Unprecedented right- and left-handed quadruplex structures formed by heterochiral oligodeoxyribonucleotides

CD and NMR studies on heterochiral oligodeoxynucleotides (d/l-ODNs) forming quadruplex structures are reported. Heterochiral ODNs, based on sequence TGGGGT, are able to form stable either right- or left-handed quadruplexes depending on d/l ratio and residues position. Results suggest that the 3′-end and the core of the G-run are more important than the 5′-end in determining the quadruplex handness. Particularly, oligonucleotide T_DG_DG_LG_LG_DT_D (L34) at low temperatures forms a well-defined left-handed quadruplex, notwithstanding it is mostly composed by natural d residues. This structure is characterized by three all-anti G-tetrads and one all-syn G-tetrad.     

Structural Studies of Heterochiral DNA/DNA,RNA/RNA,AND DNA/RNA Duplexes

Using DNA and RNA heptanucleotides containing an unnatural L-nucleotides as well as the complementary strands, effects of the introduction of an L-nucleotide on the structure of DNA/DNA, RNA/RNA, and DNA/RNA duplexes were investigated by circular dichroism experiments and RNase H-mediated RNA strand cleavage reaction. The results suggested that the substitution of the central D-nucleotide with an L-nucleotide in the duplexes causes the significant structural alterations as the duplex structures change to conformations with more B-form similarities.     

Binding ability of a thymine-functionalized oligolysine towards nucleic acids

In this Letter, we investigated the binding properties towards nucleic acids of a thymine-functionalized oligolysine, composed of nucleobase-bearing amino acid moieties and underivatized l-lysine residues alternate in the backbone. The basic nucleopeptide proved to be well soluble in water and able to interact with both DNA and RNA, as suggested by circular dichroism, UV and surface plasmon resonance studies performed on the thymine-containing oligomer with both adenine-containing DNA (dA₁₂) and RNA (rA₁₂ and poly rA) molecules. In both cases the thymine-functionalized oligolysine was proven to form complexes characterized by a 1:1 T/A stoichiometric ratio, as evidenced by CD titration. UV melting experiments revealed that the complex formed between the homothymine oligolysine and rA₁₂ RNA was more stable than the complex with dA₁₂ DNA probably due to the additional H-bonding of the 2′-OH groups in RNA, that reinforces the overall interaction with the nucleopeptide. Finally, human serum stability assays were conducted on the thymine-bearing nucleopeptide which showed a half-life of 45 min.     

Synthesis and properties of novel 2'-O-alkoxymethyl-modified nucleic acids

Novel 2'-O-modified oligoribonucleotides with alkoxymethyl skeletons were synthesized, and their ability to hybridize complementary nucleic acids and their nuclease resistance were analyzed. The hybridization ability was improved by introducing electron-withdrawing groups and the increases in melting temperature (T(m) value) was particularly high for chlorine-substituted compounds. Nuclease resistance of these 2'-O-alkoxymethylated oligomers was lower than expected, but cyano substitution resulted in a higher nuclease resistance than 2'-O-methylation.     

Diastereomer characterizations of nitroxide-labeled nucleic acids

Site-directed spin labeling (SDSL) obtains structural and dynamic information of a macromolecule using a site-specifically attached stable nitroxide radical. SDSL studies of arbitrary DNA and RNA sequences can be achieved using an efficient phosphorothioate labeling scheme, where a nitroxide is attached to a phosphorothioate substituted at a target site during chemical synthesis. The chemically introduced phosphorothioate contains two diastereomers (Rp and Sp), and nitroxides attached to each diastereomer may experience different local environments. Here, we report work on using anion-exchange HPLC to separate and characterize diastereomers in three DNA oligonucleotides and one RNA oligonucleotide. In all oligonucleotides studied, the Rp diastereomer was found to elute earlier than the Sp in the unlabeled oligonucleotides, while a reversal in the elution order (Sp earlier than Rp) was observed for nitroxide-labeled oligonucleotides. The results enable a one-step purification procedure for preparing diastereomerically pure nitroxide-labeled oligonucleotides. This expands the score of nucleic acids SDSL.     

FRET-based kinetic analysis of highly reactive heterochiral DNA toward EcoRI endonuclease

We found unusual reactivity of a heterochiral dodecadeoxynucleotide toward EcoRI that contains an unnatural l-nucleotide residue at the 3′-flanking site of the hexameric EcoRI recognition sequence. In this study, the kinetic parameters for the reaction were determined by means of a heterochiral molecular beacon that contains the EcoRI recognition sequence in the stem region and possesses the fluorescent and quenching dyes at the 5′- and 3′-termini, respectively. We found that the heterochiral molecular beacon showed large V_max and small K_m values compared to the corresponding homochiral one. This chiral modification is expected to induce a preferable conformational change for binding and catalysis by EcoRI.     

DNA hybridization as a guide to phylogeny: Chemical and physical limits

Summary The technique of forming interspecific DNA heteroduplexes and estimating phylogenetic distances from the depression in their duplex melting temperature has several physical and chemical constraints. These constraints determine the maximum phylogenetic distance that may be estimated by this technique and the most appropriate method of analyzing that distance.Melting curves of self-renatured single copy primate DNAs reveal the presence of components absent from the renaturation products of exactly paired sequences. This observation, which confirms existing literature, challenges a fundamental assumption: that orthologous (i.e., corresponding) DNA sequences in the divergent species are being compared in DNA heteroduplex melting experiments.As a model system, the thermal stabilities of heteroduplexes formed between a human alpha-globin cDNA and four alpha-like globin genes isolated from chimpanzee are qualitatively compared. The results of this comparison show that the cross-hybrids of imperfectly matched gene duplicates from divergent species can contribute to the additional components that are present in renatured single copy DNAs. Single copy DNA, as usually defined, includes sequence duplicates that will obscure phylogenetic comparisons in a mass hybridization of genomes.     

Fractionation with ethanol of nucleic acids from viroid-infected plants

A combination of high salt and low ethanol concentration allowed the fractionation of nucleic acids extracted from viroid-infected leaves. By adding 0.4-0.5 vol of ethanol to 1 vol of a solution in 2 M LiCl of nucleic acids (containing mainly DNA, 4S, 5S, 7S, and viroid RNAs), 85% of the DNA and 75% of the 4S RNA remained in solution, from where they could be recovered by increasing the ethanol concentration, whereas almost all 5S, 7S, and viroid RNAs precipitated. When this process was repeated three times a 95% elimination of the initial DNA and 4S RNA was achieved. The method can be of special interest in viroid purification considering that DNA and 4S RNA are the most abundant contaminants in the starting solution of nucleic acids. It is suggested that the highly ordered secondary structure of viroid RNA may be responsible for its particular behavior in the ethanol fractionation of nucleic acids.     

Synthesis of nucleic acids and localization of atrial natriuretic peptide in crayfish haemocytes

Three types of cells circulate in the haemolymph of the crayfish Astacus astacus, i.e., agranular haemocytes (HCs I), small-granule haemocytes (HCs II), and large-granule haemocytes (HCs III). Their proliferation, differentiation, and function remain poorly understood. Using light and electron microscopic autoradiography with [³H]-thymidine, we found that only HCs I are capable of DNA synthesis and mitosis whereas HCs II and HCs III are replicatively inactive. To verify whether HCs I are proliferating progenitor cells for granular HCs, we have analyzed autographs of the HC population 1, 2, 7, and 21 days after a single [3H]-thymidine administration. Contrary to our expectations, we have failed to find labeled HCs II and HCs III. These findings have raised doubts as to the capacity of HCs I to differentiate into two other types of HCs. With the use of ³H-uridine autoradiography, it was found that RNA synthesis was the most active in HCs I and 2 and 4 times lower in HCs II and HCs III, respectively. ANP-like immunoreactivity was revealed in large granules of the HCs III by electron microscopic immunocytochemistry. We assume that the presence of ANP in secretory granules extends the possible functions of crayfish HCs and suggests their participation in the regulation of the watersalt balance and immune response.     

Radical-generating coordination complexes as tools for rapid and effective fragmentation and fluorescent labeling of nucleic acids for microchip hybridization

DNA microchip technology is a rapid, high-throughput method for nucleic acid hybridization reactions. This technology requires random fragmentation and fluorescent labeling of target nucleic acids prior to hybridization. Radical-generating coordination complexes, such as 1,10-phenanthroline-Cu(II) (OP-Cu) and Fe(II)-EDTA (Fe-EDTA), have been commonly used as sequence nonspecific "chemical nucleases" to introduce single-strand breaks in nucleic acids. Here we describe a new method based on these radical-generating complexes for random fragmentation and labeling of both single- and double-stranded forms of RNA and DNA. Nucleic acids labeled with the OP-Cu and the Fe-EDTA protocols revealed high hybridization specificity in hybridization with DNA microchips containing oligonucleotide probes selected for identification of 16S rRNA sequences of the Bacillus group microorganisms.We also demonstrated cDNA- and cRNA-labeling and fragmentation with this method. Both the OP-Cu and Fe-EDTA fragmentation and labeling procedures are quick and inexpensive compared to other commonly used methods. A column-based version of the described method does not require centrifugation and therefore is promising for the automation of sample preparations in DNA microchip technology as well as in other nucleic acid hybridization studies.     

In situ hybridization of nucleic acids

Hybridization of radioactive nucleic acid sequences can be carried out with DNA in cytological preparations. By autoradiography distinct DNA sequences can then be localized in eukaryotic chromosomes.     

Multitasking with molecular dynamics Typhoon: quantifying nucleic acids and autoradiographs

With increased sensitivity and specificity, fluorescent assays are rapidly becoming the method of choice for nucleic acid quantification. The utility of the Typhoon scanner has now been extended to accurately measure low levels of DNA and RNA (5 ng ml^–1) with PicoGreen and RiboGreen dyes. In addition, with a few simple modifications, autoradiographic film images can be scanned and quantified with the Typhoon series of scanners.     

Electrophoretic elution of nucleic acids from acrylamide and agarose gels

A simple method for electrophoretic elution of nucleic acids from gel slices is described. The procedure utilizes a standard tube gel system and can be completed in as little as one hour. Nucleic acids are recovered in a small volume with almost 100% efficiency. The procedure is applicable equally to acrylamide and agarose gels, and small as well as large RNA and DNA molecules. The eluted nucleic acids are essentially undegraded and are suitable for a variety of structural and biological analyses.     

Studies on the retention of plasmid DNA and Escherichia coli nucleic acids by hydrophobic interaction chromatography

This work presents studies on the interactions of supercoiled plasmid DNA and Escherichia coli genomic DNA (gDNA) and RNA, with an hydrophobic interaction chromatography (HIC) gel, obtained by derivatisation of Sepharose CL-6B with 1,4-butanediol diglycidyl ether. Nucleic acids purified from E. coli were injected separately in the above HIC column and eluted with 1.5 M (NH₄)₂SO₄ in the buffer. The column was able to separate single-stranded from double-stranded nucleic acids. RNA and denatured gDNA were retarded in a different way due to the interactions of the exposed hydrophobic bases with the ligands. Supercoiled plasmid DNA, on the contrary, eluted in the flowthrough. This revised version was published online in July 2006 with corrections to the Cover Date.     

Assays for urinary biomarkers of oxidatively damaged nucleic acids

Abstract

The analysis of oxidized nucleic acid metabolites can be performed by a variety of methodologies: liquid chromatography coupled with electrochemical or mass-spectrometry detection, gas chromatography coupled with mass spectrometry, capillary electrophoresis and ELISA (Enzyme-linked immunosorbent assay). The major analytical challenge is specificity. The best combination of selectivity and speed of analysis can be obtained by liquid chromatography coupled with tandem mass spectrometric detection. This, however, is also the most demanding technique with regard to price, complexity and skills requirement. The available ELISA methods present considerable specificity problems and cannot be recommended at present. The oxidized nucleic acid metabolites in urine are assumed to originate from the DNA and RNA. However, direct evidence is not available. A possible contribution from the nucleotide pools is most probably minimal, if existing. Recent investigation on RNA oxidation has shown conditions where RNA oxidation but not DNA oxidation is prominent, and while investigation on DNA is of huge interest, RNA oxidation may be overlooked. The methods for analyzing oxidized deoxynucleosides can easily be expanded to analyze the oxidized ribonucleosides. The urinary measurement of oxidized nucleic acid metabolites provides a non-invasive measurement of oxidative stress to DNA and RNA.     

Thermal stability of oligodeoxynucleotide duplexes containing l-deoxynucleotide at termini

The effects of substituting l-deoxynucleotide for d-deoxynucleotide at duplex termini were evaluated and the terminal substitutions were found to show much less effects on duplex destabilization and to show a similar tendency in base pairing selectivity, compared with internal chiral substitutions.     

Cross-platform comparison of nucleic acid hybridization: Toward quantitative reference standards

Measuring interactions between biological molecules is vitally important to both basic and applied research as well as development of pharmaceuticals. Although a wide and growing range of techniques is available to measure various kinetic and thermodynamic properties of interacting biomolecules, it can be difficult to compare data across techniques of different laboratories and personnel or even across different instruments using the same technique. Here we evaluate relevant biological interactions based on complementary DNA and RNA oligonucleotides that could be used as reference standards for many experimental systems. We measured thermodynamics of duplex formation using isothermal titration calorimetry, differential scanning calorimetry, and ultraviolet–visible (UV–vis) monitored denaturation/renaturation. These standards can be used to validate results, compare data from disparate techniques, act as a teaching tool for laboratory classes, or potentially to calibrate instruments. The RNA and DNA standards have many attractive features, including low cost, high purity, easily measurable concentrations, and minimal handling concerns, making them ideal for use as a reference material.     

Towards a universally adaptable method for quantitative extraction of high-purity nucleic acids from soil

A universally adaptable protocol for quantitative extraction of high-purity nucleic acids from soil is presented. A major problem regarding the extraction of nucleic acids from soil is the presence of humic substances, which interfere with the extraction process itself and in subsequent analytical manipulations. By the approach described here, the humic compounds are precipitated prior to cell lysis with Al(2)(SO(4))(3), and thus eliminated prior to the nucleic acid extraction. The protocol allows for removing of a considerable content and range of humic acids and should therefore be applicable for a wide spectrum of soil types. Accordingly, reproducible results in analyses of different soil types are made possible, inclusively for quantitative comparisons.