DNA cleavage photoinduced by new water-soluble zinc porphyrins linked to 9-methoxyellipticine.

Two hybrid molecules based on a water-soluble zinc porphyrin covalently linked to 9-methoxyellipticine, 1 and 2, were studied as photoactivable DNA cleavers. The behaviour and efficiency of these photosensitizers were compared with the constitutive units of the hybrid molecules: meso-tetrakis(4-N-methylpyridiniumyl)porphyrinato-zinc(II) tetraacetate (ZnTMPyP, 3) and 9-methoxy-N2methylellipticinium acetate (9-OMe-NME, 4). On irradiation at 436 nm, the efficiency of these hybrids is similar to that of ZnTMPyP and 50-fold greater than that of haematoporphyrin derivative (HPD). This photoinduced DNA cleavage is markedly reduced in the absence of oxygen and also depends on the DNA base pair to porphyrin ratio. It is inhibited by N-acetylhistidine and sodium azide, unaffected by mannitol and superoxide dismutase (SOD) and enhanced when changing H2O for D2O. The same scavenger effects are observed on irradiation at 514 nm. At 313 nm, the efficiency of hybrids 1 and 2 is intermediate between those of ZnTMPyP and 9-OMe-NME. In these conditions, a slight inhibitory effect of mannitol is observed, suggesting the participation of radicals probably derived from partial decomposition of the porphyrins. At these three wavelengths, singlet oxygen seems to be the main species responsible for DNA cleavage. In contrast with expectation, the great affinity of these molecules for DNA does not enhance their efficiency as DNA cleavers. This effect is discussed taking into account the long lifetime of singlet oxygen which may be generated far from the target. These molecules which are only photoactivable in the presence of DNA appear to be an efficient "molecular light switch".     

DIFFERENT EFFECTS OF RAD GENES OF SACCHAROMYCES CEREVISIAE ON INCISIONS OF INTERSTRAND CROSSLINKS AND MONOADDUCTS IN DNA INDUCED BY PSORALEN PLUS NEAR UV LIGHT TREATMENT

Abstract— The RAD1, RAD2, RAD3 and RAD4 genes of Saccharomyces cerevisiae are required for incising DNA containing UV induced pyrimidine dimers or psoralen plus 360 nm light induced interstrand crosslinks. We have now determined if these genes are also required for incising DNA at psoralen plus 360 nm light induced monoadducts. For distinguishing between incision breaks occurring at crosslinks and at monoadducts. we have used the cdc9-2 mutant, defective in DNA ligase activity at the restrictive temperature, and the radl-2 cdc9-2, rad2-5 cdc9-2 , rad3-2 cdc9-2 and rad4-4 cdc9-2 double mutant combinations. We conclude that the radl, rad2 , and rad4 mutants are defective in incising DNA both at crosslinks and monoadducts, whereas the rad3 mutant is proficient in incising DNA at monoadducts but not at crosslinks.     

REPAIR OF THYMINE DIMERS AND (6–4) PHOTOPRODUCTS IN GROUP A XERODERMA PIGMENTOSUM CELL LINES HARBORING A TRANSFERRED NORMAL CHROMOSOME 9

Transfer of a normal chromosome 9 into a xeroderma pigmentosum (XP)-A cell line partially restored its DNA repair activity. XP-A cell lines harboring a transferred chromosome were much more UV-resistant than parental XP-A cells but still more UV-sensitive than normal cells. The amount of UV-induced unscheduled DNA synthesis was only one-third of that in normal cells. The repair of thymine dimers and (6-4) photoproducts in these cell lines was analyzed by using monoclonal antibodies raised against them. Although these XP-A cell lines carrying a normal chromosome 9 could repair (6-4) photoproduct with a little lower efficiency than normal cells, the repair of thymine dimers was completely absent in these cells. The present results suggest a gene-dosage effect in DNA excision repair mechanisms in human cells or a rather complicated mechanism which involves two or more pathways.     

Synthesis and DNA binding properties of bis-9-acridinyl derivatives containing mono-, di-and tetra-viologen units as a connector of bis-intercalators

Bis-9-acridinyl derivatives 1–3 containing mono-, di-and tetra-viologen units as a rigid connector were synthesized. The binding studies of these intercalators for natural and synthetic DNAs showed that these compounds act as bis-intercalators where viologen moieties lie in the minor groove of the DNA duplex. The DNA binding affinity of the intercalators was enhanced with an increase in the number of the viologen unit.     

N7-guanine as a C+ mimic in hairpin aeg/aepPNA-DNA triplex: probing binding selectivity by UV-Tm and kinetics by fluorescence-based strand-invasion assay

N7-substituted guanine (N7G) has been introduced into aminoethylglycyl bisPNA (7) as a C(+) mimic to achieve pH-independent triplex formation with complementary DNA sequences. The introduction of chiral, cationic aminoethylprolyl units with C(+) and C(+) mimic N7G in the backbone of bisPNAs (8, 9) influenced the recognition of complementary DNA in an orientation-selective manner. A simple fluorescence assay is developed to examine the process of strand invasion of target DNA duplex by these modified bisPNAs and comparative results of the study employing triplex forming polypyrimidine (C/T) (6, 8) and purine-pyrimidine (N7G/T) mixmer-bisPNAs (7, 9) are presented.     

Electronic detection of single-base mismatches in DNA with ferrocene-modified probes.

Genotyping and gene-expression monitoring is critical to the study of the association between genetics and drug response (pharmacogenomics) and the association of sequence variation with heritable phenotypes. Recently, we developed an entirely electronic method for the detection of DNA hybridization events by the site-specific incorporation of ferrocenyl derivatives into DNA oligonucleotides. To perform rapid and accurate point mutation detection employing this methodology, two types of metal-containing signaling probes with varying redox potentials are required. In this report we describe a new ferrocene-containing phosphoramidite 9 that provides a range of detectable redox potentials. Using automated DNA/RNA synthesis techniques the two ferrocenyl complexes were inserted at various positions along oligonucleotide probes. Thermal stability analysis of these metal-containing DNA oligonucleotides indicates that incorporation of 9 resulted in no destabilization of the duplex. A mixture of oligonucleotides containing compounds 9 and I was analyzed by alternating current voltammetry (ACV) monitored at the 1st harmonic. The data demonstrate that the two ferrocenyl oligonucleotide derivatives can be distinguished electrochemically. A CMS-DNA array was prepared on an array of gold electrodes on a printed circuit board substrate with a self-assembled mixed monolayer, coupled to an electronic detection system. Experiments for the detection of a single-base match utilizing two signaling probes were carried out. The results demonstrate that rapid and accurate detection of a single-base mismatch can be achieved by using these dual-signaling probes on CMS-DNA chips.     

2'-Spiro ribo- and arabinonucleosides: synthesis, molecular modelling and incorporation into oligodeoxynucleotides

We have synthesized four conformationally restricted bicyclic 2'-spiro nucleosides via 2'-C-allyl nucleosides as key intermediates. The ribo-configured 2'-spironucleosides 9b and 14b were obtained by a convergent strategy starting from 2-ketofuranose 1 whereas the arabino-configured 2'-spironucleosides 21 and 27 were obtained by a linear strategy with a 2'-ketouridine derivative as starting material. The furanose ring of 9b/14b adopts N-type conformations whereas the furanose ring of 21/27 exists as an N<==>S equilibrium. These compounds showed no anti-HIV-1 activity or cytotoxicity. Incorporation of the four 2'-spironucleosides (as monomers X4 and X5) into oligodeoxynucleotides was accomplished using the phosphoramidite approach on an automated DNA synthesizer. Irrespective of monomeric configuration, hybridization studies revealed that these 2'-spironucleotide monomers (X4 and X5) induce decreased duplex thermostabilities compared with the corresponding DNA:DNA and DNA:RNA duplexes. Molecular modelling indicated that steric constraints are a possible reason for the lowered binding affinities of the modified oligodeoxynucleotides towards complementary single-stranded DNA and single-stranded RNA complements.     

Sugar pucker modulates the cross-correlated relaxation rates across the glycosidic bond in DNA

The dependence of N1/9 and C1' chemical shielding (CS) tensors on the glycosidic bond orientation (chi) and sugar pucker (P) in the DNA nucleosides 2'-deoxyadenosine, 2'-deoxyguanosine, 2'-deoxycytidine, and 2'-deoxythymidine was studied using the calculation methods of quantum chemistry. The results indicate that these CS-tensors exhibit a significant degree of conformational dependence on chi and P structural parameters. The presented data test underlying assumptions of currently established methods for interpretation of cross-correlated relaxation rates between the N1/9 chemical shielding tensor and C1'-H1' dipole-dipole (Ravindranathan et al. J. Biomol. NMR 2003, 27, 365-75. Duchardt et al. J. Am. Chem. Soc. 2004, 126, 1962-70) and highlight possible limitations of these methods when applied to DNA.     

Acridine derivatives. VI . Redox chemistry of novel 9-anilinoacridines with antitumor activities

In order to elucidate the mechanism of deoxyribonucleic acid (DNA) strand breaks caused by 9-anilino-acridine DNA intercalators, the antitumor activity of L1210, P388 and the reduction-oxidation (redox) reaction of 9-anilinoacridines were studied. The redox reaction induced by two electrons causing structural changes in quinone diimines of 9-anilinoacridines is believed to be an important factor in DNA strand breaks and was examined by means of temperature-dependent nuclear magnetic resonance and cyclic voltammetry. The redox reaction of 9-anilinoacridines is induced by the effect of a low-energy electron transfer from the acridine to the aniline ring. We propose that the redox reaction plays an important role in the DNA strand cleavage of 9-anilinoacridine when it is intercalated into double-strand DNA.     

A DNA Origami-Based Gene Editing System for Efficient Gene Therapy in Vivo

DNA nanostructures have played an important role in the development of novel drug delivery systems. Herein, we report a DNA origami-based CRISPR/Cas9 gene editing system for efficient gene therapy in vivo. In our design, a PAM-rich region precisely organized on the surface of DNA origami can easily recruit and load sgRNA/Cas9 complex by PAM-guided assembly and pre-designed DNA/RNA hybridization. After loading the sgRNA/Cas9 complex, the DNA origami can be further rolled up by the locking strands with a disulfide bond. With the incorporation of DNA aptamer and influenza hemagglutinin (HA) peptide, the cargo-loaded DNA origami can realize the targeted delivery and effective endosomal escape. After reduction by GSH, the opened DNA origami can release the sgRNA/Cas9 complex by RNase H cleavage to achieve a pronounced gene editing of a tumor-associated gene for gene therapy in vivo. This rationally developed DNA origami-based gene editing system presents a new avenue for the development of gene therapy.     

Interaction between the DNA model base 9-ethylguanine and a group of ruthenium polypyridyl complexes: kinetics and conformational temperature dependence

The binding capability of three ruthenium polypyridyl compounds of structural formula [Ru(apy)(tpy)Ln-](ClO4)(2-n) [1a-c; apy = 2,2'-azobis(pyridine), tpy = 2,2':6',2'-terpyridine, L = Cl, H2O, CH3CN] to a fragment of DNA was studied. The interaction between each of these complexes and the DNA model base 9-ethylguanine (9-EtGua) was followed by means of 1H NMR studies. Density functional theory calculations were carried out to explore the preferential ways of coordination between the ruthenium complexes and guanine. The ruthenium-9-EtGua adduct formed was isolated and fully characterized using different techniques. A variable-temperature 1H NMR experiment was carried out that showed that while the 9-EtGua fragment was rotating fast at high temperature, a loss of symmetry was suffered by the model base adduct as the temperature was lowered, indicating restricted rotation of the guanine residue.     

New Anthraquinone Derivatives as Electrochemical Redox Indicators for the Visualization of the DNA Hybridization Process

Interactions of dsDNA and ssDNA, at the surface of gold and silver electrodes, with three novel anthraquinone derivatives: 3‐(9′,10′‐dioxo‐9′,10′‐dihydro‐anthracen‐1‐yl)‐7,11‐di(carboxymethyl)‐3,7,11‐triazatridecanedioic acid, (AQ‐1); 1‐(9′,10′‐dioxo‐9′,10′‐dihydro‐anthracen‐1yl)‐9‐carboxymethyl‐5‐methyl‐1,5,9‐triazaundecanoicacid, (AQ‐2); and N‐(2‐(9,10‐dioxo‐9,10‐dihydro‐anthracen‐1‐ylamino)ethyl)‐2‐(1,4,10,13‐tetraoxa‐7,16‐diazacyclooctadecan‐7‐yl)acetamide, (AQ‐3) are studied. These derivatives are well soluble in water and phosphate buffer solutions. The square wave voltammetric behavior of these redox indicators is described and the parameters of interactions with DNA are reported. It is also pointed out that these compounds can be employed as the hybridization indicators. The difference in the binding ability of the particular redox indicator to single and double stranded DNA can be used for the detection of the complementary nucleic acids.     

A dsDNA-lighted fluorophore for monitoring protein-ligand interaction through binding-mediated DNA protection

Because of their important roles in cellular functions, life activities, drug screening, and disease treatment, the development of efficient methods for monitoring protein-ligand interaction is essential. In this study, inspired by our previous studies on DNA conformation-selective fluorescent indicators, we developed a new sensing platform for monitoring protein-ligand interaction and detecting protein activity based on binding-mediated DNA protection and the dsDNA-lighted fluorophore, ethyl-4-[3,6-bis(1-methyl-4-vinylpyridium iodine)-9 H-carbazol-9-yl)] butanoate(EBCB). The ligand was purposefully linked to the 3?-terminal of a hairpin DNA probe to selectively bind with the target protein and protect the DNA from cleavage by exonuclease III. By virtue of EBCB's outstanding capacity to discriminate DNA conformation, the protein-ligand interaction could be effectively monitored through a fluorescence change in EBCB. A high fluorescence signal was detected when the hairpin DNA was protected in the presence of the target protein, whereas a much lower signal was observed in the presence of nontarget proteins.Our results demonstrated that the proposed strategy had high potential, such as high selectivity and relatively high sensitivity, for monitoring protein-ligand interaction and detecting protein activity. We believe these results will pave the way for applying dsDNA-lighted fluorophore EBCB as an effective signal transducer for DNA conformation transformation-mediated biochemical sensing.     

Trehalose click polymers inhibit nanoparticle aggregation and promote pDNA delivery in serum

Herein, three new glycopolymers have been synthesized via "click polymerization" to promote nucleic acid delivery in the presence of biological media containing serum. These structures were designed to contain a trehalose moiety to promote biocompatibility, water solubility, and stability against aggregation, amide-triazole groups to enhance DNA binding affinity, and an oligoamine unit to facilitate DNA encapsulation, phosphate neutralization, and interactions with cell surfaces. A 2,3,4,2',3',4'-hexa-O-acetyl-6,6'-diazido-6,6'-dideoxy-D-trehalose (4) monomer was polymerized via copper(I)-catalyzed azide-alkyne cycloaddition with a series of dialkyne-amide comonomers that contain either one, two, or three Boc-protected secondary amines (7a, 7b, or 7c, respectively). After deprotection, three water-soluble polycations (9a, 9b, or 9c) were obtained with similar degrees of polymerization (n = 56-61) to elucidate the role of amine number on nucleic acid binding, complex formation, stability, and cellular delivery. Gel electrophoresis and ethidium bromide experiments showed that 9a-9c associated with plasmid DNA (pDNA) and formed complexes (polyplexes) at N/P ratios dependent on the amine number. TEM experiments revealed that 9a-9c polyplexes were small (50-120 nm) and had morphologies (spherical and rodlike) associated with the polymer chain stiffness. Dynamic light scattering studies in the presence of media containing serum demonstrated that 9c polyplexes had a low degree of flocculation, whereas 9a and 9b polyplexesd aggregate rapidly. Further biological studies revealed that these structures were biocompatible and deliver pDNA into HeLa cells. Particularly, 9c polyplexes promoted high delivery efficacy and gene expression profiles in the presence of serum.     

Nonspecific interactions between SpCas9 and dsDNA sites located downstream of the PAM mediate facilitated diffusion to accelerate target search

RNA-guided Streptococcus pyogenes Cas9 (SpCas9) is a sequence-specific DNA endonuclease that works as one of the most powerful genetic editing tools. However, how Cas9 locates its target among huge amounts of dsDNAs remains elusive. Here, combining biochemical and single-molecule fluorescence assays, we revealed that Cas9 uses both three-dimensional and one-dimensional diffusion to find its target with high efficiency. We further observed surprising apparent asymmetric target search regions flanking PAM sites on dsDNA under physiological salt conditions, which accelerates the target search efficiency of Cas9 by ∼10-fold. Illustrated by a cryo-EM structure of the Cas9/sgRNA/dsDNA dimer, non-specific interactions between DNA ∼8 bp downstream of the PAM site and lysines within residues 1151–1156 of Cas9, especially lys1153, are the key elements to mediate the one-dimensional diffusion of Cas9 and cause asymmetric target search regions flanking the PAM. Disrupting these non-specific interactions, such as mutating these lysines to alanines, diminishes the contribution of one-dimensional diffusion and reduces the target search rate by several times. In addition, low ionic concentrations or mutations on PAM recognition residues that modulate interactions between Cas9 and dsDNA alter apparent asymmetric target search behaviors. Together, our results reveal a unique searching mechanism of Cas9 under physiological salt conditions, and provide important guidance for both in vitro and in vivo applications of Cas9.

Nonspecific interactions between DNA ∼8 bp downstream of the PAM and lysines within residues 1151–1156 of Cas9 mediate one-dimensional diffusion and cause asymmetric target search regions flanking the PAM.     

Synthesis,Characterization, Novel Interaction of DNA,Antioxidant and Antimicrobial Studies of New Water Soluble Metallophthalocyanines Posture Eight Hydroxyphenyl Moiety via 1,3,4‐oxadiazole Bridge

The new peripheral 2(3),9(10),16(17),23(24)‐tetra‐5‐[4,4′‐diphenol]‐phenyl‐[1,3,4]‐oxadiazole substituted metallophthalocyanine (MPc) complexes has been well designed and executed. Due to high conjugation and excellent solubility in water makes them potential use in DNA binding and cleavage studies. Fourier transform infrared spectroscopy, nuclear magnetic resonance, electron spin ionization mass spectra data, and elemental analysis confirmed the well‐defined saddle‐like distorted structures for these substituted MPc complexes. The successful synthesis of these novel water soluble MPc moieties were employed as an effective DNA binding with calf thymus DNA was monitored using ultraviolet?visible spectral titrations and cleavage pBR322 DNA conceded in the absence of reductant by agarose gel electrophoresis method. The results indicate that all these water soluble complexes significantly show excellent binding and modest cleavage sensitivity activity. It is noteworthy that 6 and 7 exhibit potential antimicrobial and appreciable antioxidant activity with other water soluble phthalocyanines.     

Potential DNA bis-intercalating agents: Synthesis and antitumor activity of novel,conformationally restricted bis(9-aminoacridines)

A series of bis(9-aminoacridines) bridged by conformationally restricted tethers was synthesized and evaluated against L1210 in vitro. Several of these compounds were found to be highly active in this test system, with ID₅₀ values below 10^?7 M. CPK molecular models suggest that this antitumor activity can be correlated to the ability of these bis(9-aminoacridines) to form bis-intercalative complexes with DNA.     

β-K4H3[SiW9O37(CpTi)3]·11H2O环戊二烯钛多酸衍生物与DNA作用研究

某些过渡金属配合物具有特异性催化DNA和RNA断裂的功能, 因而研究过渡金属配合物对DNA和RNA的断链反应对新型抗肿瘤、抗艾滋病化学药物的定向设计及其基因治疗和分子生物学中DNA和RNA的高度专一性定点断裂、 DNA定位诱变和构象识别具有重要意义和应用前景[1,2]. 我们对二茂钛多酸有机金属衍生物合成及抗肿瘤活性研究表明, 环戊二烯钛多氧金属酸盐衍生物具有很高的抗肿瘤活性和较好的水溶性及稳定性, 有潜在的抗肿瘤药用价值[3].     

Design of photoactivated DNA oxidizing agents: synthesis and study of photophysical properties and DNA interactions of novel viologen-linked acridines

A new series of photoactivated DNA oxidizing agents in which an acridine moiety is covalently linked to viologen by an alkylidene spacer was synthesized, and their photophysical properties and interactions with DNA, including DNA cleaving properties, were investigated. The fluorescence quantum yields of the viologen-linked acridines were found to be lower than that of the model compound 9-methylacridine (MA). The changes in free energy for the electron transfer reactions were found to be favorable, and the fluorescence quenching observed in these systems is explained by an electron transfer mechanism. Intramolecular electron transfer rate constants were calculated from the observed fluorescence quantum yields and singlet lifetime of MA and are in the range from 1.06x10(10) s(-1) for 1 a (n=1) to 6x10(8) s(-1) for 1 c (n=11), that is, the rate decreases with increasing spacer length. Nanosecond laser flash photolysis of these systems in aqueous solutions showed no transient absorption, but in the presence of guanosine or calf thymus DNA, transient absorption due to the reduced viologen radical cation was observed. Studies on DNA binding demonstrated that the viologen-linked acridines bind effectively to DNA in both intercalative and electrostatic modes. Results of PM2 DNA cleavage studies indicate that, on photoexcitation, these molecules induce DNA damage that is sensitive to formamidopyrimidine DNA glycosylase. These viologen-linked acridines are quite stable in aqueous solutions and oxidize DNA efficiently and hence can be useful as photoactivated DNA-cleaving agents which function purely by the co-sensitization mechanism.     

Preparation of a Novel Polymer‐Modified Si Surface for DNA Immobilization

The design of a novel polymer‐modified overlayer composed of PPAPE and GPMS on a silicon wafer for immobilization of DNA molecules is described. After hydroxylation of Si(100) surfaces, GPMS molecules were self‐assembled onto these surfaces. PPAPE molecules were then covalently attached to the epoxy‐terminated surfaces. The incubation time and concentration of PPAPE was found to effect both layer thickness and water CA. The type of organic solvent and the pH were found to change the nature of the PPAPE‐modified surface for DNA immobilization. It is concluded that PPAPE‐modified surfaces show advantages for DNA immobilization by electrostatic interactions between DNA molecules and positively charged free amino groups of the PPAPE‐modified surfaces at the appropriate pH values.