The Mechanism of Staining Explained on A Chemical Basis. I. The Reaction Between Dyes,Proteins and Nucleic Acid

Basic dyes cause an increase in hydrogen-ion concentration when added to a solution containing nucleic acid, the both solutions were originally at the same pH. Acid dyes have no effect on nucleic acid solutions. Basic dyes show the same behavior when treated with solutions of typical proteins. Acid dyes when treated with proteins show an analogous effect but in the opposite direction. The only adequate explanation found is that there is a definite reaction between the dye ions and the oppositely charged ions of protein or nucleic acid. The bearing of these results on the theory of staining is discussed. The growing recognition of the dominance of chemical forces in colloidal adsorption behavior is indicated, and certain of the experimental bases for this recognition are pointed out and discussed.     

The improved method in agarose gel electrophoresis of nucleic acid

In most molecular experiments, nucleic acids are subjected to agarose gel electrophoresis to determine the size of the molecule. The addition of a nucleic acid dye allows the nucleic acid to be detected under the UV image system after running the gel, so the nucleic acid dye is an integral part of the electrophoresis experiment. But when considering the mutagenicity and toxicity of nucleic acid dyes, one must be careful to insure the proper disposal of experimental waste. In this article, a new usage of nucleic acid dye in agarose gel electrophoresis is described where the nucleic acid dyes were added to the loading buffer and nucleic acid marker buffer. The results show that this method has advantages as: a smaller amount of dye can be used, there is less time in contact with the dye, and its operation is easier and reduces toxicity damage. Also the bands showed a much clearer image, having a lower background value. The improved method shows better results with lower toxicity and is superior to the traditional method.     

Quantitative approaches to monitor protein-nucleic acid interactions using fluorescent probes

Sequence-specific recognition of nucleic acids by proteins is required for nearly every aspect of gene expression. Quantitative binding experiments are a useful tool to measure the ability of a protein to distinguish between multiple sequences. Here, we describe the use of fluorophore-labeled oligonucleotide probes to quantitatively monitor protein/nucleic acid interactions. We review two complementary experimental methods, fluorescence polarization and fluorescence electrophoretic mobility shift assays, that enable the quantitative measurement of binding affinity. We also present two strategies for post-synthetic end-labeling of DNA or RNA oligonucleotides with fluorescent dyes. The approaches discussed here are efficient and sensitive, providing a safe and accessible alternative to the more commonly used radio-isotopic methods.     

Cytofluorometric studies on conformation of nucleic acids in situ. I. Restriction of acridine orange binding by chromatin proteins.

Binding of the fluorochrome acridine orange (AO) to nucleic acids in situ is studied by automated cytofluorometry in two differentiating cell systems: Friend virus-transformed murine erythroleukemia induced to differentiate by dimethyl sulfoxide, and phytohemagglutinin-stimulated human lymphocytes. The specificity of the stain for deoxyribonucleic acid is discussed on the basis of data obtained by cell treatment with nucleases. Evidence is presented that in the case of Friend leukemia cells, but not phytohemagglutinin-stimulated lymphocytes, a significant change in the number of AO-intercalating sites in DNA occurrs during differentiation. These results suggest that changes in nuclear chromatin occurring during cell differentiation may be correlated, in some but not all systems, with changes in accessibility of DNA in situ to intercalating dyes. The role of divalent cations, especially Mg2+, in the conformation of nuclear chromatin and in modulation of the accessibility of nucleic acids to AO is discussed. The method provides a tool for the study of nucleic acid-protein interaction in situ, and in some cell systems it may be applicable as a marker for recognition of cell transformation, differentiation or neoplasia.     

Models of interaction between nucleic acids and proteins. Hydrogen bonding of arginine with nucleic acid bases, phosphate groups and carboxylic acids.

Complex formation between the side chain of arginine and nucleic acid bases has been investigated by proton magnetic resonance in dimethylsulfoxide. Simultaneous formation of two hydrogen bonds leads to a selectivity of arginine interaction towards cytosine and guanine. A comparison is made of the interaction of arginine side chain with nucleic acid bases, phosphate and carboxylate anions. It is shown that interaction between carboxylate and arginine is stronger than between phosphate and arginine. These results are discussed with respect to the selective recognition of nucleic acid bases by arginine side chains and by the arginyl-glutamyl ion pair which could form in proteins interacting with nucleic acids.     

Domains required for nucleic acid binding activities in chloroplast ribonucleoproteins.

Five ribonucleoproteins (or RNA-binding proteins) from tobacco chloroplasts have been identified to date; each of these contains an acidic N-terminal domain (24-64 amino acids) and two conserved RNA-binding domains (82-83 amino acids). All five ribonucleoproteins can bind to ssDNA and dsDNA but show high specificity for poly(G) and poly(U). Here we present the nucleic acid binding activity of each domain using a series of deletion mutant proteins made in vitro from the chloroplast 29 kDa ribonucleoproteins. The acidic domain does not have a positive effect on binding activities and proteins lacking this domain show higher affinities for nucleic acids than the wild-type proteins. Mutant proteins containing single RNA-binding domains can bind to poly(G) and poly(U), though with lower affinities than proteins containing two RNA-binding domains. The spacer region (11-37 amino acids) between the two RNA-binding domains does not interact with poly(G) or poly(U) by itself, but is required for the additive activity of the two RNA-binding domains. Proteins consisting of two RNA-binding domains but lacking the spacer have the same activity as those containing only one RNA-binding domain. Possible roles for each domain in chloroplast ribonucleoproteins are discussed.     

An electron microscopic method for studying nucleic acid-protein complexes. Visualization of RNA polymerase bound to the DNA of bacteriophages T7 and T3

Double-stranded DNA can be readily adsorbed on mica or directly on carbon coated grids from the surface of solutions containing ethidium bromide, actinomine, or propidium diiodide. The DNA molecules are unfolded, well separated, and show a length distribution similar to molecules prepared by protein monolayer techniques. Since the intercalating dyes tested do not lead to an increased apparent diameter of the nucleic acid the method is useful for the study of nucleic acid–protein complexes. As a model, the binding of E. coli RNA polymerase to phage T7 and T3 DNA was examined under different conditions. The enzyme can easily be identified and its position along the DNA molecule can be mapped.     

核酸-蛋白质结合能在剪切位点识别中的应用

把最大信息原理应用到核酸序列的保守位点分析中。利用最大信息原理,推导出了核酸和蛋白质特异性结合时的结合能表达式,并且估计了和蛋白质发生相互作用的核酸序列上的位点范围。为了检验此理论是否较为成功地反映了核酸和蛋白质结合时的实际情况,把它应用到基因内含子剪切位点的识别中,识别结果达到了较高的敏感性和特异性,这说明利用最大信息原理推导结合能表达式及估计核酸序列上参与反应的位点范围的理论是较为成功的。此研究结果一方面有助于核酸和蛋白质相互作用的理解,另一方面,也有助于和蛋白质发生相互作用的各种核酸序列的计算机识别研究。     

The Use of Buffered Solutions in Staining: Theory and Practice

The importance of pH in staining tissue is emphasized. The effect of pH upon the selectivity and intensity of staining with iron hematoxylin, malachite green, and eosin Y is considered. Many difficulties may be avoided by staining in the higher alcohols and directions are given for the preparation of buffer solutions from pH 1.2-8 in alcohol. The concentration of stains, time of staining, and order of staining are discussed for progressive and regressive staining. At pH 8 in 95% alcohol very few tissues stain with malachite green at a concentration of 1/1000 saturated. At pH 6 most cytoplasmic elements stain with malachite green at a concentration of 1/1000 saturated or with eosin Y at 1/250 saturated. As the pH is lowered more tissue elements stain until the nucleus is completely stained. This behavior is in accord with the theory of chemical combination of dyes with proteins, which states that proteins combine with basic dyes on the basic side of their isoelectric points and with acid dyes on the acid side of their isoelectric points. With hematoxylin stain the pH range is much shorter. A satisfactory hematoxylin stain is composed of 0.1% hematoxylin, 0.1% FeCl₃, and HCl to bring the pH to 1.2-1.6 in 80% alcohol. With this stain, which may be used immediately, the nuclei of most tissues begin to stain at pH 1.2 and much of the cytoplasm will be stained if the pH is raised to 1.4. The shortness of this effective pH range is thought to be due to the dissociation of the hematoxylin-iron-protein complex. The use of different dyes successively at different pH values, such as hematoxylin at 1.3, malachite green at 8, and eosin at 6, permits better differentiation of the tissue elements, and intelligent variations in the staining technic.     

Role of Aromatic Amino-acid Residues in the Binding of Enzymes and Proteins to Nucleic Acids

MANY studies have been made of the specificity of interaction between nucleic acids and polypeptides, proteins and enzymes^1,2. Electrostatic forces between basic amino-acids and phosphate groups contribute to the stability of the complexes, but selective recognition requires more specific interactions which are not yet understood. The recognition of a specific region of a nucleic acid could be explained if this region has some particular conformation or if there are specific interactions between a few amino-acid residues and the bases of this region. We wish to report results which show that the aromatic amino-acids tryptophan and tyrosine can interact with nucleic acid bases in double stranded nucleic acids. They suggest that aromatic amino-acid residues of enzymes and proteins could participate in the binding to nucleic acids by intercalating between the bases and thus constraining the nucleic acid molecule to adopt a definite position with respect to the protein molecule.     

Unstainable DNA in cell nuclei. Comparison of ten different fluorochromes

Ten fluorochromes with specificity for DNA were used to compare the stainability of nuclei of exponentially growing, nondifferentiated Friend leukemia (FL) cells with that of dimethylsulfoxide-induced, fully differentiated FL cell nuclei. Decreased accessibility of DNA to several dyes, particularly pronounced in the case of some intercalators, was observed in differentiated cells. Dye binding was also compared for both sets of nuclei following extraction of nuclear proteins, mostly histones, with 0.1-N HCl. Acid extraction of nuclear proteins increased the accessibility of DNA to varying degrees, depending upon the fluorochrome. In most cases, the differences in fluorescence between differentiated and nondifferentiated nuclei stained with most intercalating dyes was abolished by acid treatment. The results are discussed in terms of the mode of interaction between DNA and the various fluorochromes and the factors associated with chromatin structure, which may affect or be associated with different degrees of proliferative activity.     

The Effect of the Chemical Nature of a Decolorizer on Its Functioning. II. the Apparent Isoelectric Point

The isoelectric point of a bacterial system is the hydrogen-ion concentration at which there is equal retention of anion and cation. Defining this point as that at which there is equal retention of acidic and basic stain when acetone is used as a decolorizer, it is shown that acidic decolorizers shift the experimentally determined point to a higher pH-value while basic decolorizers shift it to a lower value. Thus basic decolorizers show abnormally high decolorizing power toward smears stained with acid dyes, and acid decolorizers show the same abnormal behavior toward smears stained with basic dye. By basic decolorizer is meant, not one of high pH-value, but one which will form a salt with acids, as for example pyridin or anilin. This indicates an ionic chemical equilibrium as a factor in the mechanism of staining.     

Model studies of interactions between nucleic acids and proteins: hydrogen bonding of amides with nucleic acid bases.

The formation of hydrogen bonded complexes between nucleic acid bases and acetamide has been studied by nuclear magnetic resonance in CDC13 at different temperatures. Pairs of hydrogen bonds are formed when acetamide binds to nucleic acid bases. Thermodynamic parameters have been computed and compared to those obtained for the association of carboxylic acids with nucleic acid bases. The role of hydrogen bonded complexes in the association of proteins with nucleic acids is discussed.     

The binding of aminoacyl-tRNA synthetases to triazine dye conjugates.

The binding of thirteen aminoacyl-tRNA synthetases to thirty two immobilised procion dyes has been investigated. Most dyes bind one or more enzymes. The amino acid substrates are not normally potent eluants, with the notable exception of tryptophan eluting tryptophanyl-tRNA synthetase from Brown MX-5BR. Phosphate is frequently extremely effective, much more than expected by simple considerations of ionic strength, indicating that many of the dyes are able to mimic the phosphate groups of the phosphodiester backbone of the nucleic acid. Procedures for the purification of methionyl-, tryptophanyl- and tyrosyl-tRNA synthetases are presented and compared to the conventional purifications of these enzymes. The results indicate the general applicability of these dye columns to the purification of most enzymes of of nucleic acid metabolism and the necessity of investigating as many different dyes as possible for any individual enzyme.     

The extended left-handed helix: a simple nucleic acid-binding motif

The poly-proline type II extended left-handed helical structure is well represented in proteins. In an effort to determine the helix's role in nucleic acid recognition and binding, a survey of 258 nucleic acid-binding protein structures from the Protein Data Bank was conducted. Results indicate that left-handed helices are commonly found at the nucleic acid interfacial regions. Three examples are used to illustrate the utility of this structural element as a recognition motif. The third K homology domain of NOVA-2, the Epstein-Barr nuclear antigen-1, and the Drosophila paired protein homeodomain all contain left-handed helices involved in nucleic acid interactions. In each structure, these helices were previously unidentified as left-handed helices by secondary structure algorithms but, rather, were identified as either having small amounts of hydrogen bond patterns to the rest of the protein or as being "unstructured." Proposed mechanisms for nucleic acid interactions by the extended left-handed helix include both nonspecific and specific recognition. The observed interactions indicate that this secondary structure utilizes an increase in protein backbone exposure for nucleic acid recognition. Both main-chain and side-chain atoms are involved in specific and nonspecific hydrogen bonding to nucleobases or sugar-phosphates, respectively. Our results emphasize the need to classify the left-handed helix as a viable nucleic acid recognition and binding motif, similar to previously identified motifs such as the helix-turn-helix, zinc fingers, leucine zippers, and others.     

Milling Red SWB: A Textile Dye for Staining Epithelial Intercellular Bridges

Fourteen textile dyes were evaluated as histological stains for benign and malignant tissues. An acid dye, Milling red SWB (Acid Red 114, C.I. 23635) was found to possess great affinity for epithelial intercellular bridges. The intercellular bridges were demonstrated clearly when the Milling red SWB was used as a 1% solution in 1% potassium alum after hematoxylin and, also, when methylene blue ZX (Basic Blue 9, C.I. 52015) was used as a counter-stain, without hematoxylin. The intercellular bridges could not be demonstrated in pleomorphic cells which invaded the subepithelial connective tissue.     

Action of inhibition by fluorodeoxyuridine and its reversal by thymidine on the nucleic acid fractions of the overground parts of photoperiodically inducedChenopodium rubrum plants

The nucleic acid fractions obtained by chromatography on MAK columns were compared in 4 variants ofChenopodium rubrum plants treated in different ways during floral induction. The first variant was normally induced to flowering. The second one was inhibited by application of FUDR to the apical bud on the third day of induction. In the third variant the inhibitory effect of FUDR was reversed by application of THY 24 h after FUDR treatment. In the fourth variant THY was applied 24 h after the termination of induction at a time when it was no longer able to reverse the inhibition of flowering. In plants treated with FUDR, a decrease in DNA and RNA synthesis was observed. After reversal of the inhibitory effect of FUDR by THY, DNA synthesis remained somewhat lower than in the control variant but RNA synthesis reached the same level as in the controls or even surpassed it. In plants to which THY was applied at a time when it was no longer possible to reverse flowering, the results obtained from different experiments were not identical. In some experiments nucleic acid synthesis remained lower than in the variant in which THY restituted flowering. In other experiments nucleic acid synthesis was fully restituted and reached the same level as in the control variant. This phenomenon is explained by the different size of the experimental plants at the beginning of the different experiments. The results are discussed with respect to the relation of the dynamics of nucleic acids to growth and development of the plants.     

CRISPR-based DNA and RNA detection with liquid-liquid phase separation

The ability to detect specific nucleic acid sequences allows for a wide range of applications such as the identification of pathogens, clinical diagnostics, and genotyping. CRISPR-Cas proteins Cas12a and Cas13a are RNA-guided endonucleases that bind and cleave specific DNA and RNA sequences, respectively. After recognition of a target sequence, both enzymes activate indiscriminate nucleic acid cleavage, which has been exploited for sequence-specific molecular diagnostics of nucleic acids. Here, we present a label-free detection approach that uses a readout based on solution turbidity caused by liquid-liquid phase separation (LLPS). Our approach relies on the fact that the LLPS of oppositely charged polymers requires polymers to be longer than a critical length. This length dependence is predicted by the Voorn-Overbeek model, which we describe in detail and validate experimentally in mixtures of polynucleotides and polycations. We show that the turbidity resulting from LLPS can be used to detect the presence of specific nucleic acid sequences by employing the programmable CRISPR-nucleases Cas12a and Cas13a. Because LLPS of polynucleotides and polycations causes solutions to become turbid, the detection of specific nucleic acid sequences can be observed with the naked eye. We furthermore demonstrate that there is an optimal polynucleotide concentration for detection. Finally, we provide a theoretical prediction that hints towards possible improvements of an LLPS-based detection assay. The deployment of LLPS complements CRISPR-based molecular diagnostic applications and facilitates easy and low-cost nucleotide sequence detection.     

Use of fluorescent DNA-intercalating dyes in the analysis of DNA via ion-pair reversed-phase denaturing high-performance liquid chromatography

SYBR Green 1 is an asymmetrical cyanine DNA-binding dye that provides an opportunity for increasing the sensitivity of nucleic acid detection when used in conjunction with gel electrophoresis. In this paper, we summarize the general properties and specific uses of SYBR green 1 in ion-pair reversed-phase denaturing high-performance liquid chromatography (IP DHPLC). We describe several applications for the WAVE DHPLC platform that illustrate the generic potential of such intercalating dyes in mutation detection and gene expression profiling. We show that SYBR Green 1 obviates the need to use end-labeled oligodeoxynucleotides for the sensitive detection of nucleic acids during chromatography. Moreover the incorporation of SYBR Green 1 into samples and elution buffers does not impair resolution and has no significant effect on the retention times of DNA fragments compared with dye-free DHPLC.