Synthesis and Studies of Modified Oligonucleotides- Directed Triple Helix Formation at the Purine-Pyrimidine Interrupted Site

Abstract

Triple helix formation is still restricted to oligopurine-oligopyrimidine double stranded DNA target. Herein we focus on our progress achieved in nucleobase and oligonucleotide modifications area to address the chemical challenge to circumvent the recognition of a purine-pyrimidine base pair interruption in an oligopyrimidine-oligopurine DNA sequence.     

Melting of Cross-Linked DNA IV. Methods for Computer Modeling of Total Influence on DNA Melting of Monofunctional Adducts,Intrastrand and Interstrand Cross-Links Formed by Molecules of an Antitumor Drug

Abstract

A theoretical method is developed for calculation of melting curves of covalent complexes of DNA with antitumor drugs. The method takes into account all the types of chemical modifications of the double helix caused by platinum compounds and DNA alkylating agents: 1) monofunctional adducts bound to one nucleotide; 2) intrastrand cross-links which appear due to bidentate binding of a drug molecule to two nucleotides that are included into the same DNA strand; 3) interstrand cross-links caused by bidentate binding of a molecule to two nucleotides of different strands. The developed calculation method takes into account the following double helix alterations at sites of chemical modifications: 1) a change in stability of chemically modified base pairs and neighboring ones, that is caused by all the types of chemical modifications; 2) a change in the energy of boundaries between helical and melted regions at sites of chemical modification (local alteration of the factor of cooperativity of DNA melting), that is caused by all the types of chemical modifications, too; 3) a change in the loop entropy factor of melted regions that include interstrand cross-links; 4) the prohibition of divergence of DNA strands in completely melted DNA molecules, which is caused by interstrand cross-links only. General equations are derived, and three calculation methods are proposed to calculate DNA melting curves and the parameters that characterize the helix-coil transition.     

Sequence-Specific Alkylation of DNA by Duocarmycin A and Its Novel Derivatives Bearing PY/IM Polyamides

Abstract

A new class of sequence-specific DNA alkylating agents were developed based on the reactivity of duocarmycin A and the DNA-reading ability of pyrrole-imidazole polyamide. The DNA alkylation sequence specificity by duocarmycin A can be modulated by a variety of pyrrole-imidazole triamides in a predictable manner. Novel hybrids of the segment A of duocarmycin A and pyrrole-imidazole polyamides efficiently and highly selectively alkylated the target base possessing match sequences of Dervan's binding code.     

7-Deaza-2′-deoxyinosine: A Stable Nucleoside with the Ambiguous Base Pairing Properties of 2′-Deoxyinosine

Abstract

The base pairing ambiguity of 7-deaza-2′-deoxyinosine (c⁷I_d, 2) was studied and was found to be the same as that of 2′-deoxyinosine. The duplex stability decreases in the order [d(c⁷I-C) > d(c⁷I-A) > d(c⁷I-T) > d(c⁷I-G)]. Modified nucleosides were used to probe the various base pair motifs which were the same for dl and c⁷I_d. The 7-deazapurine nucleoside (2) is extremely stable against acid or base. As oligonucleotides can be prepared using phosphoramidite chemistry and DNA is accessible by enzymic polymerisation of the triphosphate of 2, the latter can be used as an universal nucleoside for the sequencing of DNA by chemical degradation and is otherwise a facile substitute of 2′-deoxyinosine when stability in acidic or alkaline solution is required.     

Chemical Synthesis of Oligonucleotides Containing Damaged Bases for Biological Studies

Since nucleic acids are organic molecules, even DNA, which carries genetic information, is subjected to various chemical reactions in cells. Alterations of the chemical structure of DNA, which are referred to as DNA damage or DNA lesions, induce mutations in the DNA sequences, which lead to carcinogenesis and cell death, unless they are restored by the repair systems in each organism. Formerly, DNA from bacteria and bacteriophages and DNA fragments treated with UV or γ radiation, alkylating or crosslinking agents, and other carcinogens were used as damaged DNA for biochemical studies. With these materials, however, it is difficult to understand the detailed mechanisms of mutagenesis and DNA repair. Recent progress in the chemical synthesis of oligonucleotides has enabled us to incorporate a specific lesion at a defined position within any sequence context. This method is especially important for studies on mutagenesis and translesion synthesis, which require highly pure templates, and for the structural biology of repair enzymes, which necessitates large amounts of substrate DNA as well as modified substrate analogs. In this review, the various phosphoramidite building blocks for the synthesis of lesion-containing oligodeoxyribonucleotides are described, and some examples of their applications to molecular and structural biology are presented.     

Imidazole-4-Carboxamide and 1,2,4-Triazole-3-Carboxamide Deoxynucleotides as Simplified DNA Building Blocks with Ambiguous Pairing Capacity

Abstract

2′-Deoxynucleosides of imidazole-4 (or 1,2,4-triazole-3)-carboxamide, ethyl imidazole-4 (or 1,2,4-triazole-3)-carboxylate were synthesized by enzymatic glycosylation using N-deoxyribosyltransferase from a lactobacterium. The base pairing properties of Y and V when placed opposite the natural DNA bases as well as their self were evaluated by thermal denaturation experiments. DNA templates containing imidazole-4-carboxamide base were used in elongation reaction catalysed by Klenow fragment.     

Structural and Molecular Biology of the eye Lens Membrane

Abstract

The DNA double helix exhibits local sequence-dependent polymorphism at the level of the single base pair and dinucleotide step. Curvature of the DNA molecule occurs in DNA regions with a specific type of nucleotide sequence periodicities. Negative supercoiling induces in vitro local nucleotide sequence-dependent DNA structures such as cruciforms, left-handed DNA, multistranded structures, etc. Techniques based on chemical probes have been proposed that make it possible to study DNA local structures in cells. Recent results suggest that the local DNA structures observed in vitro exist in the cell, but their occurrence and structural details are dependent on the DNA superhelical density in the cell and can be related to some cellular processes.     

Novel DNA Damage Mediated by Oxidation of Various Modified Nucleotide Residues

Abstract

Permanganate reaction of DNA oligomers containing an 8-oxoadenine or 5hydroxyuracil residue was studied, and the results were compared with those for an 8-oxoguanine-containing oligomers. We obtained similar results and found that the nucleotide residues neighboring the modified base were damaged and that the novel damage was induced by the oxidation of the modified base.     

Fluorescent 2-Pyrimidinone Nucleoside in Parallel-Stranded DNA

Abstract

Stretches of parallel-stranded (ps) double-helical DNA can arise within antiparallel-stranded (aps) Watson-Crick DNA in looped structures or in the presence of sequence mismatches. Here we studied an effect of a pyrimidinone-G (PG) base pair on the stability and conformation of the ps DNA to explore whether P is useful as a structural probe.     

Single-stranded DNA versus tailed duplex in sequence conversion of lacZα DNA

Abstract

Targeted DNA editing has great potential to cure some genetic diseases; however, the use of artificial nucleases such as CRISPR-Cas9 and TALEN in gene therapy can potentially cause severe side effects due to off-target DNA cleavages. Single-stranded (ss) DNAs and 5'-tailed duplexes (TDs) can achieve target base substitutions when introduced without artificial nucleases into cultured cells and mouse liver. In this study, ss DNA and TD were separately co-introduced into human U2OS cells, together with a target plasmid DNA bearing an inactivated lacZα gene, and the gene correction efficiencies were compared. Unlike the genes examined in previous studies, ss DNA and TD showed similar efficiencies. Therefore, ss DNAs might be as useful as TD for gene correction, depending on the target sequence.     

Probing the MvaI Methyltransferase Region that Interacts with DNA: Affinity Labeling with the Dialdehyde-Containing DNA Duplexes

Abstract

Affinity labeling of methyltransferase MvaI by DNA duplexes containing oxidized 2′-O-β-D-ribofuranosylcytidine or 1-(β-D-galactopyranosyl)thymine residues was performed. Partial chemical hydrolysis of the covalently bound methylase in the conjugates with the dialdehyde-containing DNA allowed us to determine the amino acid region in the C terminus of methylase MvaI that interacts with DNA.     

B-Z Junctions in Supercoiled pRW751 DNA Contain Unpaired Bases or Non-Watson-Crick Base Pairs

Abstract

Structural distortions on the boundary between right-handed and left-handed DNA segments in negatively supercoiled plasmid pRW751 (a derivative of pBR322 containing (dC-dG)₁₃ and (dC-dG)₁₆ segments) were studied by means of osmium tetroxide, pyridine and glyoxal. These two probes react preferentially with single-stranded DNA but only the latter requires non-paired bases for the reaction. Nuclease SI and testing of the inhibition of BamHI cleavage (whose recognition sequences GGATCC lie on the “outer” boundaries between the (dC-dG)_n and the pBR322 nucleotide sequence) were used to detect the site-specific chemical modification in pRW751.

As a result of glyoxal treatment BamHI cleavage was strongly inhibited in topoisomeric samples whose superhelical density was sufficiently negative to stabilize the (dC-dG)_n segments in the left-handed form. Osmium tetroxide, pyridine modification resulted in a similar inhibition of BamHI cleavage and in a formation of nuclease SI sensitive sites. The results suggest that the “outer” B-Z junctions in pRW751 contain one or few non-paired bases or non-Watson- Crick base pairs.     

A simplified chromatin dispersion (nuclear halo) assay for detecting DNA breakage induced by ionizing radiation and chemical agents

Abstract

Methods for visualizing DNA damage at the microscopic level are based on treatment of cell nuclei with saline or alkaline solutions. These procedures for achieving chromatin dispersion produce halos that surround the nuclear remnants. We improved the fast halo assay for visualizing DNA breakage in cultured cells to create a simplified method for detection and quantitative evaluation of DNA breakage. Nucleated erythrocytes from chicken blood were selected as a model test system to analyze the production of nuclear halos after treatment with X-rays or H₂O₂. After staining with ethidium bromide or Wright's methylene blue-eosin solution, nuclear halos were easily observed by fluorescence or bright-field microscopy, respectively, which permits rapid visualization of DNA breakage in damaged cells. By using image processing and analysis with the public domain ImageJ software, X-ray dose and H₂O₂ concentration could be correlated well with the size of nuclear halos and the halo:nucleus ratio. Our results indicate that this simplified nuclear halo assay can be used as a rapid, reliable and inexpensive procedure to detect and quantify DNA breakage induced by ionizing radiation and chemical agents. A mechanistic model to explain the differences between the formation of saline or alkaline halos also is suggested.     

DNA Base Pair Binding Specificity of CC-1065

Abstract

Oligomer duplexes were prepared by a solid-phase phosphoramidite triester coupling approach in order to study the DNA base pair binding specificity of the antitumor antibiotic CC-1065 by CD spectroscopy.     

Synthesis of Amide Linked Nucleosides at the 6 Position of Deoxy Inosine and Their application to DNA synthesis,Hybridization Studies.

Abstract

Synthesis of amide linked nucleosides at the 6 position of the purine base to recognize “G:C” base pair in a DNA duplex is described. Here we describe the synthesis of amide linked nucleosides containing heteroatoms N, O and C and their application to DNA synthesis and hybridization studies.     

Oligonucleotides with 2,6-Diaminopurine Base Replacing for Adenine: Synthesis and Properties

Abstract

Synthesis of three nucleoside building blocks with a benzoyl protected diaminopurine (DAP) base and their incorporation at different positions of DNA, RNA and hexitol oligomers (HNA) have been accomplished. DNA hairpins with a DAP substituted for one (or more) adenine in the loop structure were not found to be more stable. But the stability of RNA-hexitol as well as hexitol-hexitol duplexes improved when the adenine base was replaced with DAP.     

The Flexibility of A-Form DNA

Abstract

We have determined the rise per base pair and persistence length of A-form DNA in trifluoroethanol solutions for fragments 350–900 base pairs in length that best describe rotational diffusion coefficients determined by transient electric birefringence. The 2.6 A spacing between base pairs found in crystal and fiber A-form structures is preserved in solution. The persistence length is about 1500 A, or about three times longer than for B-form DNA. There is no apparent electrostatic contribution to the persistence length in the salt concentration range 0.2–2.0 mM Na cacodylate. This suggests an even closer association between DNA and its neutralizing counterions than predicted by condensation theory, perhaps due to a sheath of trifluoroethanol excluded water surrounding the A-form helix.     

Convenient Procedure for Oligonucleotide-Directed in vitro Mutagenesis of Cloned DNA

Abstract

A new modification of the oligonucleotide-mediated mutagenesis technique has been used to produce specific base changes in the double-stranded plasmid DNA.