nature of DNA can be critical for hybridization‐based detection systems. magnified image
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Oligomer restriction
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Saenger sequencing has led the advances in molecular biology, while faster and cheaper next generation technologies are urgently needed. A newer approach exploits nanopores, natural or solid-state, set in an electrical field, and obtains base sequence information from current variations due to the passage of a ssDNA molecule through the pore. A hurdle in this approach is the fact that the four bases are chemically comparable to each other which leads to small differences in current obstruction. 'Base calling' becomes even more challenging because most nanopores sense a short sequence and not individual bases. Perhaps sequencing DNA via nanopores would be more manageable, if only the bases were two, and chemically very different from each other; a sequence of 1s and 0s comes to mind. Osmylated DNA comes close to such a sequence of 1s and 0s. Osmylation is the addition of osmium tetroxide bipyridine across the C5–C6 double bond of the pyrimidines. Osmylation adds almost 400% mass to the reactive base, creates a sterically and electronically notably different molecule, labeled 1, compared to the unreactive purines, labeled 0. If osmylated DNA were successfully sequenced, the result would be a sequence of osmylated pyrimidines (1), and purines (0), and not of the actual nucleobases. To solve this problem we studied the osmylation reaction with short oligos and with M13mp18, a long ssDNA, developed a UV–vis assay to measure extent of osmylation, and designed two protocols. Protocol A uses mild conditions and yields osmylated thymidines (1), while leaving the other three bases (0) practically intact. Protocol B uses harsher conditions and effectively osmylates both pyrimidines, but not the purines. Applying these two protocols also to the complementary of the target polynucleotide yields a total of four osmylated strands that collectively could define the actual base sequence of the target DNA.
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The DNA lesions induced by free 1O2 and the biological and mutagenic consequences of 1O2-induced DNA damage have been studied. Using anion exchange HPLC, reverse-phase HPLC with electrochemical detection and 32P-postlabelling methods, we have shown that 1O2 reacts with 2'-deoxyguanine 3'-monophosphate (dGp) but not with any other dNp. Reaction with dGp yields a large number of products; one minor product was identified as 7-hydro-8-oxo-2'-deoxyguanosine 3'-monophosphate (8-oxo-dGp), and a second tentatively as a formamidopyridine derivative of dGp. 8-Oxo-dGp was also found after reaction of 1O2 with single-stranded (ss) DNA, double-stranded (ds) DNA or an oligonucleotide (16-mer) having one G. With the oligonucleotide we found a second unidentified reaction product. With ss DNA, 8-oxo-dG was a much more prominent product than in the reaction of 1O2 with free dGp and the yield was about eight-fold higher than with ds DNA. This agrees with our finding that ss M13 DNA is at least 100-fold more sensitive than ds M13 DNA to biological inactivation by 1O2. The inactivation of ss M13 DNA must be largely due to 1O2-induced lesions other than 8-oxo-dG. In agreement with the observed preferential reaction of 1O2 with dG, most of the mutations induced by 1O2 in ss or ds M13mp10 DNA occurred at a G or G/C basepair, respectively. A preference for G(C) to T(A) transversions was observed for which 8-oxo-dG might have been responsible. In ss DNA, a significant number of mutations are characterized by the fact that a G is deleted.
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Telomere is an emerging target for the treatment of human cancers. Here, we report a structure-based approach to sequence-specific cleaving of human telomeric DNA by G-quadruplex formation. Oligonucleotide with multiphosphonate [DNA-EDTP.Ce(IV)] at the 5' end binds to human telomere DNA by G-quadruplex formation and causes a sequence-specific strand break. These results provide the first proof of concept for targeting the human telomere DNA based on G-quadruplex formation, and this may serve as a starting point for the design of more efficient telomere sequence-specific cleaving reagents by G-quadruplex formation.
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The sequence specificity of photobinding to DNA of two tetrahydrobenzopsoralen derivatives has been investigated by testing the photoreactivity toward a number of self-complementary oligonucleotides. The thermodynamic constant for noncovalent binding to each DNA sequence was evaluated. The extent of photoreactivity was greatly dependent upon base composition. The two tetracyclic compounds show similar behavior in comparison to other bifunctional derivatives. Their overall rate constants were greatly enhanced in comparison to classical psoralens. However, their high efficiency of covalent binding is counterbalanced by low affinity for noncovalent interaction with DNA.
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