The content of DNA and RNA in microparticles released by Jurkat and HL-60 cells undergoing in vitro apoptosis

Microparticles are small membrane-bound vesicles that are released from apoptotic cells during blebbing. These particles contain DNA and RNA and display important functional activities, including immune system activation. Furthermore, nucleic acids inside the particle can be analyzed as biomarkers in a variety of disease states. To elucidate the nature of microparticle nucleic acids, DNA and RNA released in microparticles from the Jurkat T and HL-60 promyelocytic cell lines undergoing apoptosis in vitro were studied. Microparticles were isolated from culture media by differential centrifugation and characterized by flow cytometry and molecular approaches. In these particles, DNA showed laddering by gel electrophoresis and was present in a form that allowed direct binding by a monoclonal anti-DNA antibody, suggesting antigen accessibility even without fixation. Analysis of RNA by gel electrophoresis showed intact 18s and 28s ribosomal RNA bands, although lower molecular bands consistent with 28s ribosomal RNA degradation products were also present. Particles also contained messenger RNA as shown by RT-PCR amplification of sequences for β-actin and GAPDH. In addition, gel electrophoresis showed the presence of low molecular weight RNA in the size range of microRNA. Together, these results indicate that microparticles from apoptotic Jurkat and HL-60 cells contain diverse nucleic acid species, indicating translocation of both nuclear and cytoplasmic DNA and RNA as particle release occurs during death.     

NMR studies of dynamics in RNA and DNA by 13C relaxation

RNA and DNA molecules experience motions on a wide range of time scales, ranging from rapid localized motions to much slower collective motions of entire helical domains. The many functions of RNA in biology very often require this molecule to change its conformation in response to biological signals in the form of small molecules, proteins or other nucleic acids, whereas local motions in DNA may facilitate protein recognition and allow enzymes acting on DNA to access functional groups on the bases that would otherwise be buried in Watson-Crick base pairs. Although these statements make a compelling case to study the sequence dependent dynamics in nucleic acids, there are few residue-specific studies of nucleic acid dynamics. Fortunately, NMR studies of dynamics of nucleic acids and nucleic acids-protein complexes are gaining increased attention. The aim of this review is to provide an update of the recent progress in studies of nucleic acid dynamics by NMR based on the application of solution relaxation techniques.     

Characterization of extracellular RNAs produced by the marine photosynthetic bacterium Rhodovulum sulfidophilum

The marine photosynthetic bacterium Rhodovulum sulfidophilum produces extracellular nucleic acids that are involved in its flocculation. These were found to be produced concomitantly with cell growth. The RNA fraction of these extracellular nucleic acids was subjected to cDNA analysis by applying a micro RNA cloning method and found to contain mainly fully mature-sized tRNAs and fragments of 16S and 23S rRNAs. Analyses of modified bases and genes of the RNAs revealed no structural difference between the intracellular and extracellular RNAs. This is the first report of structural analyses of bacterial extracellular RNAs.     

Properties of an LNA-modified ricin RNA aptamer

'Locked nucleic acids' (LNAs) are sugar modified nucleic acids containing the 2'-O-4'C-methylene-β-D-ribofuranoses. The substitution of RNAs with LNAs leads to an enhanced thermostability. Aptamers are nucleic acids, which are selected for specific target binding from a large library pool by the 'SELEX' method. Introduction of modified nucleic acids into aptamers can improve their stability. The stem region of a ricin A chain RNA aptamer was substituted by locked nucleic acids. Different constructs of the LNA-substituted aptamers were examined for their thermostability, binding activity, folding and RNase sensitivity as compared to the natural RNA counterpart. The LNA-modified aptamers were active in target binding, while the loop regions and the adjacent stem nucleotides remained unsubstituted. The thermostability and RNase resistance of LNA substituted aptamers were enhanced as compared to the native RNA aptamer. This study supports the approach to substitute the aptamer stem region by LNAs and to leave the loop region unmodified, which is responsible for ligand binding. Thus, LNAs possess an encouraging potential for the development of new stabilized nucleic acids and will promote future diagnostic and therapeutic applications.     

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.     

Segregation of RNA and separate packaging of DNA and RNA in apoptotic bodies during apoptosis

Apoptosis is characterized by a complex and remarkably ordered choreography of events consisting of the preparatory and execution steps that all culminate in disposal of the cell remnants. The disposal occurs in a manner that is the least destructive to the tissue: the remains of nuclear chromatin and cytoplasm are packaged in apoptotic bodies which are then phagocytized by neighboring live cells without invoking inflammatory or autoimmune response. In the present study we describe that in the course of apoptosis cellular RNA becomes sequestered and packaged into granules and then into apoptotic bodies, separately from DNA. This separation, which appears to be initiated by the nucleolar segregation, was observed in HL-60 cells that were undergoing spontaneous apoptosis in cultures or were treated with the DNA-damaging drug, DNA topoisomerase I inhibitor camptothecin (CPT), or with the cell death ligand, tumor necrosis factor-alpha. RNA separation was also observed in apoptotic MCF-7 cells following treatment with CPT. RNA and DNA in apoptotic cells were identified histochemically, by their differential stainability with pyronin Y and Hoechst 33342 fluorochromes, respectively, and immunocytochemically, by labeling the RNA with BrU for various periods of time and detection of the incorporated precursor with fluoresceinated anti-BrU mAb; DNA was counterstained with 7-aminoactinomycin D. Over 90% of apoptotic bodies that contained RNA had no detectable DNA and vice versa, the apoptotic bodies containing DNA had no detectable RNA. Packaging RNA and DNA into separate apoptotic bodies suggests that the phagosomes of the cells that ingest these particles are specialized: some of them are responsible for DNA degradation, others for degradation of RNA. Such specialization may facilitate heterophagic degradation of nucleic acids during apoptosis.     

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”.     

Nucleic acid binding properties of the nucleic acid chaperone domain of hepatitis delta antigen

The N terminal region of hepatitis delta antigen (HDAg), referred to here as NdAg, has a nucleic acid chaperone activity that modulates the ribozyme activity of hepatitis delta virus (HDV) RNA and stimulates hammerhead ribozyme catalysis. We characterized the nucleic acid binding properties of NdAg, identified the structural and sequence domains important for nucleic acid binding, and studied the correlation between the nucleic acid binding ability and the nucleic acid chaperone activity. NdAg does not recognize the catalytic core of HDV ribozyme specifically. Instead, NdAg interacts with a variety of nucleic acids and has higher affinities to longer nucleic acids. The studies with RNA homopolymers reveal that the binding site size of NdAg is around nine nucleotides long. The extreme N terminal portion of NdAg, the following coiled-coil domain and the basic amino acid clusters in these regions are important for nucleic acid binding. The nucleic acid–NdAg complex is stabilized largely by electrostatic interactions. The formation of RNA–protein complex appears to be a prerequisite for facilitating hammerhead ribozyme catalysis of NdAg and its derivatives. Mutations that reduce the RNA binding activity or high ionic strength that destabilizes the RNA–protein complex, reduce the nucleic acid chaperone activity of NdAg.     

Characterization of nucleic acids in membrane vesicles from scrapie-infected hamster brain.

This study reports the partial characterization of nucleic acids present in gradient fractions enriched for large membrane vesicles from scrapie-infected and uninfected hamster brains. Labeling of phenol-extracted nucleic acids at the 3' or 5' ends revealed abundant amounts of low-molecular-weight RNA and little or no DNA. These nucleic acids survived nuclease treatment of membrane vesicles but were sensitive to RNase after phenol extraction. Analysis of 5'-end-labeled nucleic acids by one- and two-dimensional gel electrophoresis revealed an RNA of ca. 100 bases in preparations from scrapie-infected hamster brain that could not be detected in uninfected brain. The possibility that this apparently unique small RNA may result from tissue damage or abnormal RNA processing or may be a component of the infectious complex is discussed.     

Extracellular nucleic acids

Extracellular nucleic acids are found in different biological fluids in the organism and in the environment: DNA is a ubiquitous component of the organic matter pool in the soil and in all marine and freshwater habitats. Data from recent studies strongly suggest that extracellular DNA and RNA play important biological roles in microbial communities and in higher organisms. DNA is an important component of bacterial biofilms and is involved in horizontal gene transfer. In recent years, the circulating extracellular nucleic acids were shown to be associated with some diseases. Attempts are being made to develop noninvasive methods of early tumor diagnostics based on analysis of circulating DNA and RNA. Recent observations demonstrated the possibility of nucleic acids exchange between eukaryotic cells and extracellular space suggesting their participation in so far unidentified biological processes.     

Biochemistry of selenium-derivatized naturally occurring and unnatural nucleic acids

Selenium (Se) can provide unique biochemical and biological functions, and properties to macromolecules, including protein and RNA. Although Se has not yet been found in DNA, identification of the presence of Se in natural tRNAs has led to discovery of the naturally occurring 2-selenouridine and 5-[(methylamino)methyl]-2-selenouridine (mnm(5)se(2)U). The Se-atoms at C(2) of the modified uridines are introduced by 2-selenouridine synthase via displacement of the S-atoms in the corresponding 2-thiouridine nucleotides of the tRNAs, and selenophosphate is used as the Se donor. The research indicated that mnm(5)se(2)U is located at the first or wobble position of the anticodons in several bacterial tRNAs, including tRNA(Lys), tRNA(Glu), and tRNA(Gln). The 2-seleno functionality on this modified nucleotide probably improves the translation accuracy and/or efficiency. These observations in vivo suggest that the presence of Se can provide natural RNAs with useful properties to better function and survival. To further investigate the biochemical and structural properties of Se-derivatized nucleic acids (SeNA), we have pioneered chemical and enzymatic synthesis of Se-derivatized nucleic acids, and introduced Se into both RNA and DNA at a variety of positions by atom-specific replacement of oxygen. This review outlines the recent advancements in chemical and biochemical syntheses, and studies of SeNAs, and their potential applications in structural and functional investigation of nucleic acids and their protein complexes.     

Nucleic acid aptamers and enzymes as sensors

The function of nucleic acids has been an endless source of discovery and invention that has drastically enhanced our appreciation of DNA and RNA as multifaceted polymers. It is now widely known that nucleic acids can act as enzymes (deoxyribozymes and ribozymes) and as receptors (aptamers), and that these functional nucleic acids (FNAs) can either be found in nature or isolated from pools of random nucleic acids. The availability of many natural and artificial FNAs has opened a new horizon for the development of 'smart' molecules for a variety of chemical and biological applications. This review provides a snapshot of recent progress in the application of FNAs as novel sensors for biomolecular detection, drug discovery and nanotechnology.     

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.     

Interstrand disulfide cross-linking of internal sugar residues in duplex RNA

Disulfide cross-linking is being used increasingly more to study the structure and dynamics of nucleic acids. We have previously developed a procedure for the formation of disulfide cross-links through the sugar-phosphate backbone of nucleic acids. Here we report the preparation and characterization of an RNA duplex containing a disulfide interstrand cross-link. A self-complementary oligoribonucleotide duplex containing an interstrand cross-link was prepared from the corresponding 2'-amino modified oligomer. Selective modification of the 2'-amino group with an aliphatic isocyanate, containing a protected disulfide, gave the corresponding 2'-urea derivative in excellent yield. An RNA duplex containing an intrahelical, interstrand disulfide cross-link was subsequently prepared by a thiol disulfide exchange reaction in nearly quantitative yield as judged by denaturing polyacrylamide gel electrophoresis (DPAGE). The cross-linked RNA was further characterized by enzymatic digestion and the Structure of the cross-link lesion was verified by comparison to an authentic sample, prepared by chemical synthesis. The effect of the chemical modifications on duplex stability was determined by UV thermal denaturation experiments. The intrahelical cross-link stabilized the duplex considerably: the disulfide cross-linked oligomer had a melting temperature that was ca. 40 degrees C higher than that of the noncross-linked oligomer.     

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.     

Stable extracellular RNA fragments of Mycobacterium tuberculosis induce early apoptosis in human monocytes via a caspase-8 dependent mechanism

The molecular basis of pathogen-induced host cell apoptosis is well characterized for a number of microorganisms. Mycobacterium tuberculosis is known to induce apoptosis and it was shown that live but not heat killed M. tuberculosis stimulates this biological pathway in monocytes. The dependence of this activity on live bacilli led us to hypothesize that products released or secreted by M. tuberculosis are the primary apoptotic factors for human monocytes. Thus, the culture filtrate of in vitro grown M. tuberculosis strain H37Rv was fractioned by conventional chromatography and the apoptosis-inducing activity of individual fractions was measured on human monocytes. The tests employed included measurement of cell membrane damage, caspase activation, and cytokine release. Small molecular weight RNAs of M. tuberculosis were recognized as the predominant apoptosis inducing factors. The RNA was comprised primarily of tRNA and rRNA fragments that stably accumulate in the culture filtrate during early log-phase growth. The RNA fragments signaled through a caspase-8 dependent, caspase-1 and TNF-α independent pathway that ultimately compromised the human monocytes' ability to control M. tuberculosis infection. These studies provide the first report of bacterial RNA inducing apoptosis. They also provide a foundation to pursue pathways for secretion or release of nucleic acids from M. tuberculosis and the impact of secreted RNA fragments on pathogenesis.