Basic aspects of the biochemical reactivity of 4-hydroxynonenal

4-hydroxynonenal (HNE), a major lipid peroxidation product of n-6 polyunsaturated fatty acids, which was discovered by the late Hermann Esterbauer, is a remarkable trifunctional molecule. Both the hydroxy group and the conjugated system consisting of a C=C double bond and a carbonyl group contribute to the high reactivity of HNE. Most of the biochemical effects of HNE can be explained by its rapid reactions with thiol and amino groups. Among the primary reactants for HNE are the amino acids cysteine, histidine and lysine, which--either free or protein-bound--undergo readily Michael additions to the C=C bond. After this primary reaction, which confers rotational freedom to the C2-C3 bond, secondary reactions may occur involving the carbonyl and the hydroxy group. Primary amines may alternatively react with the carbonyl group to form Schiff bases. Reactions which do not fit into this scheme are the oxidation and the reduction respective of the carbonyl group and the epoxidation of the C=C double bond. Examples will be presented for the interaction of HNE with various classes of biomolecules such as proteins and peptides, lipids and nucleic acids and the biochemical consequences will be discussed.     

Poliovirus protein 3AB displays nucleic acid chaperone and helix-destabilizing activities

Poliovirus protein 3AB displayed nucleic acid chaperone activity in promoting the hybridization of complementary nucleic acids and destabilizing secondary structure. Hybridization reactions at 30 degrees C between 20- and 40-nucleotide RNA oligonucleotides and 179- or 765-nucleotide RNAs that contained a complementary region were greatly enhanced in the presence of 3AB. The effect was nonspecific as reactions between DNA oligonucleotides and RNA or DNA templates were also enhanced. Reactions were optimal with 1 mM MgCl(2) and 20 mM KCl. Analysis of the reactions with various 3AB and template concentrations indicated that enhancement required a critical amount of 3AB that increased as the concentration of nucleic acid increased. This was consistent with a requirement for 3AB to "coat" the nucleic acids for enhancement. The helix-destabilizing activity of 3AB was tested in an assay with two 42-nucleotide completely complementary DNAs. Each complement formed a strong stem-loop (DeltaG = -7.2 kcal/mol) that required unwinding for hybridization to occur. DNAs were modified at the 3' or 5' end with fluorescent probes such that hybridization resulted in quenching of the fluorescent signal. Under optimal conditions at 30 degrees C, 3AB stimulated hybridization in a concentration-dependent manner, as did human immunodeficiency virus nucleocapsid protein, an established chaperone. The results are discussed with respect to the role of 3AB in viral replication and recombination.     

Modification of end phosphate gruops in mono- and oligonucleotides.

A method is described for selective activation of phosphomonoester end groups of oligonucleotides and nucleosidedi-(tri) phosphates via mixed anhydrides with mesitoic acid. Mixed anhydrides are synthesized in high yield and isolated by paper or DEAE-cellulose column chromatography. The ability of such anhydrides to phosphorylate different nucleophilic agents was used for synthesis of amidates, imidazolidates, esters, thioesters and pyrophosphates of mono- and oligonucleotides. Mixed anhydrides mono-, oligonucleotides and nucleosidedi-(tri)phosphates and mesitoic acid were also applied to achieve immobilization of the mono- and oligonucleotides via their end groups on hexamethylenediamine - Sepharose support. Mixed anhydrides studied may be efficiently used for affinity labeling of proteins and nucleic acids and also as material for preparating reagents for template reactions.     

Two sides of the coin: affinity and specificity of nucleic acid interactions

During the past decade, synthetic nucleobase oligomers have found wide use in biochemical sciences, biotechnology and molecular medicine, both as research and/or diagnostic tools and as therapeutics. Numerous applications of common and modified oligonucleotides and oligonucleotide mimics rely on their ability to sequence-specifically recognize nucleic acid targets (DNA or RNA) by forming duplexes or triplexes. In general, these applications would benefit significantly from enhanced binding affinities of nucleobase oligomers in the formation of various secondary structures. However, for high-affinity probes, the selectivity of sequence recognition must also be improved to avoid undesirable associations with mismatched DNA and RNA sites. Here, we review recent progress in understanding the molecular mechanisms of nucleic acid interactions and the development of new high-affinity plus high-specificity oligonucleotides and their mimics, with particular emphasis on peptide nucleic acids.     

DNA mimics based on pyrrolidine and hydroxyproline

In recent years, a great number of analogues and mimics of nucleic acids have been developed with the aim of improving the physicochemical and biological properties of native oligonucleotides, in particular, to increase their affinity for nucleic acids, selectivity of action, and biological stability. This review summarizes the data on the synthesis and properties of DNA mimics, the analogues of peptide nucleic acids, which are the derivatives of pyrrolidine and hydroxyproline. Some physicochemical and biological properties of negatively charged mimics of this type are considered, which contain phosphonate residues in the back-bone and exhibit a high affinity for DNA and RNA, the selectivity of binding to nucleic acids, and stability in various biological systems. Examples of using these mimics as tools in molecular biology studies, in particular, functional genomics, are given. The prospects for their application in diagnosis and medicine are discussed.     

Regulation of in vitro gene expression using antisense oligonucleotides or antisense expression plasmids transfected using starburst PAMAM dendrimers.

Starburst polyamidoamine (PAMAM) dendrimers are a new type of synthetic polymer characterized by a branched spherical shape and a high density surface charge. We have investigated the ability of these dendrimers to function as an effective delivery system for antisense oligonucleotides and 'antisense expression plasmids' for the targeted modulation of gene expression. Dendrimers bind to various forms of nucleic acids on the basis of electrostatic interactions, and the ability of DNA-dendrimer complexes to transfer oligonucleotides and plasmid DNA to mediate antisense inhibition was assessed in an in vitro cell culture system. Cell lines that permanently express luciferase gene were developed using dendrimer mediated transfection. Transfections of antisense oligonucleotides or antisense cDNA plasmids into these cell lines using dendrimers resulted in a specific and dose dependent inhibition of luciferase expression. This inhibition caused approximately 25-50% reduction of baseline luciferase activity. Binding of the phosphodiester oligonucleotides to dendrimers also extended their intracellular survival. While dendrimers were not cytotoxic at the concentrations effective for DNA transfer, some non-specific suppression of luciferase expression was observed. Our results indicate that Starburst dendrimers can be effective carriers for the introduction of regulatory nucleic acids and facilitate the suppression of the specific gene expression.     

2'-O,4'-C-ethylene-bridged nucleic acids (ENA): highly nuclease-resistant and thermodynamically stable oligonucleotides for antisense drug.

To develop antisense oligonucleotides, novel nucleosides, 2'-O,4'-C-ethylene nucleosides and their corresponding phosphoramidites, were synthesized as building blocks. The 1H NMR analysis showed that the 2'-O,4'-C-ethylene linkage of these nucleosides restricts the sugar puckering to the N-conformation as well as the linkage of 2'-O,4'-C-methylene nucleosides which are known as bridged nucleic acids (BNA) or locked nucleic acids (LNA). The ethylene-bridged nucleic acids (ENA) showed a high binding affinity for the complementary RNA strand (DeltaT(m)=+5.2 degrees C/modification) and were more nuclease-resistant than natural DNA and BNA/LNA. These results indicate that ENA have better properties as antisense oligonucleotides than BNA/LNA.     

Application of unnatural oligonucleotides to chemical modification of gene expression

Chemical entities that specifically interact with biological molecules can reveal the biological role of their targets. Increasing knowledge of genomes and the intricate mechanisms that regulate their expression has aroused considerable interest in molecules that target nucleic acids. A wide variety of technologies have been developed based on unnatural oligonucleotides for gene silencing, gene-detection or bio-inspired chemical reactions, for example. In contrast to natural oligonucleotides, their unnatural counterparts can exhibit unique functions based on the chemical reactivity of the accessory molecule. This review focuses on the molecular design of chemically reactive accessory molecules of unnatural oligonucleotides, emphasising their application in specific recognition and reaction toward biological targets.     

Synthesis of steroid-containing oligonucleotides and their alkylating derivatives

New alkylating derivatives of oligonucleotides carrying a steroid (cholesterol, testosterone or ergosterol) residue have been synthesized, the residue being introduced via its hydroxyl group into the triester oligonucleotide block in the presence of triisopropylbenzenesulphonyl chloride and N-methylimidazole. Covalent attachment of steroids to oligonucleotides increases their hydrophobicity and does not influence the melting temperature of their complementary complexes. The data obtained showed that the oligonucleotide derivatives, bearing both an alkylating group of nitrogen mustard and a steroid residue, can be used as reagents for specific modification of nucleic acids.     

ADP-ribosylation of RNA and DNA: from in vitro characterization to in vivo function

The functionality of DNA, RNA and proteins is altered dynamically in response to physiological and pathological cues, partly achieved by their modification. While the modification of proteins with ADP-ribose has been well studied, nucleic acids were only recently identified as substrates for ADP-ribosylation by mammalian enzymes. RNA and DNA can be ADP-ribosylated by specific ADP-ribosyltransferases such as PARP1–3, PARP10 and tRNA 2′-phosphotransferase (TRPT1). Evidence suggests that these enzymes display different preferences towards different oligonucleotides. These reactions are reversed by ADP-ribosylhydrolases of the macrodomain and ARH families, such as MACROD1, TARG1, PARG, ARH1 and ARH3. Most findings derive from in vitro experiments using recombinant components, leaving the relevance of this modification in cells unclear. In this Survey and Summary, we provide an overview of the enzymes that ADP-ribosylate nucleic acids, the reversing hydrolases, and the substrates’ requirements. Drawing on data available for other organisms, such as pierisin1 from cabbage butterflies and the bacterial toxin–antitoxin system DarT–DarG, we discuss possible functions for nucleic acid ADP-ribosylation in mammals. Hypothesized roles for nucleic acid ADP-ribosylation include functions in DNA damage repair, in antiviral immunity or as non-conventional RNA cap. Lastly, we assess various methods potentially suitable for future studies of nucleic acid ADP-ribosylation.     

DNA mimics on the base of pyrrolidine and hydroxyproline

In order to improve physicochemical and biological properties of natural oligonucleotides in particular increasing their affinity for nucleic acids, the selectivity of action and biological sustainability, several types of DNA mimics were designed. The survey collected data on the synthesis and properties of the DNA mimics - peptide-nucleic acids analogues, which are derivatives of pyrrolidine and hydroxyproline. We examine some physicochemical and biological properties of negatively charged mimics of this type, containing phosphonate residues, and possessing a high affinity for DNA and RNA, selective binding with nucleic acids and stability in various biological systems. Examples of the use of these mimics as tools for molecular biological research, particularly in functional genomics are given. The prospects for their use in diagnostics and medicine are discussed.     

Exogenous nucleic acids aggregate in non-P-body cytoplasmic granules when transfected into cultured cells

To modulate gene expression in research studies or in potential clinical therapies, transfection of exogenous nucleic acids including plasmid DNA and small interference RNA (siRNA) are generally performed. However, the cellular processing and the fate of these nucleic acids remain elusive. By investigating the cellular behavior of transfected nucleic acids using confocal imaging, here we show that when siRNA was co-transfected into cultured cells with other nucleic acids, including single-stranded RNA oligonucleotides, single and double-stranded DNA oligonucleotides, as well as long double-stranded plasmid DNA, they all aggregate in the same cytoplasmic granules. Interestingly, the amount of siRNA aggregating in granules was found not to correlate with the gene silencing activity, suggesting that assembly of cytoplasmic granules triggered by siRNA transfection may be separable from the siRNA silencing event. Our results argue against the claim that the siRNA-aggregating granules are the functional site of RNA interference (RNAi). Taken together, our studies suggest that, independent of their types or forms, extraneously transfected nucleic acids are processed through a common cytoplasmic pathway and trigger the formation of a new type of cytoplasmic granules “transfection granules”.     

Protein-induced changes in DNA structure and dynamics observed with noncovalent site-directed spin labeling and PELDOR

Site-directed spin labeling and pulsed electron–electron double resonance (PELDOR or DEER) have previously been applied successfully to study the structure and dynamics of nucleic acids. Spin labeling nucleic acids at specific sites requires the covalent attachment of spin labels, which involves rather complicated and laborious chemical synthesis. Here, we use a noncovalent label strategy that bypasses the covalent labeling chemistry and show that the binding specificity and efficiency are large enough to enable PELDOR or DEER measurements in DNA duplexes and a DNA duplex bound to the Lac repressor protein. In addition, the rigidity of the label not only allows resolution of the structure and dynamics of oligonucleotides but also the determination of label orientation and protein-induced conformational changes. The results prove that this labeling strategy in combination with PELDOR has a great potential for studying both structure and dynamics of oligonucleotides and their complexes with various ligands.     

The peptide nucleic acids (PNAs): introduction to a new class of probes for chromosomal investigation

Peptide nucleic acids (PNAs) are synthetic DNA mimics in which the sugar phosphate backbone is replaced by repeating N-(2-aminoethyl) glycine units linked by an amine bond and to which the nucleobases are fixed. Peptide nucleic acids hybridize with complementary nucleic acids with remarkably high affinity and specificity, essentially because of their uncharged and flexible polyamide backbone. The unique physicochemical properties of PNAs have led to the development of a large variety of biological research assays, and, over the last few years, PNAs have proved their powerful usefulness in genetic and cytogenetic diagnostic procedures. Several sensitive and robust PNA-dependent methods have been designed for modulating polymerase chain reactions, detecting genomic mutation or capturing nucleic acids. The more recent applications of PNA involve their use as molecular hybridization probes. Thus, the in situ detection of several human chromosomes has been reported in various types of tissues.Communicated by E.A. Nigg     

Design and synthesis of polyacrylamide-based oligonucleotide supports for use in nucleic acid diagnostics.

Polyacrylamide supports, in a range of pore sizes, were investigated as nucleic acid affinity matrices for the detection of target DNA or RNA sequences using a sandwich hybridization format. Bromoacetyl and thiol oligonucleotide derivatives were covalently linked to sulfhydryl- and bromoacetyl-polyacrylamide supports with greater than 95% end-attachment efficiencies. These polyacrylamide-oligonucleotide supports were further derivatized with anionic residues to provide multi-functional supports which show low non-specific binding for non-complementary nucleic acids. While all the polyacrylamide-oligonucleotide supports capture complementary oligonucleotides with high affinity, the pore size was found to be a critical parameter in sandwich hybridization reactions. The superior hybridization characteristics of the Trisacryl support was ascribed to a combination of its macroporous nature, hydrophilicity and the terminal attachment of its capture oligonucleotides.     

Biotin-streptavidin-labeled oligonucleotides as probes of helicase mechanisms

Helicases use the energy from ATP hydrolysis to catalyze formation of single-stranded nucleic acids by unwinding double-stranded nucleic acids. The ATP-dependent reaction can be broken down into at least two steps: melting of the duplex and translocation of the enzyme along the nucleic acid lattice. Each step presents difficulties for study because clear end points for the reactions are not always available. For example, translocation involves the movement of the enzyme from one point along the lattice to a new position, with no net change in chemical structure of the nucleic acid. Hence, new assays have been developed in which the nucleic acid is modified to contain a "protein block" that impedes translocation of the enzyme. To prepare such protein blocks, biotin-streptavidin has been used due to the ease with which the biotin can be incorporated into nucleic acids by chemical synthesis. Several applications of oligonucleotides labeled with biotin-streptavidin for the study of helicase mechanisms are described.     

Sequencing of pools of nucleic acids on oligonucleotide arrays

In sequencing-by-hybridization methods, the nucleotide sequence of a nucleic acid is reconstructed by overlapping oligonucleotides capable of hybridizing with the nucleic acid. In their present form, the methods are hardly suitable for sequencing of long nucleic acid molecules because of the occurrence of non-unique overlaps between the oligonucleotides, and similarly to the conventional sequencing methods, it is necessary to obtain an individual molecule. In the method described here, most ambiguities in reconstruction of a sequence from the constituent oligonucleotides are eliminated by preparing on oligonucleotide arrays and separate surveying of the nucleic acid nested partials. This enables longer nucleic acids to be sequenced, and results in a high redundancy of the input data allowing most hybridization errors to be eliminated by algorithmic means. Furthermore, large pools of nucleic acid strands can be sequenced directly, without isolating individual strands.     

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 gamma 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.     

Sulfur-containing Nucleic Acids: Synthesis,Chemical Reactions in Supramolecular Biopolymer Complexes,Biological Applications

The chemical behavior of sulfur-containing oligonucleotides and their reactivity in self-assembled nucleic acids (NA) and specific NA–protein complexes is considered. Reviewed are postsynthetic approaches that allow introducing sulfur-containing linkages at preselected positions of the sugar-phosphate backbone of DNA and between neighboring nucleobases, to incorporate disulfide bridges between complementary strands of double- and triple-stranded DNAs, in large catalytic RNA, etc. Special reference is given to the site-specific chemical modifications as a tool for elucidating the structure, folding, and function of biomolecules. Structure-directed chemical reactions are shown to be helpful in detecting point mutations in DNA, targeting the modifications on specific positions of NA, probing the molecular recognition in protein–DNA interfaces, studying the conformational dynamics of nucleic acids, and discriminating between different folding models.