Article

Determination of Base Sequence in Nucleic Acids with the Electron Microscope: Visibility of a Marker

Authors:
To read the full-text of this research, you can request a copy directly from the authors.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

... Certainly, UA provides the highest value of one dye bound per base pair, compared with BAU and DP that perform a factor of 4 or 5 worse than UA. Beer and Moudrianakisen showed that attachment of three heavy markers per base pair was required for one full amplitude contrast onto thin carbon membranes in wide-field TEM, [25] whereas staining with UA could yield a maximum of only one heavy atom of uranium. This, together with phase-contrast limitations imposed by CTFs shown in Figure 5 C and D, explains the faint contrast that one obtains in normal wide-field TEM mode, for which even utilization of a DDD is not helpful to boost the contrast ( Figure 5 A and B). ...
Article
Staining compounds containing heavy elements (electron dyes) can facilitate the visualization of DNA and related biomolecules using transmission electron microscopy (TEM). However, research on the synthesis and utilization of alternative electron dyes has been limited. Here, we report the synthesis of a novel DNA intercalator molecule, bis‐acridine uranyl (BAU). Nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) confirmed the validity of our synthesis scheme and gel electrophoresis verified the binding of BAU to DNA. For TEM imaging of DNA, we use two‐dimensional DNA origami nanostructures as a robust microscopy test object. Using scanning TEM (STEM) imaging, which is favored over conventional wide‐field TEM for improved contrast and therefore quantitative image analysis, we find that the synthesized BAU intercalator can render DNA visible, even at the single‐molecule scale. For comparison, we also evaluated other staining compounds with a purported affinity towards DNA, such as dichloro platinum, cisplatin, osmium tetroxide, and uranyl acetate. We discuss the STEM contrast in terms of the DNA‐dye association constants, number of dye molecules bound per base pair, and the electron‐scattering capacity of the metal‐containing ligands. Our findings pave the way for future development of electron dyes with specific DNA‐binding motifs for high‐resolution TEM imaging.
... To resolve the issues of similar chemical structure and unreadable speed of translocation [26], we considered a different approach, namely selective labeling of ssDNA. Historically, selective labeling was evaluated in the 1960s in an effort to image DNA conjugated with contrast agents by electron microscopy in order to elucidate sequence from the images [27][28][29]. This was tested within this work and it was found that one of the contrast agents, a 1:1 mixture of osmium tetroxide and 2,2'-bipyridine, exhibited extraordinary properties, and was considered to be the "perfect" label (Scheme 1). ...
Article
Full-text available
Osmium tetroxide 2,2'-bipyridine (OsBp) is known to react with pyrimidines in ssDNA and preferentially label deoxythymine (T) over deoxycytosine (C). The product, osmylated DNA, was proposed as a surrogate for nanopore-based DNA sequencing due to OsBp's " perfect " label attributes. Osmylated deoxyoligos translocate unassisted and measurably slow via sub-2 nm SiN solid-state nanopores, as well as via the alpha-hemolysin (α-HL) pore. Both nanopores discriminate clearly between osmylated and intact nucleobase; α-HL was also shown to discriminate between osmylated T and osmylated C. Experiments presented here confirm that the kinetics of osmylation are comparable for short oligos and long ssDNA and show that pyrimidine osmylation is practically complete in two hours at room temperature with less than 15 mM OsBp. Under the proposed labeling conditions: deoxyoligo backbone degradation measures less than 1/1,000,000; false positives such as osmylated deoxyadenine (A) and osmylated deoxyguanine (G) measure less than 1/100,000; false negatives, i.e., unosmylated C measure less than 1/10,000; and unosmylated T must measure substantially lower than 1/10,000 due to the 27-fold higher reactivity of T compared to C. However, osmylated C undergoes degradation that amounts to about 1–2% for the duration of the labeling protocol. This degradation may be further characterized, possibly suppressed, and the properties of the degradation products via nanopore translocation can be evaluated to assure base calling quality in a DNA sequencing effort. 1434
... OsBp is known to add to the C5-C6 double bond of the pyrimidine ring ( Figure 2). Because Osmium is a good contrast agent for imaging by electron microscopy (EM), osmylated DNA (DNA(OsBp), Note) was proposed 60 years ago and exploited in attempts to obtain DNA sequence information by EM imaging [34][35][36]. The more recent advancement of nanopores as single molecule detection devices, and the corresponding progress in manufacturing, parallelization, and commercialization of such platforms [37], supported the idea of testing DNA(OsBp) as a surrogate for intact DNA (Figure 3). ...
... In the 1960s a far-reaching approach to whole genome sequencing was postulated and explored. The concept was to react a nucleic acid with an organo-metallic molecule that exhibited selectivity for one of the four bases [10]. Labeled DNA would then be stretched on suitable surfaces, imaged by electron microscopy to detect the position of the metal and infer the position of the labeled base [11,12]. ...
Article
Full-text available
Saenger sequencing has led the advances in molecular biology, while faster and cheaper next generation technologies are urgently needed. A newer approach exploits nanopores, natural or solid-state, set in an electrical field, and obtains base sequence information from current variations due to the passage of a ssDNA molecule through the pore. A hurdle in this approach is the fact that the four bases are chemically comparable to each other which leads to small differences in current obstruction. 'Base calling' becomes even more challenging because most nanopores sense a short sequence and not individual bases. Perhaps sequencing DNA via nanopores would be more manageable, if only the bases were two, and chemically very different from each other; a sequence of 1s and 0s comes to mind. Osmylated DNA comes close to such a sequence of 1s and 0s. Osmylation is the addition of osmium tetroxide bipyridine across the C5-C6 double bond of the pyrimidines. Osmylation adds almost 400% mass to the reactive base, creates a sterically and electronically notably different molecule, labeled 1, compared to the unreactive purines, labeled 0. If osmylated DNA were successfully sequenced, the result would be a sequence of osmylated pyrimidines (1), and purines (0), and not of the actual nucleobases. To solve this problem we studied the osmylation reaction with short oligos and with M13mp18, a long ssDNA, developed a UV-vis assay to measure extent of osmylation, and designed two protocols. Protocol A uses mild conditions and yields osmylated thymidines (1), while leaving the other three bases (0) practically intact. Protocol B uses harsher conditions and effectively osmylates both pyrimidines, but not the purines. Applying these two protocols also to the complementary of the target polynucleotide yields a total of four osmylated strands that collectively could define the actual base sequence of the target DNA.
... Existing sequencing methods read the bases one after another, progressing along the chain. A far-reaching approach in sequencing has been to have a way to simply look at the molecule and obtain structural information directly [1][2][3]. As a step toward enabling it we sought to render the pyrimidine bases visible for single-atom resolution electron microscopy (EM) by forming their osmium addition products [3]. ...
Article
Full-text available
With the recent advances in electron microscopy (EM), computation, and nanofabrication, the original idea of reading DNA sequence directly from an image can now be tested. One approach is to develop heavy atom labels that can provide the contrast required for EM imaging. While evaluating tentative labels for the respective nucleobases in synthetic oligodeoxynucleotides (oligos), we developed a streamlined CE protocol to assess the label stability, reactivity, and selectivity. We report our protocol using osmium tetroxide 2,2'-bipyridine (Osbipy) as a thymidine (T) specific label. The observed rates show that the labeling process is kinetically independent of both the oligo length, and the base composition. The conditions, i.e. temperature, optimal Osbipy concentration, and molar ratio of reagents, to promote 100% conversion of the starting oligo to labeled product were established. Hence, the optimized conditions developed with the oligos could be leveraged to allow osmylation of effectively all Ts in ssDNA, while achieving minimal mislabeling. In addition, the approach and methods employed here may be adapted to the evaluation of other prospective contrasting agents/labels to facilitate next-generation DNA sequencing by EM.
... There is simply not enough difference between the base types to be detected without suitable contrast enhancement. Various groups have worked to overcome this problem, chiefly by chemically modifying single-stranded DNA with clusters of heavy atoms~Beer & Moudrianakis, 1962;Moudrianakis & Beer, 1965;Ottensmeyer, 1979!. The approach employed here uses a standard, template-directed polymerase enzyme to incorporate heavy-atom-modified bases directly into a long DNA molecule~Fig. ...
Article
Advances in DNA sequencing, based on fluorescent microscopy, have transformed many areas of biological research. However, only relatively short molecules can be sequenced by these technologies. Dramatic improvements in genomic research will require accurate sequencing of long ~.10,000 base-pairs!, intact DNA molecules. Our approach directly visualizes the sequence of DNA molecules using electron microscopy. This report represents the first identification of DNA base pairs within intact DNA molecules by electron microscopy. By enzymatically incorporating modified bases, which contain atoms of increased atomic number, direct visual-ization and identification of individually labeled bases within a synthetic 3,272 base-pair DNA molecule and a 7,249 base-pair viral genome have been accomplished. This proof of principle is made possible by the use of a dUTP nucleotide, substituted with a single mercury atom attached to the nitrogenous base. One of these contrast-enhanced, heavy-atom-labeled bases is paired with each adenosine base in the template molecule and then built into a double-stranded DNA molecule by a template-directed DNA polymerase enzyme. This modification is small enough to allow very long molecules with labels at each A-U position. Image contrast is further enhanced by using annular dark-field scanning transmission electron microscopy ~ADF-STEM!. Further refinements to identify additional base types and more precisely determine the location of identified bases would allow full sequencing of long, intact DNA molecules, significantly improving the pace of complex genomic discoveries.
Article
Full-text available
Synthetic efforts towards nucleosides, nucleotides, oligonucleotides and nucleic acids covalently mercurated at one or more of their base moieties are summarized, followed by a discussion of the proposed, realized and abandoned applications of this unique class of compounds. Special emphasis is given to fields where active research is ongoing, notably the use of Hg(II)‐mediated base pairing to improve the hybridization properties of oligonucleotide probes. Finally, this minireview attempts to anticipate potential future applications of organomercury nucleic acids.
Article
A protein-free nucleic acid preparation method for electron microscopy is described. The basic procedure is very similar to the classical protein monolayer spreading techniques. The carrier protein (usually cytochrome c) is replaced by benzyldimethylalkylammonium chloride. Both the hypophase method and the microdiffusion or droplet method can be applied with this compound. Unlike cytochrome c, benzyldimethylalkylammonium chloride does not lead to any apparent thickening of the nucleic acid strands. Partially denatured DNA spread with this reagent shows a loosened structure with a foamy appearance in the regions previously considered to be "unmelted," which open up locally into melted loops of different size. Specifically bound proteins, such as RNA polymerase on bacteriophage T7 DNA, can be detected unambiguously.
The complexes formed between cytosine, cytidine and cytidine phosphate and U(VI), Th(IV), Ce(III) and La(III) have been investigated using spectrophotometric and conductometric methods. The apparent formation constants of these complexes have been calculated. The mode of bonding of the ligand in the solid chelates was studied by I.R. spectrophotometry which showed that chelate formation takes place through C(2)O and CN(3) groups in the case of cytosine and cytidine. The I.R. spectra of cytidine phosphate complexes indicated a tridentate ligand which coordinated via the former two groups and phosphate groups to the central metal ion.
The reaction of diazotized 2-amino-p-benzenedisulphonic acid with the nucleotides was investigated at several pH values.At pH 9 guanylic aicds react 60 times faster than any of the other common nucleotides.The reaction leads to an addition product which contains the phosphorus and the base of the nucleotides and the sulphur of the diazonium salt. At pH 2 and 22° the product decomposes over 10 h; at pH 9 it is stable for several days.The diazonium salt reacts with ribose, deoxyribose and ribose 5'-phosphate at rates comparable to that of the nucleotides other than guanylic acid, suggesting the sugar as the site of the slow reactions.These results suggest that the guanine residues of the nucleic acid molecules could selectively be labelled over the other.
Article
The systematic study of interactions between metal ions and nucleobases, the constituents of nucleic acids, as well as nucleic acids in general started some 50 years ago, around 1950. This review is an attempt to recall the developments in this field, to list metal binding patterns as established today, and to examine prospects for the future. The focus of this survey will be on the coordination chemistry of metal species with the heterocyclic parts of nucleobases.
Article
Organomercurials form stable stoichiometric complexes with thiolated nucleosides. The complexes inhibited uptake of ribonucleosides and cytosine arabinoside (CAR) in various types of normal and transformed cells. The inhibition was competitive and reversible (Ki = 3--6 micrometer). The interaction between complexes and transport system displayed a 1:1 stoichiometry. Chemical factors which contributed to the inhibitory power were evaluated with a series of S-alkylated derivatives and S--Hg--R complexes of mercaptonucleosides. The inhibitory potency was not determined exclusively by the hydrophobic nature of either the S-alkylated or the S--Hg--R moieties. Chemical modification of cells with penetrating and nonpenetrating organomercurials lead to stimulation of nucleoside uptake and to an increase in its susceptibility to inhibition by S--Hg--R complexes or S-aklylated derivatives of mercaptopurine ribosides. The kinetic and chemical data obtained with nucleoside analogs and with chemical modifiers suggested complex features of nucleoside transport systems. Four distinct classes of sites were implied: (i) a substrate binding site susceptible directly to competitive inhibition by organomercurial-mercaptonucleoside complexes, (ii) an additional site susceptible either to S-arylalkylated or S-mercuriated derivatives of 6-mercaptopurine ribosides, (iii) SH-containing modifier sites which stimulate uridine uptake upon binding of organomercurials, and (iv) SH-containing modifier sites which inhibit the function upon binding of organomercurials. From the observation that only SH sites related to stimulation were susceptible to modification by macromolecular-SH modifier probes, some conclusions can be drawn regarding the disposition of the various sites in the cell membrane in general and among membrane components in particular.
Article
Platinum binding to nucleoside phosphorothioates has been examined to determine their suitability as heavy metal labeling sites for the potential electron microscopic sequencing of nucleic acids. The complex platinum terpyridine nitrate forms a 1:1 adduct with either adenosine or uridine monophosphorothioate. Spectroscopic evidence strongly indicates the presence of platinum-sulfur bonds. Both platinum terpyridine nitrate and chloroterpyridineplatinum(II) bind to poly(sA-U), a polymer prepared from adenosine 5'-O-(1-thiotriphosphate) and UTP. Binding to the sulfur atoms of the phosphorothioate groups is quantitative, as shown by double label experiments using [35S]poly(sA-U) and [3H]chloroterpyridine-platinum(II). Similar experiments with [14C]poly(A-U) indicated no platinum binding. No evidence of nicking or loss of sulfur from poly(sA-U) could be detected after platinum binding. The phosphorothioate group is a strong, highly selective binding site for platinum in polynucleotides. Previous studies have demonstrated quantitative enzymatic incorporation of phosphorothioate groups into a polynucleotide adjacent to a specific base [Matzura, H. & Eckstein, F. (1968) Eur. J. Biochem. 3, 448-452]. The use of heavy metal-labeled phosphorothioate groups for the sequencing of nucleic acids by electron microscopy therefore appears feasible.
In this review we have tried to cover the direct mounting techniques currently in use and, through micrographs kindly supplied by our colleagues, to illustrate uses of the different methods. For the novice, it is important to select one technique, taking time to become acquainted with its use in visualizing simple test samples before applying it to examine more complex structures. We have tried to emphasize the importance of controlling the fixation for two reasons. First, confidence in the fixation step allows one to correlate structures observed by EM with structures present in solution. Second, fixation of otherwise labile samples allows one to apply powerful purification techniques.
Article
Heavy-metal ions are commonly used as staining agents. An important property in staining with metal ions is that the resulting tissue–metal complex be sufficiently stable to withstand subsequent manipulations without reversible dissociation. This stability may be either thermodynamic (that is, the complex has a sufficiently high formation constant) or kinetic (that is, the rate of dissociation is very slow as compared to the time for tissue manipulation). Where metal chelates are sufficiently stable in either of these senses, they offer the advantage of a clean staining because better washing procedures can be used and nonspecific deposition is diminished. One type of metal-containing stain relies on the simple precipitation of inorganic species, usually because of enzyme activity on an appropriate substrate. Thus, phosphatases can be visualized by the liberation of phosphate, with subsequent precipitation of lead phosphate following the addition of lead ions. One type of method commonly used for electron staining of tissues with heavy-metal complexes depends on the adjustment of conditions so that the metal complex in the solution is near the limit of its stability. Immersion of tissue sections in the solution allows binding groups on the tissue to compete successfully for some of the metal ion present and hence, to undergo staining.
Article
The reaction of platinum (II) - dimethylsulphoxide complex with the bases of the nucleic acids were investigated with a new towards their use as heavy atom markers for base sequence determination by electron microscopy. Both at pH 6.0 and pH 7.5 one platinum atom was bound simultaneously to the pyrimidines and two to adenine, while at the lower pH one platinum, and at the higher pH, two platinum atoms were bound to guanine. The stain therefore appears to be useful to determine the guanine and adenine sequence in single strands of RNA and DNA. Where complementary strands are available the complete sequence determination of all four bases should be possible.
Article
This chapter focuses on the medicinal chemistry of 1,2,3-benzotriazines (2), benzotriazines (3) fused at the c bond to an azino- or azolo-ring (x), and the important series of hetero-fused 1,2,3-triazines (4). Some of the chemical and biological properties of diazonium compounds have been summarized in the chapter. The close parallel between the chemical properties of aryldiazonium compounds and 1,2,3-triazines fused to an aromatic carbocyclic or heterocyclic ring has been repeatedly stressed in this chapter. A medicinally desirable diazonium compound would test the skills of the most talented pharmaceutical formulator because of the inherent instability of this structural type: fused 1,2,3-triazines on the other hand are, in general, stable crystalline compounds readily amenable to manipulation yet lacking little in chemical versatility. Only a small proportion of the many structurally diverse 1,2,3-triazines that have been prepared over the years have been examined for biological activity.
Article
A series of complexes of Au(III) with nucleosides and nucleotides and their methyl derivatives in different stoichiometry have been prepared. Ultraviolet, visible, ir, and nmr studies have been performed to determine the site of binding of these ligands with the metal ion. In (1:4) Au(III): guanosine complex, N7 is the binding site, whereas at 1:1 complex, a bidentate type of chelation through C6O and N7 is observed. C6-NH2 is favored over N1 as coordinating site at all stoichiometry in the adenosine complex. Inosine binds through N1 at r = 1. In cytidine, N1 is the binding site, whereas thymidine reacts only at high pH. In the case of nucleotides a bidentate type of chelation through the phosphate and the ring nitrogen occurs. The phosphate binding ability of Au(III) was further confirmed by studying the interaction of Au(III) with dimethyl phosphate—a conformational analog of the phosphate backbone in DNA chain.
Article
We have synthesized his(pyridine)oxoosmium(VI) and 2,2′-bipyridyloxoosmium(VI) esters of the common nucleosides in which osmium is bonded through the 2′- and 3′-hydroxyl groups of the sugar residue. We have also prepared the 2,2′-bipyridyloxoosmium(VI) esters of uridine, cytidine, and thymidine which result from addition of OSO4 to the 5,6 double bond of the pyrimidine ring. Kinetic studies of the formation of the sugar esters from the nucleoside and the Os(VI) dimer, Os2O6py4, give the apparent rate law, v = k[S][Os(VI)][Py]-1[OH-]0.6-0.8, in which the hydroxyl ion term reflects hydroxyl ion promoted dissociation of the Os(VI) dimer to monomeric species. The true rate law probably involves three terms, one zero-order, one half-order, and one first-order in hydroxyl ion. The bis(pyridine) esters undergo relatively rapid transesterification reactions with free glycols whereas the 2,2′-bipyridyl esters are much more inert. Kinetic studies of the transesterification reactions give the rate law, v = k [osmate ester] [glycol] [OH-] [py]-1.
Article
Ultra-fine grids with holes of a few hundred angstroms in diameter were made from tropomyosin for specimen supporting media. DNA was mounted on the grid. Microscope images were obtained demonstrating one double-stranded DNA filament bridging a hole. Using mercurated DNA a few filamentous images were observed in a bridge, and series of faint fine sports were recognized along an edge of a hole. These spots were interpreted as the images of single mercury atoms bound to the filament. The control experiment was made on unstained DNA, some images of which showed only uniform intensity over the bridge, whereas others were disturbed to some extent by noise presumably due to extraneous molecules coating the filament.
Article
A new method for applying nucleic acid and nucleoprotein specimens to grid-mounted carbon support films is described. The novel feature of this method involves drawing the sample solution into a thin film of liquid by means of a wire loop prior to its application to the carbon film. The advantages of this “loop film” technique are: (a) the layer of liquid applied is so thin that backgrounds are very clean and no staining or rinsing of the specimen is necessary, thereby eliminating the possibility of selective removal of specimen components by rinsing; (b) single stranded nucleic acid retains a structure which, although shrunken, is related to its condensed native configuration in solution, thereby facilitating the distinction between single and double stranded forms; (c) no extraneous protein is added to the specimen for spreading, so that proteins of biological interest which may be adsorbed to the nucleic acid should be readily detectable.
Article
DNA molecules bind to carbon films placed on the surface of aqueous solutions and are extended and oriented by the flow forces on removal from the surface. The number of molecules bound increases with ionic strength suggesting that the bonding is hydrophobic. If carbon films are subjected to ionic bombardment in a flow discharge, they can be modified so that molecules bind strongly, and are deposited in a tangled, random-coil configuration, uninfluenced by flow forces. The strength of binding is decreased by reducing the discharge time and current, and by addition of salt (NaCl), suggesting that the bonding is ionic. When weakly bound, the molecules are condensed by ethanol. The strength of binding can be adjusted so that the molecules are largely untangled by flow, and in a form suitable for the mapping of bound enzymes, or other identifiable markers. Aggregation and extension of molecules, that occur on unmodified films, and the binding of cytochrome c, that occurs in methods based on that of Kleinschmidt & Zahn (1959) are avoided. Both double and single-stranded molecules can be deposited and seen after rotary shadowing with Pt. The discharge can be performed in a vacuum produced by a standard oil-filled rotary pump.
Article
Thymine and its derivatives react rapidly in aqueous solution with osmium tetroxide and pyridine to form stable bis(pyridine) osmate esters. We have studied these esters with the following pyrimidines: thymine, thymidine, thymidine-5′-phosphate, and uracil, using pyridine, 3-picoline, 3-chloropyridine, and 3-pyridyl mercuric acetate as the pyridine components. We have examined the kinetics of these reactions as a function of the concentration of substrate, pyridine, osmium tetroxide, and pH. The kinetic dependencies suggest the following rate expression:We report values for β1, β2, κ1, and κ2.
Article
Complexes of a series of alkylated xanthine derivatives in which ruthenium(II and III) is bound to the N7 or C8 sites of the purine are described. The N-bound ruthenium(III) complexes exhibit two ligand-to-metal charge transfer bands, which provide information about the energies of molecular orbitals on the xanthine ligand. The ruthenium(II) complexes exhibit metal-to-ligand charge transfer transitions in the ultraviolet. Reduction potentials for the neutral and deprotonated ligand complexes are reported. Measurements of pKa values for both N- and C-bound Ru(II and III) complexes are presented. Evidence for a trans labilization effect in the series of C8-xanthinylideneruthenium(III) complexes is described.
Article
The theories of the helix–coil transition of Lifson, Lifson and Allegra, and Allegra for random copolymers are compared with the exact results of Lehman and McTague, as was done by Fink and Crothers in an earlier test of the approximate theories of Fixman and Zeroka, Reiss et al, and Montroll and Goel. The Fixman-Zeroka theory is the best approximation to the exact result (under the conditions used), and the Allegra theory is next best, the calculated slope of the transition curve in the Allegra theory being too small by a factor of two. An alternative derivation of the Allegra approximation, using the method of sequence generating functions, is given, and the resulting equations are of very simple form. A hybrid method, based on the work of Lifson and Allegra, is also developed here; it involves a technique of successive approximations, and reduces to the Allegra theory in first order. The fourth-order approximation gives a slope that is too small by 10%; however, the value of the slope, extrapolated to infinite order of approximation, converges to the exact result- of Lehman and Mc-Tague. By using illustrative calculations of helix–coil transition curves, some physical insight into the behavior of a copolymer in the transition region is provided: it is found that an important feature determining the shape of the transition curve is the variation in composition over the correlation length. The question of the application of copolymer theory to DNA is discussed.
Article
Zusammenfassung Durch Alkali nachFreifelder undDavison einsträngig gemachte DNA aus Forellengonaden wird mittels Spreitung einer DNA/Protein-Lösung elektronenmikroskopisch dargestellt. Ihre Konfiguration, gemessen durch das mittlere Quadrat der Abstände der Molekülenden als Funktion der Konturenlänge, ist flexibler als bei zweisträngiger DNA unter gleichen Bedingungen, weicht aber wie diese infolge ihres Polyelektrolytcharakters vom statistischen Knäuel merklich ab. Die exponentielle Verteilung der Konturenlängen wird als statistische Verteilung der Bruchstellen gedeutet.
Chapter
Considerable light is shed on the problems of reactivity and chemical modification of nucleic acids by reactions involving exocyclic substituents of the purine and pyrimidine bases, i. e., the amino groups of cytosine, adenine, or guanine, and the carbonyl groups of uracil, guanine, xanthine, and their derivatives, and also reactions of the sulphur atom of thio derivatives (minor components of RNA). As was mentioned previously (see Chapter 3), n electrons of the nitrogen atoms of amino groups and of the oxygen atom of carbonyl groups of the nucleic acid bases (and their derivatives) react strongly with the π electron system of the heterocyclic ring, and consequently the properties of the corresponding components of the nucleic acids differ greatly from the properties of simple amines, amides, or thioamides.
Article
Die Mononucleotide Guanyl-, Adenyl- und Cytidylsäure bilden mit diazotierter Sulfanilsäure bei pH 10–11 Farbstoffe, die im Bereich von 370–440 nm starke Absorption zeigen. Guanylsäure reagiert am raschesten. Uridyl- und Thymidylsäure geben keine Reaktion. Die pH-Optima wurden für den Umsatz mit den einzelnen Nucleotiden ermittelt. Alle Farbstoffe, am raschesten jedoch der mit Adenylsäure gebildete, spalten bei pH 3 in die Ausgangskomponenten zurück. Die optischen pKA-Werte der Farbstoffe wurden bestimmt. Die Farbstoffbildung ist in Gegenwart von Formaldehyd inhibiert. Bei den Farbstoffen handelt es sich also um Diazoaminoverbindungen. An RNA und DNA verläuft die Reaktion mit diazotierter Sulfanilsäure ähnlich; Cytidylsäure reagiert jedoch nicht. Die Möglichkeiten für einen Guanylsäure-spezifischen Umsatz werden diskutiert.
Chapter
The development of STEM is outlined from the first developments by Baron Manfred von Ardenne, through the first successful field emission gun STEM by Albert Crewe and his collaborators, to its widespread application today in the era of aberration correction. The review focuses on the development and understanding of incoherent imaging and electron energy loss spectroscopy at atomic resolution and will not include details on microanalysis, low loss imaging, or specialized modes such as cathodoluminescence. Although it attempts to cover all the major advances in approximately chronological order, undoubtedly there are omissions and an overemphasis on developments that the author is most familiar with from his own history.
Article
Full-text available
In an attempt to understand the factors that govern the rates of reaction of the complexes [Pt(en)Cl(NH2R)]+NO3− (en = ethylene diamine) with guanosine monophosphate (dGMP) a series of amine complexes, where RC8H9NO2 (benzo[d][1,3]dioxol-5-ylmethanamine) (1), C8H11N (phenethylamine) (2), C7H9N (benzylamine) (3), C6H7N (aniline) (4), C6H6IN (p-iodo-aniline) (5) C3H9NO (2-methoxy-ethylamine) (6) and C6H13N (cyclohexylamine) (7), were synthesized and their reactions with deoxyguanosine monophosphate (dGMP) were followed by 1H NMR. Compound 1 was initially chosen because it showed significant water solubility. Compound 1 reacted quantitatively but slowly with dGMP and a subsequent Transmission Electron Microscopy (TEM) study of the binding 1 to a GATC DNA repeat gave a TEM micrograph that showed selective labeling of DNA at guanine, using a technique that allowed the laying down of a straight single strand of DNA on a carbon platform. The TEM suggested a possible side reaction with adenine and so a study of the reaction of 1 with adenine was performed and showed slow and what appeared to be non-specific binding to deoxyadenosine monophosphate (dAMP). The reactions of compounds 2–7 with dGMP were then studied by 1H NMR and it was found that 2 reacted much faster than 1 with dGMP while the remaining complexes reacted more slowly. No reaction of 2 with dAMP was observed in the same time frame. The ultimate goal of the project was to bind a third row transition metal cluster to guanine and given the effective binding of 1 to DNA the synthesis of the complex [Os3(CO)11PPh2(CH2)2NH2(en)PtCl]NO3 (9) is also reported that contains Pt as a linker to label guanine. The synthesis was performed by reacting Os3(CO)10(CH3CN)2 with Ph2PCH2CH2NH2 which gave an η2 chelate complex Os3(CO)10PPh2(CH2)2NH2 (8). Complex 8 was reacted with [Pt(en)Cl(DMF)]NO3 in a CO atmosphere to give 9. 1H and 195Pt NMR indicate formation of an adduct with dGMP but too slowly to be of use in labeling DNA. The solid-state structure of 8 is also reported.
Treatment of poly G with diazotised p-aminobenzene disulphonic acid gives a polymer in which 30 % of the guanine residues are substituted at C-8. This polynucleotide absorbs in the visible region, possesses a hydrogen-bonded secondary structure with the substituted residues looped out and forms a complex with poly C, in which all residues are hydrogen bonded. Protonation gives rise to a new structure as in the case of poly G·poly C. The same reaction has been applied to poly N2-dimethylguanylic acid.RésuméLe poly G traité par le sel de diazonium de l'acide disulfonique conduit à un copolymère où 30 % des résidus guanine se trouvent substitués en position 8. Le polymère analogue est jaune et absorbe jusqu'à 450 mμ. Il possède une structure secondaire où les résidus de guanine modifiés ne sont pas liés par des liaisons hydrogène et donne un complexe à double brin avec le poly C où tous les résidus sont liés par des liaisons hydrogène. A pH acide, ce complexe présente une nouvelle conformation comme celle du poly G·poly C protoné. Le traitement de l'acide poly donne un produit similaire à celui obtenu avec le poly G.
Article
The reactions have been studied of the nucleic acid bases, adenine, cytosine, guanine, purine, hypoxanthine, mercaptopurine and the nucleosides, guanosine, cytidine and adenosine with sodium molybdate in aqueous solutions. Compounds of the type (AH+)4 Mo8O26·4H2O (where A = adenine, cytosine, guanine, hypoxanthine, purine, mercaptopurine, adenosine and cytidine) were isolated at pH values around 4 and compounds of the type (AH+)2Mo6O19 (where A = Adenosine, cytidine or guanosine) at around pH 2. The compounds prepared were characterized by elemental analysis, infrared, Raman, electronic and 1H NMR spectroscopy, conductivity measurements and pH tirations.
Article
The synthesis of N3, N7, and N9 linkage isomers of a series of pentaammineruthenium(II)- and -(III)-hypoxanthine complexes is reported. Physical measurements of these compounds were made to gain information which might be employed in separating and identifying the metabolites of heavy metals which bind to nucleic acids or their constituent bases. The ruthenium(III) complexes exhibit broad ligand to metal charge-transfer transitions which can be used to assign the isomers. Reduction potentials for these complexes over a broad pH range are presented and are also of use in identifying the complexes. Measurements of the pKa values of the complexes are reported and were utilized in separating the linkage isomers. A novel N3 to N9 isomerization of one of the complexes has been observed to be acid catalyzed.
Article
Absorptions of purine and pyrimidine derivatives by Co- and Ni- montmorillonite at pH < 6 and by Cu-montmorillonite at pH < 3 are similar to their absorption by Ca-montmorillonite and take place primarily by a cation exchange process. In the weakly acidic to weakly alkaline range, absorption is due to complex formation with the inorganic cations, and decrease in the order Cu ≫ Ni > Co ≫ Ca. Adenine, 7-methyladenine, hypoxanthine, and purine are strongly absorbed, 9-methyladenine, 6-chloropurine, and cytosine are weakly absorbed, and thymine and uracil are not absorbed. At pH < 5, the nucleosides are absorbed by Co-, Ni-, and Cu-montmorillonite in approximately the same manner as by Ca-montmorillonite, but at pH > 6 their absorptions decrease in the order Cu ≫ Ni > Co > Ca. Fe(III)-montmorillonite behaves quite differently from the other montmorillonites studied. With purines and pyrimidines, there is strong absorption from pH 3 to pH 7-8; with the nucleosides, the absorption varies considerably with the compounds considered decreasing in the order adenosine > cytidine ≫ guanosine ≫ inosine.
Patent
The chapter discusses various synthetic polynucleotides. Synthetic polynucleotides are of considerable interest from the biological viewpoint of code cracking, and, also, as grossly simplified models for the study of the numerous physical properties, manifested by nucleic acids. This chapter is discusses the physical studies of the structure in synthetic polynucleotides, including homopolymers of naturally occurring nucleotides and of various analogs. The chapter very briefly reviews some technical aspects of the physical chemistry of polynucleotides. The various random coil and structured forms of polynucleotides, containing a single naturally occurring purine or pyrimidine base, are discussed in this chapter. Under suitable conditions of pH or salt concentration, all such polymers form hydrogen-bonded, multi stranded, secondary structures. The chapter discusses various polynucleotide complexes, such as complexes between Poly G and Poly C. All homopolynucleotides are derived from natural or almost natural nucleotides form multistranded secondary structures under appropriate conditions. In case of the “basic” (or 6-amino) nucleosides (A and C), slightly acidic conditions are necessary, whereas the “acidic” (or 6-keto) polymers (G, U, I, X), all give defined secondary structures at pH 7 albeit, with vast different stabilities.
Article
Several complexes of Mn2+, Fe2+, Zn2+, Pd2+, Cd2+, Pb2+, Ce2+, Pr3+, Sm3+, Tb3+, and Uo22+ metal ions with adenosine 5'-monophosphate have been isolated. The stoichiometry of all the complexes prepared were 1:1 metal to mononucleotide, respectively, and they were characterized by elemental analysis, infrared, electronic and fluorescence spectroscopy, conductivity, and magnetic measurements. Similarities among the spectra of the substances with structurally known metal-mononucleotide complexes suggest that the metal ions interact directly or indirectly with the N7 of the purine ring and the phosphate group of the mononucleotide.
Article
The action of acid, alkali, hydrazine, hydroxylamine, alkylating and oxidizing agents and certain other chemical reagents on DNA is reviewed. In particular, there is discussion of methods which permit the determination of the distribution of nucleotide residues to be made. The mode of action of some chemical mutagens is also described.RésuméOn passe en revue l'action sur l'ADN des acides, des bases, de l'hydrazine, de l'hydroxylamine , d'agents alkylants et oxydants et de quelques autres réactifs chimiques. On discute en particulier les mðhodes qui permettent de déterminer la dictribution des résidus de nucléotides. On décrit également l'action de quelques mutagénes chimiques.ResumenVienne esaminata l'azione chimica sul DNA di acidi, basi, idrazine, idrosilamine, agenti alchilanti e ossidanti e di diversi altri reattivi chimici. In particolare, vengono discussi i metodi che permettono di fare la determinazione della distribuzione dei residui nucleotidici. Viene anche discussa l'azione di alcuni mutageni chimici.ZusammenfassungEs wird eine Übersicht über die Einwirkung von Säure, Alkali, Hydrazin, Hydroxylamin, alkylierende und oxidierende Agentien, sowie bestimmte andere chemische Reagentien auf DNA gegeben. Insbesondere werden Methoden diskutiet, die eine Bestmmung der Verteilung der Nucleotidreste erlauben. Die Wirkungsweise einiger chemischer Mutagene wird gleichfalls beschrieben.
Article
Advances in DNA sequencing, based on fluorescent microscopy, have transformed many areas of biological research. However, only relatively short molecules can be sequenced by these technologies. Dramatic improvements in genomic research will require accurate sequencing of long (>10,000 base-pairs), intact DNA molecules. Our approach directly visualizes the sequence of DNA molecules using electron microscopy. This report represents the first identification of DNA base pairs within intact DNA molecules by electron microscopy. By enzymatically incorporating modified bases, which contain atoms of increased atomic number, direct visualization and identification of individually labeled bases within a synthetic 3,272 base-pair DNA molecule and a 7,249 base-pair viral genome have been accomplished. This proof of principle is made possible by the use of a dUTP nucleotide, substituted with a single mercury atom attached to the nitrogenous base. One of these contrast-enhanced, heavy-atom-labeled bases is paired with each adenosine base in the template molecule and then built into a double-stranded DNA molecule by a template-directed DNA polymerase enzyme. This modification is small enough to allow very long molecules with labels at each A-U position. Image contrast is further enhanced by using annular dark-field scanning transmission electron microscopy (ADF-STEM). Further refinements to identify additional base types and more precisely determine the location of identified bases would allow full sequencing of long, intact DNA molecules, significantly improving the pace of complex genomic discoveries.
Article
I shall review the work on the electron microscopic determination of base sequence in DNA restricting myself to the approach in which one kind of base is selectively marked. The requirements for success are 1. Appropriate staining reaction: stain must be detectable yet sufficiently compact to allow resolution of adjaconts. Reactions must be sufficiently selective and sufficiently gentle to preserve the.polynucleotide chain. 2. Deposition of polynucleotide chains in untangled and sufficiently extended form. 3. Means for accurately enumerating unmarked bases between the marked ones.
Article
The synthesis, characterization, and x-ray crystal structure analysis of 2-hydroxyethanethiolato(2,2′,2″-terpyridine)-platinum(II) nitrate, [Pt(terpy)(SCH2CH2OH)]NO3, are reported. Solution studies reveal the presence of both monomers and stacked dimers in aqueous media above 10-4 M. The red complex crystallizes in the triclinic space group P1 with two formula units per unit cell of dimensions a = 10.487 (2) Å, b = 10.718 (2) Å, c = 9.131 (2) Å, α = 82.72 (1)°, β = 111.96 (1)°, and γ = 112.53 (1)°. From 3224 unique observed reflections collected on an automated four-circle diffractometer, the structure was solved and refined to final values for the discrepancy indices of R1 = 0.028, R2 = 0.033. The platinum in the slightly distorted square planar complex is bound to the three nitrogen atoms of terpyridine and to the sulfur atom of mercaptoethanol with N-Pt-N angles of 80.6 (2) and 80.8 (2)° and N-Pt-S angles of 100.4 (1) and 98.1 (2)°. The Pt-N bond to the middle nitrogen atom of the terpyridine ligand is shorter, 1.968 (5) Å, than those to the other nitrogen atoms, 2.023 (5) and 2.030 (5) Å. The two methylene carbon atoms of the mercaptoethanol ligand are disordered. There is a hydrogen bond between the hydroxyl proton on the mercaptoethanol ligand and an oxygen atom of the nitrate anion. Two types of stacking interactions are seen in the crystal, a direct head-to-tail overlap, and an overlap involving only two of the three aromatic rings of the terpyridine ligand. A comparison is made of the stacking properties of [Pt(terpy)(SCH2CH2OH)]NO3 with those of ethidium bromide. Both compounds are known to bind to double stranded DNA by intercalation. The syntheses and preliminary characterization of several related metallointercalation reagents in the class [Pt(terpy)(SCH2R)]n+ are reported.
Article
It has been found that protein stained with p-chloromercuriphenyl sulphonic acid followed by counter-staining with uranyl acetate is visible in the electron microscope. Furthermore, individual particles have been observed that are somewhat similar in appearance and size to particles observed in shadowed preparations of the same material. The possible usefulness of this reagent to stain proteins and in studies on individual molecules is discussed.
Chemical studies have been carried out on the interaction of DNA with uranyl salts. The effect of variations in pH, salt concentration, and structural integrity of the DNA on the stoichiometry of the salt-substrate complex have been investigated. At pH 3.5 DNA interacts with uranyl ions in low concentration yielding a substrate metal ion complex with a UO2++/P mole ratio of about ½ and having a large association constant. At low pH's (about 2.3) the mole ratio decreases to about ⅓. Destruction of the structural integrity of the DNA by heating in HCHO solutions leads to a similar drop in the amount of metal ion bound. Raising the pH above 3.5 leads to an apparent increase in binding as does increasing the concentration of the salt solution. This additional binding has a lower association constant. Under similar conditions DNA binds about seven times more uranyl ion than bovine serum albumin, indicating useful selectivity in staining for electron microscopy.
Article
The pH of uranyl perchlorate solutions has been measured over a wide concentration range at various temperatures and ionic strengths. The results can be explained in terms of two hydrolytic reactions, leading respectively to the formation of the monomelic UO2·OH+ and the dimeric UO2·UO32+ ion, ΔH for formation of the former being considerably larger than for the latter. The effect of complex formation with anions is discussed and a new method of detecting perchlorate-complex formation is suggested.
Article
The contrast due to elastic and inelastic scattering of electrons in the electron microscope is calculated on a wave-optical basis. Various approximations are made, especially in the case of inelastic scattering where additional scatterin data is required for a rigorous analysis. It is shown that the major contribution to contrast arises from the gap left in the illuminating wavefront by the inelastically scattered electrons. The next most important effect is by phase contrast with elastic scattering. In-phase or amplitude contrast is very small, and the inelastically scattered electrons have little effect except for extended objects.
Article
DNA fibrils on staining in uranyl acetate or nitrate at pH 3·5 become dense in the electron microscope. Fibrils obtained from solutions containing 5 μg/ml. or less of DNA measure 20 ± 5 Å perpendicular to the plane of the supporting membrane. On staining, they appear as a dense line measuring 20 ± 5 Å in the plane of the supporting membrane. At stain concentrations of 0·0012 M, 0·012 M and 0·12 M the corresponding contrast is 5 ± 2, 5 ± 2, and 9 ± 3% respectively. This contrast variation parallels the extent of attachment of uranyl ions to DNA in solution. On the assumption that DNA adsorbed to the supporting membrane complexes with uranyl, as does DNA in solution, the 5±2% contrast can be attributed to DNA stained with one uranyl ion per base-pair. The above assumption and the significance of the results in nucleic acid sequence determination are discussed.
Article
A method is described for transferring DNA from its solution to supporting films for electron microscopy as unbroken straight single molecules. The membrane used is a copolymer of styrene and vinylpyridine which has weakly basic ion exchange properties. DNA is transferred to the membrane when it is streaked along the solution. The amount transferred depends on the pH, ionic strength and concentration of the DNA solution.
Chemical studies have been carried out on the interaction of DNA with uranyl salts. The effect of variations in pH, salt concentration, and structural integrity of the DNA on the stoichiometry of the salt-substrate complex have been investigated. At pH 3.5 DNA interacts with uranyl ions in low concentration yielding a substrate metal ion complex with a UO(2) (++)/P mole ratio of about (1/2) and having a large association constant. At low pH's (about 2.3) the mole ratio decreases to about (1/3). Destruction of the structural integrity of the DNA by heating in HCHO solutions leads to a similar drop in the amount of metal ion bound. Raising the pH above 3.5 leads to an apparent increase in binding as does increasing the concentration of the salt solution. This additional binding has a lower association constant. Under similar conditions DNA binds about seven times more uranyl ion than bovine serum albumin, indicating useful selectivity in staining for electron microscopy.
  • M Beer
4 Beer, M., J. Mol. Biol., 3, 263 (1961).
  • F Ahnued
  • D J W Cruikshank
Ahnued, F., and D. J. W. Cruikshank, Acta Cryst., 5, 852 (1952).
This work was supported by grants from the National Science Foundation (G-12323) and the National Institutes of Health (RG 8968) One of us (E. N. M.) is a holder of a NATO predoctoral fellowship and a fellowship from the Greek Atomic Energy Commission. I Zobel
* This work was supported by grants from the National Science Foundation (G-12323) and the National Institutes of Health (RG 8968). One of us (E. N. M.) is a holder of a NATO predoctoral fellowship and a fellowship from the Greek Atomic Energy Commission. I Zobel, C. R., and M. Beer, J. Biophys. and Biochem. Cytol., 10, 335 (1961). 2 Beer, M., and C. R. Zobel, J. Mol. Biol., 3, 717 (1961).
  • J M Broomhead
  • A D I Nicol
Broomhead, J. M., and A. D. I. Nicol, Acta Cryst., 1, 88 (1948).
The Aromatic Diazo Compounds
  • K H Saunders
Saunders, K. H., The Aromatic Diazo Compounds (London: Edward Arnold and Co., 1936).
The Electron Microscope
  • M E Haine
Haine, M. E., The Electron Microscope (London: E. and F. N. Spon Ltd., 1961).
  • I Zobel
  • M Beer
I Zobel, C. R., and M. Beer, J. Biophys. and Biochem. Cytol., 10, 335 (1961).
  • E N Moudrianakis
  • M Beer
  • C A Thomas
Moudrianakis, E. N., and M. Beer, to be published. 10 Berns, K. I., and C. A. Thomas, J. Mol. Biol., 3, 289 (1961).