Basic Protein Film Methods in the Electron Microscopy of Nucleic Acids

1978 ◽  
Vol 36 (3) ◽  
pp. 587-594
Author(s):  
N. Davidson
Keyword(s):  
Electron Microscopy ◽  
Nucleic Acid ◽  
Nucleic Acids ◽  
Electrostatic Interactions ◽  
Basic Protein ◽  
Electrolyte Concentration ◽  
Protein Film ◽  
Increased Strength ◽  
Film Method ◽  
Positively Charged

I wish to discuss applications of the basic protein film method for mounting nucleic acids for electron microscopy. In this method, the nucleic acid and an excess of a positively charged low molecular weight globular protein (cytochrome-c is commonly used) are dissolved in a suitable electrolyte solution (ammonium acetate or Tris works well). The electrolyte concentration should be high enough so that the electrostatic binding of the positively charged protein to the negatively charged nucleic acid does not cause precipitation. This solution is layered on to a hypophase which contains a lower concentration of the electrolyte. Some of the protein denatures and forms a film on the surface of the hypophase. Because of the increased strength of the electrostatic interactions at the reduced electrolyte concentration, some of the nucleic acid molecules bind to the positively charged protein film.

Electron Microscopy of Nucleic Acids

1974 ◽  
Vol 32 ◽  
pp. 302-303
Author(s):  
Norman Davidson
Keyword(s):  
Electron Microscopy ◽  
Nucleic Acids ◽  
Basic Protein ◽  
Molecular Biologist ◽  
Topological Properties ◽  
Protein Film ◽  
Polynucleotide Chain

The basic protein film technique for mounting nucleic acids for electron microscopy has proven to be a general and powerful tool for the working molecular biologist in characterizing different nucleic acids. It i s possible to measure molecular lengths of duplex and single-stranded DNAs and RNAs. In particular, it is thus possible to as certain whether or not the nucleic acids extracted from a particular source are or are not homogeneous in length. The topological properties of the polynucleotide chain (linear or circular, relaxed or supercoiled circles, interlocked circles, etc. ) can also be as certained.

Nucleic Acid Molecules Prepared by Monolayer Techniques

1972 ◽  
Vol 30 ◽  
pp. 178-179
Author(s):  
Dimitrij Lang
Keyword(s):  
Electron Microscopy ◽  
Standard Deviation ◽  
Nucleic Acid ◽  
Cytochrome C ◽  
Nucleic Acids ◽  
Contour Length ◽  
Sample Standard Deviation ◽  
Molecular Weights ◽  
Length Measurements ◽  
Protein Monolayer

The success of the protein monolayer technique for electron microscopy of individual DNA molecules is based on the prevention of aggregation and orientation of the molecules during drying on specimen grids. DNA adsorbs first to a surface-denatured, insoluble cytochrome c monolayer which is then transferred to grids, without major distortion, by touching. Fig. 1 shows three basic procedures which, modified or not, permit the study of various important properties of nucleic acids, either in concert with other methods or exclusively:1) Molecular weights relative to DNA standards as well as number distributions of molecular weights can be obtained from contour length measurements with a sample standard deviation between 1 and 4%.

scholarly journals Quantitative Evaluation of Nucleic Acid Degradability of Copper Alloy Surfaces and Its Correlation to Antibacterial Activity

Antibiotics ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 1439
Author(s):  
Akiko Yamamoto ◽  
Shinji Tanaka ◽  
Keiichiro Ohishi
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Copper Ion ◽  
Antibacterial Activities ◽  
Ion Release ◽  
Antibacterial Materials ◽  
Cu Content ◽  
Cu Alloys ◽  
Film Method

Copper (Cu) and its alloys have bactericidal activity known as “contact killing” with degradation of nucleic acids inside the bacteria, which is beneficial to inhibit horizontal gene transfer (HGF). In order to understand the nucleic acid degradability of Cu and its alloy surfaces, we developed a new in vitro method to quantitatively evaluate it by a swab method under a “dry” condition and compared it with that of commercially available antibacterial materials such as antibacterial stainless steel, pure silver, and antibacterial resins. As a result, only Cu and its alloys showed continuous degradation of nucleic acids for up to 6 h of contact time. The nucleic acid degradability levels of the Cu alloys and other antibacterial materials correlate to their antibacterial activities evaluated by a film method referring to JIS Z 2801:2012 for Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria. Nucleic acid degradation by copper (I) and (II) chlorides was confirmed at the ranges over 10 mM and 1–20 mM, respectively, suggesting that the copper ion release may be responsible for the degradation of the nucleic acids on Cu and its alloy surfaces. In conclusion, the higher Cu content in the alloys gave higher nucleic acid degradability and higher antibacterial activities.

A Critical Assessment of RNA-Mediated Materials Synthesis

2010 ◽  
Vol 1272 ◽  
Author(s):  
Stefan Franzen ◽  
Donovan Leonard
Keyword(s):  
Electron Microscopy ◽  
Catalytic Activity ◽  
Nucleic Acid ◽  
Nucleic Acids ◽  
In Vitro Selection ◽  
Water Soluble ◽  
Cds Nanoparticles ◽  
Materials Synthesis ◽  
Rna And Dna

AbstractRNA- and DNA-mediation or templating of materials has been used to synthesize nanometer scale wires, and CdS nanoparticles. However, RNA and DNA have the potential to act as catalysts, which could be valuable tools in the search for new routes to materials synthesis. RNA has the ability to catalyze splicing and cutting of other RNA molecules. Catalytic activity has been extended to more general classes of reactions for both RNA and DNA using in vitro selection methods. However, catalytic activity in materials synthesis is a more recent idea that has not yet found great application. The first example of RNA-mediated evolutionary materials synthesis is discussed with specific data examples that show incompatibility of reagents in the solvent system utilized. The hydrophobic reagent Pd2(DBA)3, used as a metal precursor, was observed to spontaneously form nanostructures composed of Pd2(DBA)3 or Pd(DBA)3 rather than palladium nanoparticles, as originally reported 1. A case study of this materials synthesis example is described including the complimentary use of multi-length scale techniques including transmission electron microscopy (TEM), selected area electron diffraction (SAED), scanning TEM (STEM), electron energy loss spectroscopy (EELS), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS) and optical microscopy (OM). This example raises important questions regarding the extent to which non-aqueous solvents should be used in nucleic acid-mediated processes, the nature of selections in enzyme and materials development, and the requirement for chemical compatibility of the precursor molecules. The importance of good characterization tools at every stage of an in vitro selection is illustrated with concrete examples given. In order to look at the way forward for nucleic acid-mediated materials synthesis, an examination of the chemical interaction of nucleic acids with various precursors is considered. Application of density functional theory calculations provides one means to predict reactivity and compatibility. The repertoire of chemical interactions in the nucleic acids is considered vis-à-vis common metals and metal chalcogenides. The case is made for the need for water-soluble syntheses and well-controlled kinetics in order to achieve the control that is theoretically possible using nucleic-acids as a synthetic tool.

scholarly journals Removing the Polyanionic Cargo Requirement for Assembly of Alphavirus Core-Like Particles to Make an Empty Alphavirus Core

Viruses ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 846
Author(s):  
Julie M. Button ◽  
Suchetana Mukhopadhyay
Keyword(s):  
Nucleic Acid ◽  
Capsid Protein ◽  
Electrostatic Interactions ◽  
Wild Type ◽  
N Terminus ◽  
Charged Residues ◽  
Size And Morphology ◽  
Positively Charged

The assembly of alphavirus nucleocapsid cores requires electrostatic interactions between the positively charged N-terminus of the capsid protein (CP) and the encapsidated polyanionic cargo. This system differs from many other viruses that can self-assemble particles in the absence of cargo, or form “empty” particles. We hypothesized that the introduction of a mutant, anionic CP could replace the need for charged cargo during assembly. In this work, we produced a CP mutant, Minus 38 (M38), where all N-terminal charged residues are negatively-charged. When wild-type (WT) and M38 CPs were mixed, they assembled into core-like particles (CLPs). These “empty” particles were of similar size and morphology to WT CLPs assembled with DNA cargo, but did not contain nucleic acid. When DNA cargo was added to the assembly mixture, the amount of M38 CP that was assembled into CLPs decreased, but was not fully excluded from the CLPs, suggesting that M38 competes with DNA to interact with WT CPs. The composition of CLPs can be tuned by altering the order of addition of M38 CP, WT CP, and DNA cargo. The ability to produce alphavirus CLPs that contain a range of amounts of encapsidated cargo, including none, introduces a new platform for packaging cargo for delivery or imaging purposes.

scholarly journals Hydrogels of Polycationic Acetohydrazone-Modified Phosphorus Dendrimers for Biomedical Applications: Gelation Studies and Nucleic Acid Loading

Pharmaceutics ◽  
2018 ◽  
Vol 10 (3) ◽  
pp. 120 ◽  
Author(s):  
Evgeny K. Apartsin ◽  
Alina E. Grigoryeva ◽  
Audrey Malrin-Fournol ◽  
Elena I. Ryabchikova ◽  
Alya G. Venyaminova ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Polyethylene Glycol ◽  
Nucleic Acid ◽  
Nucleic Acids ◽  
Biomedical Applications ◽  
Structural Units ◽  
Transmission Electron ◽  
Phosphate Buffered Saline ◽  
Acid Loading

In this work, we report the assemblage of hydrogels from phosphorus dendrimers in the presence of biocompatible additives and the study of their interactions with nucleic acids. As precursors for hydrogels, phosphorus dendrimers of generations 1–3 based on the cyclotriphosphazene core and bearing ammonium or pyridinium acetohydrazones (Girard reagents) on the periphery have been synthesized. The gelation was done by the incubation of dendrimer solutions in water or phosphate-buffered saline in the presence of biocompatible additives (glucose, glycine or polyethylene glycol) to form physical gels. Physical properties of gels have been shown to depend on the gelation conditions. Transmission electron microscopy revealed structural units and well-developed network structures of the hydrogels. The hydrogels were shown to bind nucleic acids efficiently. In summary, hydrogels of phosphorus dendrimers represent a useful tool for biomedical applications.

Terminal phosphate group influence on DNA - TiO2 nanoparticle interactions

2009 ◽  
Vol 1236 ◽  
Author(s):  
Zachary Rice ◽  
Nathaniel C Cady ◽  
Magnus Bergkvist
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Electrostatic Interactions ◽  
Phosphate Group ◽  
Covalent Bonds ◽  
Metal Oxide Surfaces ◽  
Sensor Applications ◽  
Terminal Phosphate Group ◽  
Rna Backbone ◽  
Initial Adsorption

AbstractImmobilization of DNA/RNA, onto various metal and metal oxide surfaces is of great importance for the development of future microarray, gene mapping, DNA sequencing, nanoparticle targeting, and sensor applications. Attachment of DNA to solid interfaces typically occurs through either electrostatic interactions or covalent bonds to functional groups introduced to nucleic acid termini. Previously, we and others have demonstrated that alkanephosphates and terminal phosphate groups present on nucleic acids play an important role in the interaction with group IV metal oxides such as zirconium and hafnium, providing a stable linkage to the surface. Titanium dioxide (TiO2), which is frequently employed in various nanoscale applications, belongs to the same group and similar interactions with phosphate are expected. Various adsorption studies have demonstrated binding of nucleic acids to TiO2 surfaces, although the influence of terminal phosphate versus electrostatic interaction (via the DNA/RNA backbone) on the surface interaction is unclear. The research presented here investigates the effect of nucleic acid length, presence of terminal phosphates, and differences between dsDNA and ssDNA on their binding to TiO2 nanoparticles. TiO2 nanoparticles (20 nm) were used to study the adsorption of Lambda DNA (˜48 kbp), and shorter (21 bp) ssDNA and dsDNA oligonucleotides with and without a 5’ phosphate group. Initial adsorption of DNA to nanoparticles was calculated via UV absorption. Results showed that all types of nucleic acids (Lamda DNA, ssDNA and dsDNA) initially bind to nanoparticles, independent of molecular weight single/double strandedness, or phosphorylation state. The total amount of DNA initially adsorbed to nanoparticles (ng/particle) differs between ssDNA and dsDNA, as well as the length of the DNA used. These results show that nucleic acid interactions with TiO2 nanoparticles are not dependent upon the presence of a terminal phosphate group. These results provide valuable data for future applications based on DNA-nanoparticle constructs including nanoelectronics, photovoltaics, and biotemplated synthesis of semiconducting materials.

Structure and Interaction of Protamine by Dark Field Electron Microscopy

1976 ◽  
Vol 34 ◽  
pp. 160-161
Author(s):  
D.P. Bazett-Jones ◽  
F.P. Ottensmeyer
Keyword(s):  
Electron Microscopy ◽  
Dark Field ◽  
Amino Acid Sequences ◽  
Dimensional Structure ◽  
3 Dimensional ◽  
Field Electron ◽  
Proposed Model ◽  
Field Electron Microscopy ◽  
Dark Field Electron ◽  
Positively Charged

It has been shown for some time that it is possible to obtain images of small unstained proteins, with a resolution of approximately 5Å using dark field electron microscopy (1,2). Applying this technique, we have observed a uniformity in size and shape of the 2-dimensional images of pure specimens of fish protamines (salmon, herring (clupeine, Y-l) and rainbow trout (Salmo irideus)). On the basis of these images, a model for the 3-dimensional structure of the fish protamines has been proposed (2).The known amino acid sequences of fish protamines show stretches of positively charged arginines, separated by regions of neutral amino acids (3). The proposed model for protamine structure (2) consists of an irregular, right-handed helix with the segments of adjacent arginines forming the loops of the coil.

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