Synthesis of photo-responsive acridine-modified DNA and its application to site-selective RNA scission

Photo-responsive phosphoramidite monomers, which bear an azobenzene between acridine and the phosphoramidite unit, were synthesized, and incorporated into oligonucleotides. Upon UV irradiation, the azobenzene in the modified DNA efficiently isomerized from the trans isomer into the cis isomer. Although the T(m) values of their duplexes with complementary DNA were not much changed by the isomerization, site-selective RNA scission was significantly accelerated by the UV irradiation when Mn(II) ion was used as the catalyst for RNA scission.     

Design of phosphoramidite monomer for optimal incorporation of functional intercalator to main chain of oligonucleotide

Chirally pure phosphoramidite monomers bearing 9-amino-6-chloro-2-methoxyacridine were synthesized from D- or L-threoninol and omega-aminocarboxylic acid, and incorporated into oligonucleotides. These acridine-DNA conjugates formed stable duplexes with complementary RNA because of intercalation of the acridine to DNA/RNA heteroduplexes. The stability of duplexes was not very dependent on either the chirality of the central carbon bearing the acridine or the length of the side chain. However, the ability for site-selective activation of the phosphodiester linkage in front of the acridine, which induced Lu(III)-promoted RNA scission, was strongly dependent on these two factors. The largest activation was achieved when the monomer unit was prepared from L-threoninol and 4-aminobutyric acid and the acridine was bound to the amino group. By attaching the more acidic 9-amino-2-methoxy-6-nitroacridine to this optimized scaffold, a quite effective acridine-DNA conjugate for site-selective RNA scission was obtained.     

Photo-regulation of DNA/RNA duplex formation by azobenzene-tethered DNA towards antisense strategy.

Modified DNA carrying an azobenzene was successfully applied to the photo-regulation of DNA/RNA hybridization. When the azobenzene was isomerized from trans- to cis-form on UV-irradiation, the melting temperature of the duplex was significantly lowered. This process was totally reversible so that the Tm increased by cis-->trans isomerization induced by visible light irradiation.     

Chemical synthesis of oligodeoxyribonucleotides containing the Dewar valence isomer of the (6-4) photoproduct and their use in (6-4) photolyase studies

The pyrimidine(6–4)pyrimidone photoproduct, a major UV lesion formed between adjacent pyrimidine bases, is transformed to its Dewar valence isomer upon exposure to UVA/UVB light. We have synthesized a phosphoramidite building block of the Dewar photoproduct formed at the thymidylyl(3′–5′)thymidine site and incorporated it into oligodeoxyribonucleotides. The diastereoisomers of the partially protected dinucleoside monophosphate bearing the (6–4) photoproduct, which were caused by the chirality of the phosphorus atom, were separated by reversed-phase chromatography, and the (6–4) photoproduct was converted to the Dewar photoproduct by irradiation of each isomer with Pyrex-filtered light from a high-pressure mercury lamp. The Dewar photoproduct was stable under both acidic and alkaline conditions at room temperature. After characterization of the isomerized base moiety by NMR spectroscopy, a phosphoramidite building block was synthesized in three steps. Although the ordinary method could be used for the oligonucleotide synthesis, benzimidazolium triflate as an alternative activator yielded better results. The oligonucleotides were used for the analysis of the reaction and the binding of Xenopus (6–4) photolyase. Although the affinity of this enzyme for the Dewar photoproduct-containing duplex was reportedly similar to that for the (6–4) photoproduct-containing substrate, the results suggested a difference in the binding mode.     

Conformational and phase transitions in DNA—photosensitive surfactant solutions: Experiment and modeling

DNA binding to trans‐ and cis‐isomers of azobenzene containing cationic surfactant in 5 mM NaCl solution was investigated by the methods of dynamic light scattering (DLS), low‐gradient viscometry (LGV), atomic force microscopy (AFM), circular dichroism (CD), gel electrophoresis (GE), flow birefringence (FB), UV–Vis spectrophotometry. Light‐responsive conformational transitions of DNA in complex with photosensitive surfactant, changes in DNA optical anisotropy and persistent length, phase transition of DNA into nanoparticles induced by high surfactant concentration, as well as transformation of surfactant conformation under its binding to macromolecule were studied. Computer simulations of micelles formation for cis‐ and trans‐isomers of azobenzene containing surfactant, as well as DNA‐surfactant interaction, were carried out. Phase diagram for DNA‐surfactant solutions was designed. The possibility to reverse the DNA packaging induced by surfactant binding with the dilution and light irradiation was shown. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 109–122, 2015.     

Selenium derivatization and crystallization of DNA and RNA oligonucleotides for X-ray crystallography using multiple anomalous dispersion

We report here the solid phase synthesis of RNA and DNA oligonucleotides containing the 2′-selenium functionality for X-ray crystallography using multiwavelength anomalous dispersion. We have synthesized the novel 2′-methylseleno cytidine phosphoramidite and improved the accessibility of the 2′-methylseleno uridine phosphoramidite for the synthesis of many selenium-derivatized DNAs and RNAs in large scales. The yields of coupling these Se-nucleoside phosphoramidites into DNA or RNA oligonucleotides were over 99% when 5-(benzylmercapto)-1H-tetrazole was used as the coupling reagent. The UV melting study of A-form dsDNAs indicated that the 2′-selenium derivatization had no effect on the stability of the duplexes with the 3′-endo sugar pucker. Thus, the stems of functional RNA molecules with the same 3′-endo sugar pucker appear to be the ideal sites for the selenium derivatization with 2′-Se-C and 2′-Se-U. Crystallization of the selenium-derivatized oligonucleotides is also reported here. The results demonstrate that this 2′-selenium functionality is suitable for RNA and A-form DNA derivatization in X-ray crystallography.     

DNA and RNA strand scission by copper, zinc and manganese superoxide dismutases

Copper/zinc (Cu/ZnSOD) and manganese (MnSOD) superoxide dismutases which catalyze the dismutation of toxic superoxide anion, O _inf2 ^sup– , to O₂ and H₂O₂, play a major role in protecting cells from toxicity of oxidative stress. However, cells overexpressing either form of the enzyme show signs of toxicity, suggesting that too much SOD may he injurious to the cell. To elucidate the possible mechanism of this cytotoxicity, the effect of SOD on DNA and RNA strand scission was studied. High purity preparations of Cu/ZnSOD and MnSOD were tested in an in vitro assay in which DNA cleavage was measured by conversion of phage X174 supercoiled double-stranded DNA to open circular and linear forms. Both types of SOD were able to induce DNA strand scission generating single- and double-strand breaks in a process that required oxygen and the presence of fully active enzyme. The DNA strand scission could be prevented by specific anti-SOD antibodies added directly or used for immunodepletion of SOD. Requirement for oxygen and the effect of Fe(II) and Fe(III) ions suggest that cleavage of DNA may be in part mediated by hydroxyl radicals formed in Fenton-type reactions where enzyme-bound transition metals serve as a catalyst by first being reduced by superoxide and then oxidized by H₂O₂. Another mechanism was probably operative in this system, since in the presence of magnesium DNA cleavage by SOD was oxygen independent and not affected by sodium cyanide. It is postulated that SOD, by having a similar structure to the active center of zinc-containing nucleases, is capable of exhibiting non-specific nuclease activity causing hydrolysis of the phosphodiester bonds of DNA and RNA. Both types of SOD were shown to effectively cleave RNA. These findings may help explain the origin of pathology of certain hereditary diseases genetically linked to Cu/ZnSOD gene.     

RNA polymerase II large subunit is cleaved by caspases during DNA damage-induced apoptosis

UV radiation induces DNA lesions that are repaired by the nucleotide excision repair (NER) pathway. Cells that are NER deficient such as those derived from xeroderma pigmentosum (XP) patients are susceptible to apoptosis after 10J/m(2) UV radiation, a dose largely survivable by repair proficient cells. Herein, we report that RNA polymerase II large subunit (RNAP II-LS) undergoes caspase-mediated cleavage, yielding a 140kDa C-terminal fragment in XP lymphoblasts but not NER proficient lymphoblasts after 10J/m(2) UV irradiation. Cleavage could also be induced by cisplatin or oxaliplatin, but not transplatin, an isomer of cisplatin that does not induce DNA adducts. The cleavage of RNAP II-LS was blocked by a panel of caspase inhibitors but not by proteasomal inhibitors or inhibitors of other proteases. In vitro cleavage with caspase 8 yielded the same 140kDa RNAP II-LS fragment observed in vivo. Using site-directed mutagenesis, the RNAP II-LS cleavage site was localized to an LETD sequence ending at residue 1339, which is near its C-terminal domain.     

Chemical synthesis,DNA incorporation and biological study of a new photocleavable 2′-deoxyadenosine mimic

The phototriggered cleavage of chemical bonds has found numerous applications in biology, particularly in the field of gene sequencing through photoinduced DNA strand scission. However, only a small number of modified nucleosides that are able to cleave DNA at selected positions have been reported in the literature. Herein, we show that a new photoactivable deoxyadenosine analogue, 3-nitro-3-deaza-2′-deoxyadenosine (d(3-NiA)), was able to induce DNA backbone breakage upon irradiation (λ > 320 nm). The d(3-NiA) nucleoside was chemically incorporated at desired positions into 40-mer oligonucleotides as a phosphoramidite monomer and subsequent hybridization studies confirmed that the resulting modified duplexes display a behaviour that is close to that of the related natural sequence. Enzymatic action of the Klenow fragment exonuclease free revealed the preferential incorporation of dAMP opposite the 3-NiA base. On the other hand, incorporation of the analogous 3-NiA triphosphate to a primer revealed high enzyme efficiency and selectivity for insertion opposite thymine. Furthermore, only the enzymatically synthesized base pair 3-NiA:T was a substrate for further extension by the enzyme. All the hybridization and enzymatic data indicate that this new photoactivable 3-NiA triphosphate can be considered as a photochemically cleavable dATP analogue.     

Photocontrol of kinesin ATPase activity using an azobenzene derivative

Azobenzene is a photochromic molecule that undergoes rapid and reversible isomerization between the cis- and trans-forms in response to ultraviolet (UV) and visible (VIS) light irradiation, respectively. Here, we introduced the sulfhydryl-reactive azobenzene derivative 4-phenylazophenyl maleimide (PAM) into the functional region of kinesin to reversibly regulate the ATPase activity of kinesin by photoirradiation. We prepared five kinesin motor domain mutants, A247C, L249C, A252C, G272C and S275C, which contained a single reactive cysteine residue in loops L11 and L12. These loops are considered to be key regions for the functioning of kinesin as a motor protein. PAM was stoichiometrically incorporated into the cysteine residues in the loops of the mutants. The PAM-modified S275C mutant exhibited reversible alterations in ATPase activity accompanied by cis-trans isomerization upon UV and VIS light irradiation. The ATPase activity exhibited by the cis-isomer of the PAM bound to the mutant was two times higher than that of the trans-isomer. Further, the PAM-modified L249C mutant exhibited reversible alterations in ATPase activity on UV-VIS light irradiation but exhibited the opposite effect on UV and VIS light irradiation. Using a photochromic azobenzene derivative, we have demonstrated that the ATPase activity of the motor protein kinesin is photoregulated.     

Repetitious and unique sequences in the heterogeneous nuclear and cytoplasmic messenger RNA of mammalian and insect cells

Initiation of DNA synthesis has been followed in mouse myeloma cells grown in suspension culture. In cells labeled with ³H-thymidine for short times, label first appears in short fragments of DNA which can be chased into bulk DNA (>50 S) upon further incubation in unlabeled thymidine. In a 15 min pulse, DNA fragments with a sedimentation coefficient of 30 S tend to accumulate. Our results support the contention that DNA synthesis is discontinuous in myeloma cells.However, a search for RNA associated with nascent DNA in the myeloma system was unsuccessful. Newly synthesized DNA was isolated on a benzoylated naphthoylated DEAE cellulose column. After heat denaturation, this fraction was centrifuged to equilibrium in a Cs₂SO₄ density gradient. The nascent DNA displays no shift in density greater than the density of the bulk DNA. When cells were pulse labeled with ³H-uridine and the nascent DNA fraction analyzed on Cs₂SO₄ density gradients, no ³H-labeled RNA was found associated with the DNA peak or at intermediate densities that would be indicative of a RNA-DNA molecule, covalently linked. Unless scission of the RNA primers occurs immediately after the initiation of DNA synthesis, our results indicate that DNA synthesis commences without RNA primers in myeloma cells.     

Synthesis and hybridization properties of oligonucleotides containing 6-membered azasugar nucleotides

A modified nucleoside was synthesized with adenine and a 6-membered azasugar, and it was converted to the phosphoramidite which was used for the incorporation into oligonucleotides. The hybridization properties of the modified oligonucleotides with DNA and RNA were studied.     

5-(phenylthiomethyl)-2'-deoxyuridine as an efficient photoreactive precursor to generate single and multiple lesions within DNA fragments

5-(Phenylthiomethyl)-2'-deoxyuridine was successfully incorporated into DNA oligomers by automated DNA synthesis using phosphoramidite chemistry. UV exposure of the latter thionucleoside containing oligonucleotides under anaerobic and aerobic conditions gives rise to specific base lesions. The photoproducts have been isolated and further characterized on the basis of NMR and mass spectrometric analyses.     

5-(PHENYLTHIOMETHYL)-2′-DEOXYURIDINE AS AN EFFICIENT PHOTOREACTIVE PRECURSOR TO GENERATE SINGLE AND MULTIPLE LESIONS WITHIN DNA FRAGMENTS

5-(Phenylthiomethyl)-2′-deoxyuridine was successfully incorporated into DNA oligomers by automated DNA synthesis using phosphoramidite chemistry. UV exposure of the latter thionucleoside containing oligonucleotides under anaerobic and aerobic conditions gives rise to specific base lesions. The photoproducts have been isolated and further characterized on the basis of NMR and mass spectrometric analyses.     

Effect of oxygen and UV irradiation on nucleic acid and protein syntheses in Bacteroides fragilis.

Macromolecular synthesis in Bacteroides fragilis was decreased by oxygen. DNA degradation and synthesis were inhibited by UV irradiation and oxygen, but RNA and protein syntheses were relatively unaffected.     

Efficacy of UV Irradiation in Inactivating Cryptosporidiumparvum Oocysts

To evaluate the effectiveness of UV irradiation in inactivating Cryptosporidium parvum oocysts, the animal infectivities and excystation abilities of oocysts that had been exposed to various UV doses were determined. Infectivity decreased exponentially as the UV dose increased, and the required dose for a 2-log₁₀ reduction in infectivity (99% inactivation) was approximately 1.0 mWs/cm² at 20°C. However, C. parvum oocysts exhibited high resistance to UV irradiation, requiring an extremely high dose of 230 mWs/cm² for a 2-log₁₀ reduction in excystation, which was used to assess viability. Moreover, the excystation ability exhibited only slight decreases at UV doses below 100 mWs/cm². Thus, UV treatment resulted in oocysts that were able to excyst but not infect. The effects of temperature and UV intensity on the UV dose requirement were also studied. The results showed that for every 10°C reduction in water temperature, the increase in the UV irradiation dose required for a 2-log₁₀ reduction in infectivity was only 7%, and for every 10-fold increase in intensity, the dose increase was only 8%. In addition, the potential of oocysts to recover infectivity and to repair UV-induced injury (pyrimidine dimers) in DNA by photoreactivation and dark repair was investigated. There was no recovery in infectivity following treatment by fluorescent-light irradiation or storage in darkness. In contrast, UV-induced pyrimidine dimers in the DNA were apparently repaired by both photoreactivation and dark repair, as determined by endonuclease-sensitive site assay. However, the recovery rate was different in each process. Given these results, the effects of UV irradiation on C. parvum oocysts as determined by animal infectivity can conclusively be considered irreversible.     

Copper-dependent DNA damage induced by hydrazobenzene,an azobenzene metabolite

Hydrazobenzene is carcinogenic to rats and mice and azobenzene is carcinogenic to rats. Hydrazobenzene is a metabolic intermediate of azobenzene. To clarify the mechanism of carcinogenesis by azobenzene and hydrazobenzene, we investigated DNA damage induced by hydrazobenzene, using ³²P-5′-end-labeled DNA fragments obtained from the c-Ha-ras-1 proto-oncogene and the p53 tumor suppressor gene. Hydrazobenzene caused DNA damage in the presence of Cu(II). Piperidine treatment enhanced the DNA damage greatly, suggesting that hydrazobenzene caused base modification and liberation. However, azobenzene did not cause DNA damage even in the presence of Cu(II). Hydrazobenzene plus Cu(II) caused DNA damage frequently at thymine residues. Catalase and a Cu(I)-specific chelator inhibited Cu(II)-mediated DNA damage by hydrazobenzene. Typical ^·OH scavengers did not inhibit the DNA damage. The main active species is probably a metal oxygen complex, such as Cu(I)-OOH. Formation of 8-oxo-7, 8-dihydro-2′-deoxyguanosine was increased by hydrazobenzene in the presence of Cu(II). Oxygen consumption and UV-Visible spectroscopic measurements have shown that hydrazobenzene is autoxidized to azobenzene with H₂O₂ formation. It is considered that the metal-mediated DNA damage by hydrazobenzene through H₂O₂ generation may be relevant for the expression of carcinogenicity of azobenzene and hydrazobenzene.     

Cytological evidence for DNA chain elongation after UV irradiation in the S phase

Human cells irradiated with UV light synthesize lower molecular weight DNA than unirradiated cells. This reduction in molecular weight is greater in xeroderma pigmentosum (XP) cells than in normal cells. The molecular weight of DNA is further reduced by the addition of caffeine to XP cells. By several hours after irradiation, DNA fragments are barely detectable. Cells from excision-proficient and excision-deficient XP patients were studied autoradiographically to produce cytological evidence of DNA chain elongation. Replicate cultures with and without caffeine were synchronized and irradiated with UV light during the S phase. Caffeine was removed in G₂, and the cells were labeled with ³H-thymidine. Results showed significantly increased labeling during G₂ of excision-deficient XP cells. Labeling was dependent on both time of irradiation and presence of caffeine. The XP variant cells had no increase in labeling for any irradiation time.Published with the approval of the Director of the West Virginia Agricultural Experiment Station as Scientific Paper No. 1608. Supported by N.I.C. Grant TO1CA05170-10.