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%.

Electron Microscope Study of RNA's of Paramyxoviruses

1972 ◽  
Vol 30 ◽  
pp. 196-197
Author(s):  
Ruchama Baum ◽  
J.T. Seto
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Electron Microscope Study ◽  
Sendai Virus ◽  
Sodium Dodecyl ◽  
Rna Molecules ◽  
Virus Particles ◽  
Molecular Weights ◽  
Length Measurements

The ribonucleic acid (RNA) of paramyxoviruses has been characterized by biochemical and physiochemical methods. However, paramyxovirus RNA molecules have not been studied by electron microscopy. The molecular weights of these single-stranded viral RNA molecules are not known as yet. Since electron microscopy has been found to be useful for the characterization of single-stranded RNA, this investigation was initiated to examine the morphology and length measurements of paramyxovirus RNA's.Sendai virus Z strain and Newcastle disease virus (NDV), Milano strain, were used. For these studies it was necessary to develop a method of extracting RNA molecules from purified virus particles. Highly purified Sendai virus was treated with pronase (300 μg/ml) at 37°C for 30 minutes and the RNA extracted by the sodium dodecyl sulfate (SDS)-phenol procedure.

Observation of Fast Sedimenting, Replicating Bacteriophage T7 DNA by Negative Staining

1980 ◽  
Vol 38 ◽  
pp. 538-539
Author(s):  
P. Serwer
Keyword(s):  
Electron Microscopy ◽  
Cytochrome C ◽  
Dna Packaging ◽  
Negative Staining ◽  
Bacteriophage T7 ◽  
Dna Complexes ◽  
Sufficient Detail ◽  
Protein Monolayer

To package the DNA of bacteriophage T7, a preformed, DNA-free capsid (capsid I) with an envelope thicker than the envelope of bacteriophage T7:(a) binds DNA, (b) converts to a capsid (capsid II) with a bacteriophage-like envelope prior to packaging DNA and (c) draws in DNA (1). During attempts to understand T7 DNA packaging, complexes of capsids with mature T7 DNA and complexes of capsids with longer than mature, linear T7 DNA have been isolated (2). Objects with capsid-like dimensions were observed on a fast sedimenting, replicating complex of T7 DNA (100S+ DNA) prepared for electron microscopy using a protein monolayer-shadowing technique (3). This procedure for preparation of specimens does not, however, reveal sufficient detail to rigorously identify an object as a capsid. To better visualize objects bound to 100S+ T7 DNA, this DNA has been prepared for electron microscopy using the aqueous technique for the negative staining of capsid-DNA complexes more recently described (4) (the DNA is coated with cytochrome c and is revealed in an extended configuration).

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.

HELIX: a new modular nucleic acid crystallization screen

2014 ◽  
Vol 47 (3) ◽  
pp. 948-955 ◽  
Author(s):  
Julia Viladoms ◽  
Gary N. Parkinson
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Data Bank ◽  
Diverse Range ◽  
X Ray Crystallography ◽  
Molecular Weights ◽  
Anomalous Data ◽  
Crystallization Conditions ◽  
Nucleic Acid Structures ◽  
Screen Layout

Crystallization of nucleic acids remains a bottleneck to their structural characterization by X-ray crystallography. A new 96-well-format initial screen for nucleic acids, called HELIX, has been developed at UCL School of Pharmacy, London, on the basis of a detailed analysis of the crystallization conditions from 1450 nucleic acid structures deposited in the Protein Data Bank (PDB), combined with observations and experience acquired in the authors' nucleic acids crystallography laboratory during the crystallization of DNA/RNA quadruplexes and ligand complexes. Despite using traditional buffers, precipitants and salts, the resulting modular screen is designed to offer a variety of approaches to enhance successful crystallization of oligonucleotides with a diverse range of topologies, sequences and molecular weights. HELIX includes a set of 24 conditions divided into four sets that can be mixed (inter- and intra-set) to provide a customizable orthogonal screening tool for experienced users, termed VariX. Additionally, mindful of synchrotron anomalous data collection, cacodylate buffers are avoided in the formulations and an optimized cryocrystallization module is included. This article reviews the crystallization trends and data derived from the PDB and discusses the HELIX screen layout, formulation and results from in-house crystallization trials.

A Micro-diffusion Technique for Visualizing Nucleic Acid Molecules

1968 ◽  
Vol 26 ◽  
pp. 94-95
Author(s):  
H. D. Mayor ◽  
L. E. Jordan
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Osmotic Shock ◽  
Single Observation ◽  
Molecular Weights ◽  
Topological Parameters ◽  
Diffusion Technique ◽  
Specialized Equipment ◽  
Teflon Sheet ◽  
Shadow Cast

Contour lengths, molecular weights, and topological parameters of single molecules of nucleic acids can be studied accurately by electron microscopy. However, specialized equipment and highly purified samples of DNA or RNA in an amount of 10-100 μgm have been required. We have developed a simple micro-diffusion technique which uses less than 0.01 (μgm of nucleic acid. Droplets containing the nucleic acid are placed on a clean teflon sheet. Cytochrome C powder is added by needle to form a monofilm. After 20-30 minutes diffusion time, there is usually enough material attached for the film to be picked up on a suitable specimen grid, shadow-cast or stained, and examined in the electron microscope. Each droplet provides material for a single observation, but experiments may be carried out contiguously on the sheet.The method has been developed for viral nucleic acids, but can also be applied to solutions of virus particles where the genomes can be released by osmotic shock or by other physical procedures. Less than 1010 particles per ml are required for this modification. Examples of nucleic acids liberated from papovaviruses, SV40, rabbit papilloma, human papilloma, and from adeno-associated satellite virus will be presented. Viral nucleic acids (DNAs from SV40 and adenovirus, RNA from bacteriophage R17) prepared by conventional techniques will also be shown.

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 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.

Individual macromolecules: preparation and recent results with DNA

1971 ◽  
Vol 261 (837) ◽  
pp. 151-158 ◽  
Keyword(s):  
Electron Microscopy ◽  
Diffusion Coefficient ◽  
Nucleic Acid ◽  
Physical Mapping ◽  
Messenger Rna ◽  
Rna Synthesis ◽  
Strand Breaks ◽  
Single Strand Breaks ◽  
Protein Monolayer ◽  
Adenine Thymine

Protein-monolayer techniques are described which permit visualization of individual nucleic acid molecules by electron microscopy. The range of application is demonstrated by examples of quantitative observations concerning intrinsic and artificially introduced properties of DNA molecules, namely size and shape; binding of the drug ethidium bromide; diffusion coefficient; physical mapping of genetic deletions, adenine-thymine rich regions, single-strand breaks, and sites of messenger RNA synthesis.

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