Determination of nanogram quantities of osmium-labeled nucleic acids by stripping (inverse) voltammetry

Modification of nucleic acids with OSO4 in the presence of pyridine results in a formation of a covalently bound electroactive center in a polynucleotide chain detectable by polarographic (voltammetric) methods. It has been shown that DNA modified with osmium (DNA-Os) accumulates at the hanging mercury-drop electrode during a waiting time in a wide range of potentials between 0 and -1.0 V (against the saturated calomel electrode) and produce at neutral pH a well-developed reduction peak at about -1.2 V due to scanning in the cathodic direction. Using the differential-pulse stripping (inverse) voltammetry, nanogram quantities of single-stranded DNA-Os can be determined at relatively short waiting times (1-3 min). Double-stranded DNA is modified with osmium to a much lesser extent as compared to single-stranded polynucleotides. The degree of modification of double-helical DNA is influenced by the presence of single-stranded and distorted double-stranded regions in the DNA molecules and by the environmental conditions which influence the DNA conformation. Osmium can thus be used as a probe of the DNA structure, and a few micrograms of double-helical DNA sample suffice for the voltammetric analysis.     

Stem-loop probe with universal reporter for sensing unlabeled nucleic acids

In this article, we present the design principles and application of a motif composed of a stem-loop probe (SP) hybridized to a fluorescently labeled universal reporter (UR) for sensing unlabeled nucleic acids. At room temperature, SP-UR is in the hairpin-closed form in which the fluorophore of UR is in proximity to the G bases of the hairpin, where consequently the fluorescent emission is quenched significantly. On hybridization with target, SP-UR is trapped in the hairpin-opened configuration in which the fluorophore and the G quenchers are apart. This turns off quenching, increases emission intensity, and signals the presence of target. Compared with the common approach that employs an oligonucleotide probe with a covalently linked fluorophore, the use of a fluorescently labeled universal reporter strand hybridized to an unlabeled stem-loop probe provides a more efficient approach to the fabrication of nucleic acid sensors and microarrays potentially useful for real-time analysis.     

Liquid chromatographic analysis of nanogram quantities of ascorbate in brain tissue

A simple and accurate analysis for brain ascorbate at nanogram levels is described. The analysis employs anion exchange resin high performance liquid chromatography with electrochemical detection. The procedure is rapid and involves only homogenization, centrifugation and mixing of the supernatant extract with an internal standard followed by direct quantitation via liquid chromatography. The ascorbate levels of mouse, rat, and guinea pig brains analysed by this method compare well with existing literature data. The sensitivity is such that small animal brain parts can be analysed readily.     

Assay for nanogram quantities of DNA in cellular homogenates.

The DNA concentration of a crude cellular homogenate can be measured accurately in the nanogram range using the fluorescence enhancement of 4′,6-diamidino-2-phenylindole (DAPI) or bisbenzimidazole (Hoechst H 33258) complexed with DNA. A simple assay has been devised including an internal standard, which allows reliable measurement and compensates for any quenching due to cellular components or buffer. The fluorescence enhancement is highly specific for DNA; no other cell component produces significant fluorescence. The response is linear over a broad dynamic range making the measurement of unknown DNA concentrations convenient.     

Quantitation of nanogram amounts of nucleic acids in the presence of proteins by the ethidium bromide staining technique.

Modification of the method for determining low amounts of RNA and DNA is proposed. It consists in nucleic acid staining in solution with EtBr (1 microgram/ml) followed by photography of 10 microliters drops on a UV-transparent plate under UV illumination. Densitometric measurements of the Polaroid negatives were used to construct standard concentration curves in the range of 1-16 micrograms/ml of DNA or RNA. This permitted to determine nucleic acid in amounts as little as 10 micrograms. The measurements were not influenced by the presence of proteins such as bovine serum albumin, DNase, RNase or proteinase K, thus the method proposed may be useful in determining the nucleic acid content of very small samples or of scarce biological material.     

Tables of critical values for examining compositional non-randomness in proteins and nucleic acids

A binomially distributed statistic pchi2i is defined which in conjunction with a set of critical tables permits, for peptides or proteins of arbitrary lengths, a well-defined answer to the question: Does the proportion of a particular amino acid iota present in that protein deviate significantly from random expectation? An analogous statistic is defined for nucleic acids. This statistic is simply related to the classical chi-squared test. The classical chi2 and the pchi2i are supplementary in that the former permits one to determine that a non-randomness in amino acid composition exists in a protein, while the latter permits one to localize that non-randomness to particular amino acids. The pchi2i statistic takes into account explicity the compositional fluctuations imposed by the finite length of proteins. The tables are more exact than any hitherto existing, and require no intermediate calculations for their use: from the direct experimental measurement of the number of residues of amino acid iota, one immediately reads from the tables whether the number observed is within random expectation or not. These statistics are used to analyze eight proteins of diverse length, function, and origin in an accompanying paper.     

Interaction of nucleic acids. VI. Interaction of purine with nucleic acids

The interaction of purine with DNA, tRNA, poly A, poly C, and poly A. poly U complex was investigated. In the presence of purine, the nucleic acids in coil form (such as denatured DNA, poly A and poly C in neutral solutions, or tRNA) have lower optical rotations. In addition, hydrodynamic studies indicate that in purine solutions the denatured DNA has a higher viscosity and a decreased sedimentation coefficient. These findings indicate that through interaction with purine, the bases along the poly-nucleotide chain are unstacked and are separated farther from each other, resulting in increased assymmetry (and possibly volume) of the whole polymer. Thus, the de-naturation effect of purine reported previously can be explained by this preferential interaction of purine with the bases of nucleic acids in coil form through a hydrophobic-costacking mechanism. Results from studies on optical rotation and helix-coil transition show that the interaction of purine is greater with poly A than with poly C. The influence of temperature, Mg⁺⁺ concentration, ionic strength, and purine concentration on the effect of purine on nucleic acid conformation has also been investigated. In all these situations the unraveling of nucleic acid conformation occurs at much lower temperatures (20–40°C lower) in the presence of purine (0.2–0.6M).     

An NADH-coupled assay for femtogram or nanogram quantities of chymotrypsin

Chymotrypsin can be determined with an NADH-coupled assay. Hydrolysis of the substrate benzoyltyrosine ethyl ester is monitored by coupling the liberation of ethanol to the production of NADH and determining the NADH spectrophotometrically or fluorometrically. Nanogram quantities of chymotrypsin can be determined in milliliter volumes. With these microfluorescence methods this assay can be performed in a final volume of less than a nanoliter, allowing determination of femtogram quantities of chymotrypsin, the amount present in an individual zymogen granule.     

A spectrophotometric assay for nanogram quantities of biotin and avidin

Parameters and conditions of an enzyme based assay for biotin and avidin are presented. Biotinylated glucose-6-phosphate dehydrogenase when complexed with avidin becomes inactivated. Thus it was possible to construct a competitive assay system for biotin. The assay is sensitive between 100-500 ng/ml and could detect as little as 10 ng in 0.1 ml with a between run error of 2.4%. It requires a 60 min incubation at 21 degrees C and 5 min to assay. The avidin assay, based on the degree of inactivation of biotinylated-glucose-6-phosphate dehydrogenase in relation to the concentration of avidin, could detect as little as 0.25 ng in 0.1 ml or 2.5 ng/ml with an assay time of 10 min with a between run error of 3.9%. Both assays are rapid with significant improvements over other non-isotopic methods in sensitivity and comparable to radioisotopic methods in sensitivity with the added advantage of ease of method.     

The analysis of nanogram levels of free amino acids by reverse-phase high-pressure liquid chromatography.

A simple method and apparatus are described for the efficient recovery of proteins from sodium dodecyl sulfate-polyacrylamide gel systems after electrophoretic resolution. This procedure provides for high yields of proteins which are free of sodium dodecyl sulfate and in certain cases, exhibit significant levels of biological activity.