Guanine modification during chemical DNA synthesis.

Base modification during solid-phase phosphoramidite synthesis of oligodeoxynucleotides has been investigated. We have discovered chemical modification that converts dG and dG-containing oligomers to a fluorescent form. This modification has been linked to N,N-dimethylaminopyridine (DMAP), an acylation catalyst, which can displace phosphate triester adducts at the 6-position of guanine. Further, we have found that this fluorescent intermediate can be converted in ammonium hydroxide solution to 2,6 diaminopurine deoxyribonucleoside (2,6 DAP), a potentially mutagenic nucleoside analog. We have shown that N-methylimidazole (NMI) in place of DMAP eliminates the fluorescent species and reduces 2,6 DAP contamination.     

Site-directed modification of DNA duplexes by chemical ligation.

The efficiency of chemical ligation method have been demonstrated by assembling a number of DNA duplexes with modified sugar phosphate backbone. Condensation on a tetradecanucleotide template of hexa(penta)- and undecanucleotides differing only in the terminal nucleoside residue have been performed using water-soluble carbodiimide as a condensing agent. As was shown by comparing the efficiency of chemical ligation of single-strand breaks in those duplexes, the reaction rate rises 70 or 45 times if the 3'-OH group is substituted with an amino or phosphate group (the yield of products with a phosphoramidate or pyrophosphate bond is 96-100% in 6 d). Changes in the conformation of reacting groups caused by mismatched base pairs (A.A, A.C) as well as the hybrid rU.dA pair or an unpaired base make the template-directed condensation less effective. The thermal stability of DNA duplexes was assayed before and after the chemical ligation. Among all of the modified duplexes, only the duplex containing 3'-rU in the nick was found to be a substrate of T4 DNA ligase.     

Reactivity of parallel-stranded DNA to chemical modification reagents

Four 25-nt long oligonucleotides containing dA and dT (D1, D2, D3, and D4) which are capable of forming parallel-stranded (ps) or antiparallel-stranded (aps) duplexes have been synthesized [Rippe, K., Ramsing, N. B., & Jovin, T. M. (1989) Biochemistry 28, 9536-9541]. In the present study, the OsO4-pyridine complex (Os,py), diethyl pyrocarbonate (DEPC), KMnO4, and the 1,10-phenanthroline-cuprous complex [(OP)2Cu+] were used to investigate the conformation-dependent reactivity of ps, aps, and single-stranded (ss) oligonucleotides. The products were analyzed by polyacrylamide gel electrophoresis with single-nucleotide resolution. The results confirm the duplex nature of the ps combinations of oligonucleotides and reveal structural differences in comparison with the aps molecules. Under conditions in which ss-DNA is substantially sensitive to Os,py, both the ps and aps duplexes are very unreactive. A similar result was observed with KMnO4 and DEPC, although with the latter reagent the modification pattern of the labeled strands D1* and D4* was slightly different for the parallel than for the antiparallel duplex. The (OP)2Cu+ complex efficiently cleaves the aps but not the ps duplex and shows a preference for TAT steps. We also tested the effect of monovalent and divalent cation concentrations on the chemical reactivity of the ps, aps, and ss species. Elevated NaCl concentration leads to a dramatic increase in the Os,py and KMnO4 modification of ss molecules and the ps, but not the aps, duplex. We attribute the apparent reaction with ps-DNA to a destabilization of this conformation under the conditions of reaction. In contrast, all reactions with DEPC are somewhat depressed at high salt concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence-specific chemical modification of double-stranded DNA with alkylating oligodeoxyribonucleotide derivatives

Chemical modification of double-stranded (ds) DNA with alkylating oligodeoxynucleotide (oligo) derivatives, 5'-p(N-2-chloroethyl-N-methylamino) benzylamides of oligos, has been investigated. In contrast to relaxed plasmid DNAs, the superhelical molecules interact with the oligo derivatives and specific alkylation of the DNAs occurs at the regions complementary to the oligo reagents. Alkylating derivatives of oligocytidylates and pT(pCpT)6 react with corresponding homopyrimidine-homopurine tracts within ds DNA fragments due to triple helix formation.     

A new method of studying DNA conformation by chemical modification

A new method of discrimination of double-stranded (ds) and single-stranded (ss) regions in DNA molecules has been developed. It makes use of two alkylating reagents, a voluminous and a small-sized, the former being sensitive to the DNA conformation. A bulky reagent, N,N,N'-tri(beta-chloroethyl)-N'-(p-formylphenyl) propylendiamine-1,3 (TFP), was used to detect the hairpin structure in the palindrome-containing DNA fragment 373 nucleotides long prepared from the ds EcoRI-BamHI fragment of the plasmid pBR322. The fragment was modified by TFP and cleaved by piperidine at the alkylated guanine residues according to the Maxam-Gilbert procedure. Guanine residues in the hairpin formed by palindrome were protected from the TFP action, while dimethylsulfate modified all guanines. Application of the method for the identification of loops, stem-and-loop structures, and unwinded regions of DNA is discussed.     

Surface modification and chemical surface analysis of biomaterials

The chemical composition of the surface layers of synthetic biomaterials used for human medical devices and in biotechnology plays a key role in determining interfacial interactions between biological media (such as protein solutions, cells, tissue) and the synthetic material. Accordingly, considerable research efforts focus on improving the 'biocompatibility' of biomaterials by applying various surface modification and thin film coating approaches. Here we focus on the patterning of surface chemistries, often designed to exercise spatial control over events such as cell attachment and spreading. Secondly, we review recent developments in chemical characterisation of biomaterials surfaces, which is essential both for verifying the success of intended surface modification strategies and for reliable interpretation of observed biological responses. Biomaterials surface analysis by imaging ToF-SIMS and XPS and compositional depth profiling are discussed, as is the emerging complementary technique of Metastable Induced Electron Spectroscopy.     

Immunocytochemical analysis of in vivo DNA modification

In the past decades a large number of DNA adducts induced in the intact animal by alkylating agents have been identified. The formation and repair of these adducts are important determinants, not only of mutagenesis, tumor initiation and DNA-mediated toxicity but probably also of tumor progression. Most studies on in vivo DNA modification have been performed on isolated bulk DNA.

More recently, methods have been developed to study the distribution of DNA adducts at the level of either the individual gene or the individual cell. This paper reviews immunocytochemical methods to study the formation and repair of DNA adducts and other DNA modifications at the level of the individual cell. DNA modifications induced by alkylating agents and a variety of other agents including ultraviolet radiation, aromatic amines, polycyclic aromatic hydrocarbons and platinum anti-cancer drugs will be discussed.

Up to now, immunocytochemical analysis of in vivo modified DNA has largely concentrated on experimental animals. These studies have revealed striking heterogeneities with regard to formation and/or repair of DNA adducts in tissues from rat, hamster and mouse. Immunocytochemical adduct analysis can be used to identify in a convenient, fast and detailed way cell types, cell stages and sites in which biological effects of the adducts might be expressed. More recently, immunocytochemical analysis of DNA adducts also proved to be feasible on in situ exposed human samples.

A number of existing and potential applications in the field of chemical carcinogenesis, experimental chemotherapy and molecular epidemiology are discussed.     

Theoretical analysis of epigenetic cell memory by nucleosome modification

Chromosomal regions can adopt stable and heritable alternative states resulting in bistable gene expression without changes to the DNA sequence. Such epigenetic control is often associated with alternative covalent modifications of histones. The stability and heritability of the states are thought to involve positive feedback where modified nucleosomes recruit enzymes that similarly modify nearby nucleosomes. We developed a simplified stochastic model for dynamic nucleosome modification based on the silent mating-type region of the yeast Schizosaccharomyces pombe. We show that the mechanism can give strong bistability that is resistant both to high noise due to random gain or loss of nucleosome modifications and to random partitioning upon DNA replication. However, robust bistability required: (1) cooperativity, the activity of more than one modified nucleosome, in the modification reactions and (2) that nucleosomes occasionally stimulate modification beyond their neighbor nucleosomes, arguing against a simple continuous spreading of nucleosome modification.     

Sequence-specific chemical modification of chromatin DNA with reactive derivatives of oligonucleotides

Chemical modification of the chromatin DNA with alkylating derivatives of oligothymidylate (pT)₁₆ and oligoadenylate (pA)₁₆ bearing 4-(N-2-chloroethyl-N-methylamino)benzylphosphamide group at the 5-phosphate has been investigated. It was found that the derivatives do react with DNA in chromatin. The reactions occur presumably at the complementary sequences of the DNA since the reaction of the oligothymidylate derivative is inhibited by oligonucleotide (pT)₁₆ taken in excess and is not influenced by hexadecanucleotide of a random structure. Isolated DNA does not react with the oligothymidylate derivative. It is concluded that in chromatin, DNA is partially unwound or possesses some sites which can be opened easily in the presence of complementary oligonucleotides.     

On the chemical nature of DNA and RNA modification by a hemin model system.

In order to model the interaction of hemin with DNA and other polynucleotides, we have studied the degradation of DNA, RNA, and polynucleotides of defined structure by [meso-tetrakis(N-methyl-4-pyridyl)porphinato]manganese(III) (MnTMPP) + KHSO5. The activated porphyrin was shown to release adenine, thymine, and cytosine from DNA; RNA degradation afforded adenine, uracil, and cytosine. The same products were obtained from single- and double-stranded DNA oligonucleotides of defined sequence, and also from single-stranded DNA and RNA homopolymers. The overall yield of bases from the dode-canucleotide d(CGCT3A3GCG) was equal to 14% of the nucleotides present initially, indicating that each porphyrin catalyzed the release of approximately 4 bases. Although no guanine was detected as a product from any of the substrates studied, the ability of MnTMPP + KHSO5 to degrade guanine nucleotides was verified by the destruction of pGp, and by the appearance of bands corresponding to guanosine cleavage following treatment of 32P end labeled DNA restriction fragments with activated MnTMPP. Inspection of a number of sites of MnTMPP-promoted cleavage indicated that the process was sequence-selective, occurring primarily at G residues that were part of 5'-TG-3' or 5'-AG-3' sequences, or at T residues. Also formed in much greater abundance were alkali-labile lesions; these were formed largely at guanosine residues. Also studied was the degradation of a 47-nucleotide RNA molecule containing two hairpins. Degradation of the 5'-32P end labeled RNA substrate afforded no distinct, individual bands, suggesting that multiple modes of degradation may be operative.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA methylation: bisulphite modification and analysis

DNA methylation is an important epigenetic modification of DNA in mammalian genomes. DNA methylation patterns are established early in development, modulated during tissue-specific differentiation and disrupted in many disease states, including cancer. To understand further the biological functions of these changes, accurate and reproducible methods are required to fully analyze the DNA methylation sequence. Here, we describe the 'gold-standard' bisulphite conversion protocol that can be used to re-sequence DNA from mammalian cells in order to determine and quantify the methylation state of a gene or genomic region at single-nucleotide resolution. The process of bisulphite treatment exploits the different sensitivities of cytosine and 5-methylcytosine (5-MeC) to deamination by bisulphite under acidic conditions--in which cytosine undergoes conversion to uracil, whereas 5-MeC remains unreactive. Bisulphite conversion of DNA, in either single tubes or in a 96-well format, can be performed in a minimum of 8 h and a maximum of 18 h, depending on the amount and quality of starting DNA.     

Localization of melted regions in supercoiled DNA by means of chemical modification

Diethyl pyrocarbonate was used as a probe in mapping early melting stages in supercoiled DNA. It was shown that in the process of early melting of pAO3 DNA two denatured regions (about 15 b.p.) arouse near the left and right boundaries of the cruciform structure. In course of further melting denatured regions appeared within AT-rich stretches and the cruciform structure itself disappeared.     

Effects of specific chemical modification of actin.

G-actin has been nitrated with tetranitromethane in conditions that lead to the modification of one tyrosine residue. The reactive residue was found by earlier workers to be Tyr-69. The nitrated actin is conformationally similar to native G-actin, as judged by sedimentation velocity and circular dichroism analysis. A small proportion only is in the form of covalently linked dimers and trimers. The nitrated G-actin will polymerise to form filaments, indistinguishable in the electron microscope from those of native F-actin, but the polymerisation process is slower. Reduction of the nitrophenol group to the corresponding aminophenol leaves the properties of the protein in respect of polymerisation unchanged. When a dansyl group is introduced at the same point, however, the ability of the actin to polymerise is lost. The nitrated actin and its reduced counterpart will also bind heavy meromyosin, and the characteristic arrowhead formation of the bound molecules along the filaments can be seen in the electron microscope. Neither of the modified F-actins, however, significantly activates or inhibits the myosin ATPase activity. The fluorescence of nitrated actin is strongly quenched through the presence of the nitrophenol chromophore. In soluble complexes with heavy meromyosin the fluorescence is indistinguishable from the sum of the separate contributions of the two protein components. There is thus no measurable excitation transfer between any tryptophan residues on the myosin heads, such as that inferred to be present in the ATPase site, and the nitrotyrosine in position 69 of the actin sequence. Implications of this observation are considered in relation to the different interaction sites in myosin and in actin. The activation of heavy meromyosin ATPase by copolymers containing actin and nitroactin in different proportions has been measured, and is not proportional to the fraction of native actin. The results are consistent with the view that the function of actomyosin depends on the interaction of the myosin heads with more than one actin subunit.     

Novel site-specific DNA modification in Streptomyces: analysis of preferred intragenic modification sites present in a 5.7 kb amplified DNA sequence.

Both Streptomyces lividans and Streptomyces avermitilis encode similar systems of post-replicative DNA modification which act site-specifically on closely opposed guanines on either strand. The modifications can be detected since they react in vitro with an oxidative derivative of Tris, resulting in strand cleavage. Previous analysis of the preferred modification site of plasmid pIJ101 indicated that extensive amounts of flanking sequence, including direct and inverted repeat structures, are required to direct modification in vivo within a central 6 bp palindrome. We have now examined the preferred modification sites of a chromosomal element, the 5.7 kb amplified DNA sequence (ADS5.7) found in certain S. lividans mutants. In contrast to the pIJ101 site, each of the ADS5. 7sites is intragenic and modified with a 10-fold reduced frequency. However, similar extents of flanking sequence are required for authentic double-strand modification; deletion mutants exhibited different modification profiles, including displaced double-stranded or single-stranded modi-fication. Comparison of different modification sites reveals conservation of the central core sequence, but no significant similarities between flanking sequences. Enhanced modification was detected in a cloned region of the ADS5.7, suggesting that local DNA topology, probably influenced by both DNA supercoiling and the nature of flanking sequences, can influence the modifying activity.     

siRNA function in RNAi: a chemical modification analysis

Various chemical modifications were created in short-interfering RNAs (siRNAs) to determine the biochemical properties required for RNA interference (RNAi). Remarkably, modifications at the 2'-position of pentose sugars in siRNAs showed the 2'-OHs were not required for RNAi, indicating that RNAi machinery does not require the 2'-OH for recognition of siRNAs and catalytic ribonuclease activity of RNA-induced silencing complexes (RISCs) does not involve the 2'-OH of guide antisense RNA. In addition, 2' modifications predicted to stabilize siRNA increased the persistence of RNAi as compared with wild-type siRNAs. RNAi was also induced with chemical modifications that stabilized interactions between A-U base pairs, demonstrating that these types of modifications may enhance mRNA targeting efficiency in allele-specific RNAi. Modifications altering the structure of the A-form major groove of antisense siRNA-mRNA duplexes abolished RNAi, suggesting that the major groove of these duplexes was required for recognition by activated RISC*. Comparative analysis of the stability and RNAi activities of chemically modified single-stranded antisense RNA and duplex siRNA suggested that some catalytic mechanism(s) other than siRNA stability were linked to RNAi efficiency. Modified or mismatched ribonucleotides incorporated at internal positions in the 5' or 3' half of the siRNA duplex, as defined by the antisense strand, indicated that the integrity of the 5' and not the 3' half of the siRNA structure was important for RNAi, highlighting the asymmetric nature of siRNA recognition for initiation of unwinding. Collectively, this study defines the mechanisms of RNAi in human cells and provides new rules for designing effective and stable siRNAs for RNAi-mediated gene-silencing applications.     

Novel post-replicative DNA modification in Streptomyces: analysis of the preferred modification site of plasmid pIJ101.

Both Streptomyces lividans and Streptomyces avermitilis have the ability to site specifically modify their DNA, rendering it susceptible to in vitro Tris-dependent double-strand cleavage. We have cloned a 160 bp fragment containing the preferred modification site of plasmid pIJ101 and, employing an in vitro primer extension assay, determined that the modifications occur at guanine residues on either strand separated by 3 bp. These guanines are located within a 6 bp palindromic 'core' sequence. A cloned copy of a 35 bp region of the plasmid containing this core sequence was not recognized by the modifying activity in vivo. To further investigate the nature of the site specificity a set of deletion mutants of the 160 bp sequence were analysed. This revealed that a substantial portion of this sequence is essential for authentic modification. The essential region contains three 13 bp direct repeats, the central one containing the core sequence, while the left-hand and right-hand copies overlap two potential stem-loop structures. Deletion of either left- or right-hand repeat structures abolishes modification within the core sequence, although the left-hand deletion resulted in modification at a secondary site within the right-hand direct repeat. These data support a post-replicative mechanism of modification, underlined by the observation that the modifications are not detected in single-stranded plasmid replication intermediates.     

Theoretical analysis of the kinetics of DNA hybridization with gel-immobilized oligonucleotides.

A new method of DNA sequencing by hybridization using a microchip containing a set of immobilized oligonucleotides is being developed. A theoretical analysis is presented of the kinetics of DNA hybridization with deoxynucleotide molecules chemically tethered in a polyacrylamide gel layer. The analysis has shown that long-term evolution of the spatial distribution and of the amount of DNA bound in a hybridization cell is governed by "retarded diffusion," i.e., diffusion of the DNA interrupted by repeated association and dissociation with immobile oligonucleotide molecules. Retarded diffusion determines the characteristic time of establishing a final equilibrium state in a cell, i.e., the state with the maximum quantity and a uniform distribution of bound DNA. In the case of cells with the most stable, perfect duplexes, the characteristic time of retarded diffusion (which is proportional to the equilibrium binding constant and to the concentration of binding sites) can be longer than the duration of the real hybridization procedure. This conclusion is indirectly confirmed by the observation of nonuniform fluorescence of labeled DNA in perfect-match hybridization cells (brighter at the edges). For optimal discrimination of perfect duplexes from duplexes with mismatches the hybridization process should be brought to equilibrium under low-temperature nonsaturation conditions for all cells. The kinetic differences between perfect and nonperfect duplexes in the gel allow further improvement in the discrimination through additional washing at low temperature after hybridization.     

Specific chemical modification of cytidine in T4 DNA by a spin label

A procedure for selective modification of DNA from T4 phage non-glucosylated mutant by the spin label--N(2,2',5,5') tetramethyl-3-carboxypyrrolidine-1-oxyl)-imidazole was developed. The spin label was shown to interact with hydroxyl groups of 5-hydroxymethyl-2 deoxycytidines. The modification does not affect the secondary structure of DNA, its conformation or template properties in a cell-free system of RNA synthesis.