Water-elutability of nucleic acids from metal-chelate affinity adsorbents: enhancement by control of surface charge density

Immobilized metal affinity chromatography (IMAC) is widely used for purification of proteins, especially "hexahistidine-tagged" recombinant proteins. We previously demonstrated the application of IMAC to selective capture of nucleic acids, including RNA, selectively-denatured genomic DNA, and PCR primers through interactions with purine bases exposed in single-stranded regions. We also found that the binding affinity of nucleic acids for IMAC adsorbents can be increased several-fold by addition of 20 volume% of neutral additives such as ethanol or DMSO. In the present work, it is demonstrated that bound nucleic acids can be effectively eluted with water instead of the usual imidazole-containing competitive eluants, when the surface density of negative charges is enhanced by operation at alkaline pH, or by deliberate metal-underloading of the anionic chelating ligands. With enhanced negative surface charge density, nucleic acid adsorption can be made strongly dependent on the presence of adsorption-promoting additives and/or repulsion-shielding salts, and removal of these induces elution. Complete water-elutability is demonstrated for baker's yeast RNA bound to 10% Cu(II)- underloaded IDA Chelating Sepharose in a binding buffer of 20 mM HEPES, 240 mM NaCl, pH 7. Water elutability will significantly enhance the utility of IMAC in nucleic acid separations.     

Chromatographic properties of cytosine,cytidine and their synthetic analogues

A direct reversed-phase high-performance liquid chromatographic (RP-HPLC) assay was used for the study of the effects of methanol concentration, pH, flow-rate of the mobile phase and column temperature on the retention of the natural nucleic acid components cytosine and cytidine and their synthetic 1-β-d-arabinofuranosyl, 5-aza and 6-aza analogues. The pK_a values were also determined. The greatest changes were observed with changes in pH. The relationship between the capacity factors and the hydrophobicity of the compounds studied was also investigated.     

Single DNA molecule stretching measures the activity of chemicals that target the HIV-1 nucleocapsid protein

We develop a biophysical method for investigating chemical compounds that target the nucleic acid chaperone activity of HIV-1 nucleocapsid protein (NCp7). We used an optical tweezers instrument to stretch single lambda-DNA molecules through the helix-coil transition in the presence of NCp7 and various chemical compounds. The change in the helix-coil transition width induced by wild-type NCp7 and its zinc finger variants correlates with in vitro nucleic acid chaperone activity measurements and in vivo assays. The compound-NC interaction measured here reduces NCp7's capability to alter the transition width. Purified compounds from the NCI Diversity set, 119889, 119911, and 119913 reduce the chaperone activity of 5 nM NC in aqueous solution at 10, 25, and 100 nM concentrations respectively. Similarly, gallein reduced the activity of 4 nM NC at 100 nM concentration. Further analysis allows us to dissect the impact of each compound on both sequence-specific and non-sequence-specific DNA binding of NC, two of the main components of NC's nucleic acid chaperone activity. These results suggest that DNA stretching experiments can be used to screen chemical compounds targeting NC proteins and to further explore the mechanisms by which these compounds interact with NC and alter its nucleic acid chaperone activity.     

Separation of nucleosides and nucleotides by reversed-phase high-performance liquid chromatography with volatile buffers allowing sample recovery

Reversed-phase high-performance liquid chromatography using a C18 column with volatile buffers as the eluant was applied to the separation of a number of nucleosides and nucleotides. Groups of seven nucleosides and five nucleoside monophosphates were separated isocratically employing 0.1 M trimethylammonium acetate and 2% acetonitrile at pH 7.0. Groups of seven nucleoside diphosphates and seven nucleoside triphosphates were separated with 0.1 M triethylammonium bicarbonate and 2% acetonitrile titrated to a pH of 7.1 with acetic acid. The techniques described give resolution and separations comparable to nonvolatile buffers. Moreover, the eluant trimethylammonium acetate or triethylammonium bicarbonate buffer can easily be removed in vacuo from the column effluent, making the technique useful for preparative separations of these compounds. The observed elution pattern of nucleoside phosphates suggests that "paired-ion" chromatography is involved in the separation.     

Radiation-induced decomposition of the purine bases within DNA and related model compounds

This survey focuses on recent developments in the radiation chemistry of purine bases in nucleic acids and related model compounds. Both direct and indirect effects of ionizing radiation are investigated with special emphasis on the structural characterization of the final decomposition products of nucleic acid components. Available assays for monitoring radiation-induced base lesions are critically reviewed.     

Recent aspects of the photochemistry of nucleic acids and related model compounds

This survey focuses on recent developments in the far ultraviolet photochemistry of nucleic acids and related model compounds. The photoproducts discussed are the cyclobutidipyrimidines, the pyrimidine-pyrimidone adducts, the purine-pyrimidine adducts and the addition products of amino acids to pyrimidine bases. The specific aspects of the high-intensity laser photochemistry of nucleic acid components are also briefly reviewed.     

Microemulsion electrokinetic chromatographic analysis of some polar compounds

This study presents the optimization of a microemulsion electrokinetic chromatographic (MEEKC) electrolyte solution by using UV detection and with the method, simultaneous separations of chemically, biochemically and pharmaceutically related anionic and cationic compounds. Representatives of the compound groups were from isoflavonoids, benzodiazepines, metanephrines, diuretics and peptide hormones. The MEEKC separations under basic conditions were first optimized using a two-component isoflavonoid mixture as the sample and an electrolyte containing 10 mM tetraborate as the main buffer (pH 9.5). The stable microemulsion phase was adjusted with various amounts of octane, 1-butanol and sodium dodecyl sulfate (SDS). An only acidified electrolyte solution used in the study was made of phosphoric acid (pH 1.8) containing octane, SDS and ethyl acetate. The analyses with isoflavonoids showed that electrophoretic mobilities of the investigated compounds were highly related to the concentrations of SDS and 1-butanol with linear and parabolic correlation, respectively. However, addition of octane gave linear correlation only at low concentrations. In most cases four to six structurally related compounds and even 13 diuretics with various polar properties were separated from each other in basic microemulsion medium. The acidified MEEKC electrolyte gave good resolution for anionic metanephrines.     

Volumetric properties of nucleic acids

Volumetric studies can yield useful new information on a myriad of intra- and intermolecular interactions that stabilize nucleic acid structures. In particular, appropriately designed volumetric measurements can characterize the conformation-dependent hydration properties of nucleic acids as a function of solution conditions, including temperature, pressure, ionic strength, pH, and cosolvent concentration. We have started to accumulate a substantial database on volumetric properties of DNA and RNA, as well as on related low molecular weight model compounds. This database already has provided unique insights into the molecular origins of various nucleic acid recognition processes, including helix-to-coil and helix-to-helix conformational transitions, as well as drug-DNA interactions. In this article, we review recent progress in volumetric investigations of nucleic acids, emphasizing how these data can be used to gain insight into intra-and intermolecular interactions, including hydration properties. Throughout this review, we underscore the importance of volume and compressibility data for characterizing the hydration properties of nucleic acids and their constituents. We also describe how such volumetric data can be interpreted at the molecular level to yield a better understanding of the role that hydration can play in modulating the stability and recognition of nucleic acids.     

Studies on the Autolysis of Aspergillus oryzae

The conditions of autolysis of washed mycelia of Aspergillus oryzae were systematically examined as for temperature, pH, aeration, energy supply, and chemicals which stimulate autolysis. Below 45°C, the higher the temperature the faster was the rate of autolysis. Optimum pH of autolysis with special reference to the excretion of nucleic acid components and amino acids was 5. With the optimum conditions of autolysis settled by us, 90 to 100% of nucleic acids, 75% of protein, and 20% of sugars in the mycelia were excreted into the medium within three days.

In the presence of lipophilic compounds such as toluene and sodium salts of fatty acids, autolysis occurred much faster than in distilled water. Autolysis was inhibited by the addition of glucose and aeration.

Mycelia of Aspergillus oryzae were autolyzed in distilled water, in toluene-saturated water, or in acetate buffer, pH 5.4, at 30°C. The cytoplasmic materials disappeared from cells during autolysis, but the cell wall retained its shape even after autolysis. The disappearance of the cytoplasmic materials started from the inner part under an aerobic condition and from the outer part under an anaerobic condition. During the autolysis, 15% of the cellular proteins was excreted as free amino acids (60%) and peptides (15%). Glucose, ribose, glucosamine, and three unidentified sugars were found in autolyzate. After eighteen hours of autolysis stimulated by toluene, 81% of the cellular nucleic acids was excreted as uridine (28%), xanthine (24%), hypoxanthine (17%), and two other nucleosides or bases.     

Effects of nucleic acid compounds on viability and cell composition of Bdellovibrio bacteriovorus during starvation

The effects of various exogenous nucleic acid compounds on the viability and cell composition of Bdellovibrio bacteriovorus starved in buffer were measured. In decreasing order of effectiveness, these compounds were found to decrease the rate of loss of viability and the loss of cell carbon, cell ribonculeic acid, and cell protein: glutamate > ribonucleoside monophosphates > ribonucleosides > deoxyribonucleoside monophosphates. Similar sparing effects were not observed with nucleic acid bases, deoxyribonucleosides, ribose, ribose-5-phosphate, deoxyribose, and deoxyribose-5-phosphate. Appreciable increases in the respiration rate over the endogenous rate did not occur when cell suspensions were incubated with individual or mixtures of nucleic acid compounds. Formation of ¹⁴CO₂ by cell suspensions incubated with carbon 14-labeled nucleic acid compounds indicated ribonucleosides and ribonucleoside monophosphates were respired and to a small extent, were incorporated into cell material of non-growing cells. The respired ¹⁴CO₂ was derived mainly from the ribose portion of these molecules. No respired ¹⁴CO₂ or incorporated carbon 14 was found with bdellovibrios incubated with other nucleic acid compounds tested, including free ribose. During growth of B. bacteriovorus on Escherichia coli in the presence of exogenous UL-¹⁴C-ribonucleoside monophosphates, 10–16% of the radioactivity was in the respired CO₂ and of the radioactivity incorporated into the bdellovibrios, only 40 to 50% resided in the cell nucleic acids. However, during growth on ¹⁴C-adenine,-uracil, or-thymidine labeled E. coli, only trace amounts of ¹⁴CO₂ were found and 90% or more of the incorporated radioactivity was in the bdellovibrio nucleic acids. It is concluded that bdellovibrio can use ribonucleoside monophosphates during growth and starvation as biosynthetic precursors for synthesis of both nucleic acids and other cell materials as well as catabolizing the ribose portion for energy purposes.Abbreviations HM buffer 5 mM N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid (pH 7.6) containing 0.1 mM CaCl₂ and MgCl₂ - DNA deoxyribonucleic acid - RNA ribonucleic acid - Ar, Cr, Gr, Ur ribonucleosides of adenine, cytosine, guanine, uracil, respectively - dTr deoxythymidine - AMP, CMP, GMP, UMP ribonucleoside monophosphates of adenine, cytosine, guanine, and uracil, respectively - dTMP deoxythymidine monophosphate - ATP adenosine triphosphate - PFU plaque-forming units     

From cisplatin to artificial nucleases — the role of metal ion-nucleic acid interactions in biology

Metal ions and metal coordination compounds bind to nucleic acids in a variety of ways, ranging from weak electrostatic interactions via hydrogen bonding and/or van der Waals forces to strong covalent binding. Metal ions naturally take part in the formation and the degradation of nucleic acids, and the propensity of certain metal coordination compounds to bind to nucleic acids, notably DNA, is enploited in cancer chemotherapy. Moreover, metal compounds have a wide potential as chemical probes for nucleic acid structures and as tools for nucleic acid processing.     

Simple Process for the Reduction in the Nucleic Acid Content in Yeast

A simple one-step process for the nucleic acid reduction in Rhodotorula glutinis is described. The process consists of submitting the yeast cells to a heat treatment in an acidic (pH 2) spent medium. The optimal temperature for pH 2 medium is 90 C and the final nucleic acid content in treated yeasts was 1.2%. Heat treatment at acidic pH is preferred to that at alkaline pH because it offers a better protection for amino acids and crude protein, while being more efficient in lowering the nucleic acid level. The new process is economic and rapid and could be easily used for industrial application.     

Additional components of bovine heart cytochrome c oxidase demonstrated by high-resolution polyacrylamide-gel electrophoresis in the presence of chloral hydrate.

We have shown that aq. 100% (w/v) chloral hydrate (2,2,2-trichloroethane-1,1-diol) dissociates bovine heart cytochrome c oxidase. We have developed new procedures of polyacrylamide-gel electrophoresis in the presence of chloral hydrate that permit variation in the pH of the separation, and, by using these procedures, we have observed 15 components in preparations of the enzyme. This number contrasts with the eight bands that were seen on electrophoresis in the presence of SDS (sodium dodecyl sulphate) and urea. We have isolated material from these eight bands and have characterized each by electrophoresis in the presence of chloral hydrate. Twelve of the fifteen components that were seen by electrophoresis in chloral hydrate were identified as constituents of the eight bands seen by electrophoresis in the presence of SDS and urea. Two-dimensional electrophoretic separations confirmed these identifications ans showed that the other three components which were resolved as discrete bands by electrophoresis in the presence of chloral hydrate appeared to be diffusely present in the electrophoretic separations performed in the presence of SDS and urea, which suggested anomalous behaviour in that detergent. Trypsin treatment of cytochrome c oxidase caused total loss, as observed by electrophoretic separations in the presence of chloral hydrate, of a number of components. The trypsin-sensitive components included all of those that behaved anomalously in the presence of SDS and urea. Chloral hydrate is a potent non-ionic dissociating agent for cytochrome c oxidase and its use in polyacrylamide-gel electrophoresis, with variation in the pH of the gel, permits charge-dependent separations that should have general application in the analysis of membrane proteins.     

Hybridization-based affinity partitioning of nucleic acids using PEG-coupled oligonucleotides.

Polyethylene glycol (PEG)-coupled oligonucleotides are partitioned in an aqueous two-phase system PEG/dextran. The affinity of the oligonucleotide for the PEG-rich phase increases proportionally to the length of the coupled PEG polymer. After hybridization, the PEG-coupled oligonucleotide is able to force a complementary nucleic acid strand into the PEG-rich phase. This property can be used for the sequence-specific isolation of nucleic acids through hybridization-based affinity partitioning. The dependence of the partition coefficient in this system on various parameters is described. The application of this principle to multistage chromatographic separations is demonstrated.     

Histochemical applications of two phenanthridinium compounds

The fluorescent compounds ethidium monoazide and ethidium bromide were found to react intensely with nucleic acids of fixed, paraffin embedded tissues of rat and mouse. For routine staining, 10(-5) M solutions of ethidium bromide and its monoazide analogue were virtually identical in their reactions. Fresh frozen sections of the tissues reacted in the same manner as fixed, paraffin embedded samples. Fluorescence of DNA and RNA in rat pancreas could be selectively abolished by taking advantage of the greater sensitivity of RNA to acid hydrolysis. Hydrolysis in aqueous solutions (1 N HCl at 55-60 C) abolished RNA fluorescence in 5 min, whereas 20 min or longer were required to destroy DNA fluorescence. DNA fluorescence was selectively abolished by 3 hr in 0.1 N HCl in anhydrous methanol while the RNA remained unaffected. Rat pancreas stained with the 10(-5) M ethidium compounds below pH 5.0 showed reduced RNA fluorescence, but the DNA continued to fluoresce brightly at pH 0.6. Reducing the pH of the staining solution to pH 1.0, therefore, was an additional method of selectively abolishing RNA fluorescence. Ethidium solutions in 5.0 M NaCl at pH 5.0 had little effect on DNA or RNA fluorescence. This new method of examining nucleic acids in fixed tissue samples opens new approaches to the histochemistry of these substances. The method also offers new possibilities for the study of mutagenic drug-DNA interactions.     

Histochemical Applications of Two Phenanthridinium Compounds

The fluorescent compounds ethidium monoazide and ethidium bromide were found to react intensely with nucleic acids of fixed, paraffin embedded tissues of rat and mouse. For routine staining, 10^-5 M solutions of ethidium bromide and its monoazide analogue were virtually identical in their reactions. Fresh frozen sections of the tissues reacted in the same manner as fixed, paraffin embedded samples. Fluorescence of DNA and RNA in rat pancreas could be selectively abolished by taking advantage of the greater sensitivity of RNA to acid hydrolysis. Hydrolysis in aqueous solutions (1 N HCl at 55-60 C) abolished RNA fluorescence in 5 min, whereas 20 min or longer were required to destroy DNA fluorescence. DNA fluorescence was selectively abolished by 3 hr in 0.1 N HCl in anhydrous methanol while the RNA remained unaffected. Rat pancreas stained with the 10^-5 M ethidium compounds below pH 5.0 showed reduced RNA fluorescence, but the DNA continued to fluoresce brightly at pH 0.6. Reducing the pH of the staining solution to pH 1.0, therefore, was an additional method of selectively abolishing RNA fluorescence. Ethidium solutions in 5.0 M NaCl at pH 5.0 had little effect on DNA or RNA fluorescence. This new method of examining nucleic acids in fixed tissue samples opens new approaches to the histochemistry of these substances. The method also offers new possibilities for the study of mutagenic drug-DNA interactions.     

Nucleic acid induced unfolding of recombinant prion protein globular fragment is pH dependent

Nucleic acid can catalyze the conversion of α‐helical cellular prion protein to β‐sheet rich Proteinase K resistant prion protein oligomers and amyloid polymers in vitro and in solution. Because unfolding of a protein molecule from its ordered α‐helical structure is considered to be a necessary step for the structural conversion to its β‐sheet rich isoform, we have studied the unfolding of the α‐helical globular 121–231 fragment of mouse recombinant prion protein in the presence of different nucleic acids at neutral and acid pH. Nucleic acids, either single or double stranded, do not have any significant effect on the secondary structure of the protein fragment at neutral pH; however the protein secondary structure is modified by the nucleic acids at pH 5. Nucleic acids do not show any significant effect on the temperature induced unfolding of the globular prion protein domain at neutral pH which, however, undergoes a gross conformational change at pH 5 as evidenced from the lowering of the midpoint of thermal denaturation temperatures, Tm, of the protein. The extent of Tm decrease shows a dependence on the nature of nucleic acid. The interaction of nucleic acid with the nonpolar groups exposed from the protein interior at pH 5 probably contributes substantially to the unfolding process of the protein.     

Aphelenchoides rutgersi: Growth requirements for heme and purine nucleotides in crude chick embryo extract

The crude chick embryo extract (CEE) at 20% concentration (v/v) in the basal medium (M-10), enabled the growth, reproduction, and continuous culturing of Aphelenchoides rutgersi Hooper and Myers. No such growth promoting effect was found with chick embryo extract (Ultra filtrate), human gamma globulin and beta lipoprotein (Cohn fraction IV-4) when used either alone or in combination with hemin.The nematode growth factor(s) in the CEE were stable to trypsinization and temperature of 100 C for 5 min but not for 15 min. After acid treatment to pH 3.0 with sulfuric acid, the supernatant retained only partial activity. But, after treatment with perchloric acid to pH 3.0 or with activated charcoal, the CEE was completely ineffective as a growth supplement. The activity of charcoal-treated CEE has been completely recovered by addition of hemin (10 μg/ml M-10).Deletion of nucleic acid supplements from M-10 but not from the CEE did not affect the reproduction of the nematodes at all. However, the reproduction was significantly reduced when the nucleic acids were removed both from the M-10 and the CEE. Selective deletion of deoxyribonucleotides and purine-, and pyrimidinenucleotides of ribose series indicated only limited nutritional requirement for purine compounds.