Synthesis of ferrocenylcarbodiimide as a convenient electrochemically active labeling reagent for nucleic acids

Ferrocenylcarbodiimides carrying different redox potentials, 1 and 2, were designed and synthesized as convenient electrochemically active labeling reagents for nucleic acids, which may be used as dually labeling reagents of nucleic acids like Cy3 and Cy5 dyes. These reagents could react with the imino unit of thymine or guanine base on DNA or of uracil base on RNA under a basic buffer condition to yield a labeled product quantitatively in a short period of time. The current responses of the labeled DNAs in square wave voltammetric (SWV) measurement showed a good linear correlation with the amount of the hybridized ones. DNAs labeled with the two different reagents, 1 and 2, could be detected electrochemically at different potentials after hybridization with a DNA probe-immobilized gold electrode.     

1-, 2-, and 4-ethynylpyrenes in the structure of twisted intercalating nucleic acids: structure, thermal stability, and fluorescence relationship

A postsynthetic, on-column Sonogashira reaction was applied on DNA molecules modified by 2- or 4-iodophenylmethylglycerol in the middle of the sequence, to give the corresponding ortho- and para-twisted intercalating nucleic acids (TINA) with 1-, 2-, and 4-ethynylpyrene residues. The convenient synthesis of 2- and 4-ethynylpyrenes started from the hydrogenolysis of pyrene that has had the sulfur removed and separation of 4,5,9,10-tetrahydropyrene and 1,2,3,6,7,8-hexahydropyrene, which were later converted to the final compounds by successive Friedel-Crafts acetylation, aromatization by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone, and a Vilsmeier-Haack-Arnold transformation followed by a Bodendorf fragmentation. Significant alterations in thermal stability of parallel triplexes and antiparallel duplexes were observed upon changing the attachment of ethynylpyrenes from para to ortho in homopyrimidine TINAs. Thus, for para-TINAs the bulge insertion of an intercalator led to high thermal stability of Hoogsteen-type parallel triplexes and duplexes, whereas Watson-Crick-type duplexes were destabilized. In the case of ortho-TINA, both Hoogsteen and Watson-Crick-type complexes were stabilized. Alterations in the thermal stability were highly influenced by the ethynylpyrene isomers used. This also led to TINAs with different changes in fluorescence spectra depending on the secondary structures formed. Stokes shift of approximately 100 nm was detected for pyren-2-ylethynylphenyl derivatives, whereas values for 1- and 4-ethynylpyrenylphenyl conjugates were 10 and 40 nm, respectively. In contrast with para-TINAs, insertion of two ortho-TINAs opposite each other in the duplex as a pseudo-pair resulted in formation of an excimer band at 505 nm for both 1- and 4-ethynylpyrene analogues, which was also accompanied with higher thermal stability.     

High-capacity composite adsorbents for nucleic acids

Cytopore™ is a bead-shaped, macroporous and easily compressible cellulose-based anion-exchange material intended for cultivation of anchor-dependent animal cells. Reticulated vitreous carbon (RVC) is a strong, non-compressible, high voidage (97%) matrix material that can be cut to desired geometrical shapes. Cytopore and RVC were combined to cylindrical composites (25 mm × 10 mm) fitted inside chromatography columns. The composite combined the advantageous properties of both its constituents, making it suitable for column chromatography. The composite could withstand very high flow rates without compaction of the bed (>25 column volumes/min; 4000 cm h⁻¹). Chromatography runs with tracers showed a low HETP value (0.3 mm), suggesting that pore flow was in operation. The dynamic binding capacities (10% breakthrough) per gram of dry weight Cytopore were determined for several compounds including DNA and RNA and were found to be 240–370 mg/g. The composite was used to isolate pUC 18-type plasmids from a cleared alkaline lysate in a good yield. Confocal microscopy studies showed that plasmids were bound not only to the surface of the Cytopore material but also within the matrix walls, thus offering an explanation to the very high binding capacities observed. The concept of using a composite prepared from a mechanically weak, high-binding material and a strong scaffold material may be applied to other systems as well.     

Pulse-polarographic analysis of nucleic acids and proteins

Nucleic acids and proteins were studied by means of derivative and normal pulse polarography, and d.c. and a.c. polarography in connection with the dropping mercury electrode. It was shown that natural ribonucleic acids, as transfer, ribosomal and viral RNAs yield derivative pulse-polarographic peaks; from their heights and potentials conclusions can be made about their content of ordered structure in solution, similarly as in the case of deoxyribonucleic acids studied earlier. Synthetic single-stranded polyribo-cytidylic acid yields a well developed peak, whereas in the double-helical complex with polyriboguanylie acid it is inactive when using either derivative pulse polarography or d.c. polarography. Well developed peaks were obtained also with albumin (a protein containing reducible?S?S? groups), while only an inflex was observed on the d.c. polarogram. Proteins were also studied in media containing cobalt (Brdi?ka's solution) or nickel and it was shown that derivative pulse polarography due to its high sensitivity and accuracy enables us to carry out the measurements even in less common media than Brdi?ka's solution. This fact could be exploited in clinical chemistry as well as in the investigation of the nature of catalytic currents of proteins. The currents of double-helical polynucleotides obtained by means of normal pulse polarography exhibit a marked dependence on the initial potential and cannot represent a reliable indicator of structural changes of biopolymers in solution. They can however, be used in studies of the influence of the polynucleotide adsorption at different potentials on the subsequent reduction.     

On the stability of double stranded nucleic acids

We present the first pressure-versus-temperature phase diagram for the helix-to-coil transition of double stranded nucleic acids. The thermodynamic stability of a nucleic acid duplex is a complex function of temperature and pressure and strongly depends on the denaturation temperature, T(M), of the duplex at atmospheric pressure. Depending upon T(M), pressure, and temperature, the phase diagram shows that pressure may stabilize, destabilize, or have no effect on the conformational state of DNA. To verify the phase diagram, we have conducted high-pressure UV melting experiments on poly(dIdC)poly(dIdC), a DNA duplex, poly(rA)poly(rU), an RNA duplex, and poly(dA)poly(rU), a DNA/RNA hybrid duplex. The T(M) values of these duplexes have been modulated by altering the solution ionic strength. Significantly, at low salt, these three duplexes have helix-to-coil transition temperatures of 50 degrees C or less. In agreement with the derived phase diagram, we found that the polymeric duplexes were destabilized by pressure if the T(M) is < approximately 50 degrees C. However, these duplexes were stabilized by pressure if the T(M) is > approximately 50 degrees C. The DNA/RNA hybrid duplex, poly(dA)poly(rU), with a T(M) of 31 degrees C in 20 mM NaCl undergoes a pressure-induced helix-to-coil transition at room temperature. This is the first report of pressure-induced denaturation of a nucleic acid duplex and provides new insights into the molecular forces stabilizing these structures.     

Polymeric fluorescent dyes for labeling of proteins and nucleic acids

In order to increase the sensitivity of fluorescence labeling in biochemical reactions and diagnostic procedures a labeling technique with polymeric fluorescence dyes was established and tested for its applicability. The fluorescence dye is based on the fluorophor coumarine and was covalently linked to the model proteins strepavidine and IgG. The dye was synthesized by radical polymerization of three different types of functional monomers to ensure water solubility, covalent coupling to proteins, and fluorescence. The molecular weight range was between 20 and 200 kDa. Fractions of narrow molecular weight distribution were prepared by gel filtration on Superdex 200. The relationship between size and charge of the different fractions was analyzed by gel electrophoresis. Covalent conjugation to proteins was carried out by formation of a peptide bond between a carboxylic group of the functional monomers and an amino group of the protein mediated by 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide (EDC). A novel type of gel electrophoresis was developed in order to analyze and optimize the conjugation reaction; the results were in agreement with those from analytical ultracentrifugation with fluorescence detection. Hydrodynamic studies of the uncoupled dye and the protein-dye conjugates exhibited a drastic decrease of Stokes radius of the dye due to the coupling to the protein. Under optimum conditions the fluorescence intensity of a protein-polymeric dye conjugate was enhanced 40-fold compared to a monomeric dye. Biotin binding to the protein streptavidin was not affected significantly by the conjugation with the polymeric dye. At present, the applicability of the polymeric dye in biochemical and diagnostic reactions seems to be limited due to strong but unspecific hydrophobic interactions which might be overcome by using fluoresceine as monomeric dye.     

Immobilization of proteins as a tool for studying primary structure around their cysteinyl residues

The primary structure around the single cysteinyl residue of chicken pepsin was investigated by binding the protein via this residue to an insoluble carrier. Carriers stable towards reagents used for the fragmentation of proteins and sequence analysis were prepared by coupling a spacer arm to polyN-hydroxymethyl acrylamide using a thioether bond that is potentially cleavable by mercuric ions (1). Phenacyl bromide group, attached to the free end of the spacer, reacted rapidly and specifically with the cysteinyl residue of chicken pepsin. Up to 300 mg of the enzyme were bound to 1 g of carrier. The polymer-bound protein was cleaved by trypsin or by cyanogen bromide or by a sequence of both. Fragments of 40–120 amino acid residues, depending on the method of cleavage, remained attached to the polymer through the cysteinyl residue. The compositions and partial sequences of these fragments revealed that the cysteinyl residue is located within or in the vicinity of a loop in the molecule formed by a disulfide bond.     

Variable temperature infrared spectroscopy: a convenient tool for studying the thermodynamics of weak solid-gas interactions

This tutorial review describes the use of variable temperature infrared spectroscopy of adsorbed species (VTIR), a recent method for studying the thermodynamics of weak solid-gas interactions. Examples show how a fundamental relationship of thermodynamics (the van't Hoff equation, used long since in several fields of physical chemistry) can describe equilibrium processes at the solid-gas interface. The VTIR method is fully exploited by measuring absorbance of an IR band, temperature and pressure over a wide temperature range: an estimation of the interaction energy is, however, possible even ignoring the equilibrium pressure. Precise thermodynamic characterization of solid-gas interactions is required in several fields: on the applied side, gas sensing, separation and storage, which involve such areas as work-place security, air pollution control and the energy sector; regarding fundamental knowledge, weak solid-gas interactions are relevant to a number of fields, including hydrogen bonding, coordination chemistry and surface phenomena in a broad sense.Infrared (IR) spectroscopy of (gas) molecules adsorbed on a solid is frequently used to characterize both, the adsorbed species and the adsorbing centres at the solid surface. The potential of the technique can be greatly enhanced by obtaining IR spectra over a temperature range, and simultaneously measuring IR absorbance, temperature and equilibrium pressure. When this is done, variable temperature infrared (VTIR) spectroscopy can be used not only for a more detailed surface characterization, but also for precise studies on the thermodynamics of solid-gas interactions. Furthermore, when weak interactions are concerned, the technique shows favourable features compared to adsorption calorimetry, or to other classical methods. The potential of the VTIR method is highlighted by reviewing recently reported studies on dihydrogen, dinitrogen and carbon monoxide adsorption on zeolites. To facilitate understanding, an outline of the basis of the method is also given, together with an appraisal of the critical points involved in its practical use.     

Thermo-biolithography: a technique for patterning nucleic acids and proteins

We describe a "biolithographic" technique in which the unique properties of biopolymeric materials and the selective catalytic activities of enzymes are exploited for patterning surfaces under simple and bio-friendly conditions. We begin by coating a reactive film of the polysaccharide chitosan onto an inorganic surface (glass or silicon wafer). Chitosan's pH-responsive solubility facilitates film deposition, while the nucleophilic properties of this polysaccharide allow simple chemistries or biochemistries to be used to covalently attach species to the film. The thermally responsive protein gelatin is then cast on top of the chitosan film, and the gelatin gel serves as a sacrificial "thermoresist". Pattern transfer is accomplished by applying a heated stamp to melt specific regions of the gelatin thermoresist and selectively expose the underlying chitosan. Finally, molecules are conjugated to the exposed chitosan sublayer and the sacrificial gelatin layer is removed (either by treating with warm water or protease). To demonstrate the concept, we patterned a reactive dye (NHS-fluorescein), a model 20-base oligonucleotide (using standard glutaraldehyde coupling chemistries), and a model green fluorescent protein (using tyrosinase-initiated conjugation). Because gelatin can be applied and removed under mild conditions, sequential thermo-biolithographic steps can be performed without destroying previously patterned biomacromolecules. These studies represent the first step toward exploiting nature's exquisite specificity for lithographic patterning.     

Ab initio force field for simulations of proteins and nucleic acids

We present a new self-consistent set of ab initio analytical pair potential to predict specific nonbonded interactions of protein with nucleic acid, of protein with protein, and of nucleic acid with nucleic acid. The purpose of this study is to represent the interaction between biological molecules with an accuracy equivalent to the ab initio molecular orbital calculations, which are used as reference data to obtain the pair potentials. Atoms in nucleic acids and proteins are classified according to their chemical environments. An “effective charge,” a modification of a charge obtained from the Mulliken population analysis, is introduced and used to represent the electrostatic energy. More than 30,000 SCF interaction energies have been calculated to provide the reference data for the fitting procedure that we have adopted in the parameterization of the potentials. The standard deviation is 1.61 kcal/mol for interaction energies spanning the range from about ?220 kcal/mol to +20 kcal/mol. Molecular dynamics simulations, using the above new set of force field, have been performed successfully for the systems where adequate treatments of specific interactions are required: The stability of α-helix of C-peptide and the interaction of spermine with oligonucleotide are examined as preliminary examples.     

Lipophilic nucleic acids — A flexible construction kit for organization and functionalization of surfaces

Lipophilic nucleic acids have become a versatile tool for structuring and functionalization of lipid bilayers and biological membranes as well as cargo vehicles to transport and deliver bioactive compounds, like interference RNA, into cells by taking advantage of reversible hybridization with complementary strands. This contribution reviews the different types of conjugates of lipophilic nucleic acids, and their physicochemical and self-assembly properties. Strategies for choosing a nucleic acid, lipophilic modification, and linker are discussed. Interaction with lipid membranes and its stability, dynamic structure and assembly of lipophilic nucleic acids upon embedding into biological membranes are specific points of the review. A large diversity of conjugates including lipophilic peptide nucleic acid and siRNA provides tailored solutions for specific applications in bio- and nanotechnology as well as in cell biology and medicine, as illustrated through some selected examples.     

A convenient method for the synthesis of α-silylacetic acids

A method is described for the preparation of α-silylacetic acids of the type R₃SiCH₂CO₂H by treating trimethylsilyl acetate with LDA followed by quenching with chlorosilanes.     

Spectroscopic study of the interaction of pazelliptine with nucleic acids

The antitumor drug pazelliptine (PZE) binds to natural and synthetic DNA sequences at 100 mM NaCl, pH 7.0, as deduced from the absorption and fluorescence data. Scatchard plots constructed from the results obtained with poly(dG-dC)-poly(dG-dC) give binding constants of base pairs in the range (2–6) × 10⁵ M⁻¹. The modifications in the absorption and fluorescence spectra observed when PZE binds to various polynucleotides, namely poly(dA-dT)-poly(dA-dT), poly(dA)-poly(dT), poly(dG-dC)-poly(dG-dC) and calf thymus DNA. reveal a change in the protonation state of the drug upon binding, increasing the apparent pK_a of its 9-N nitrogen atom. The PZE excited state properties serve as a sensitive probe to distinguish between homo and hetero A-T sites as well as between AT and GC sites. Fluorescence studies reveal that energy transfer occurs from polynucleotide bases to the bound PZE chromophore, a result consistent with an intercalative mode of binding of the drug to DNA. The emission is enhanced when PZE is bound to A-T base pairs ( 30% increase of φ_F) whereas it is quenched in the vicinity of G-C base pairs ( 90% decrease of φ_F). Furthermore, the fluorescence spectrum obtained with calf thymus DNA is hardly distinguishable from that obtained with poly(dG-dC)-polu(dG-dC), suggesting a binding of PZE to G-C rich regions.     

A convenient synthesis of phosphonothioic acids

A method for the convenient synthesis of phosphonothioates, or phosphonothioic acids, is reported. A significant advantage of the method is the alleviation of the need for purification of intermediates, other than washing with water. No chromatography is needed and the only purification step is the crystallization of the final product. The method uses standard reagents and should be applicable to the synthesis of phosphonothioic acids bearing a range of functional groups.     

Quantum dots as donors in fluorescence resonance energy transfer for the bioanalysis of nucleic acids,proteins, and other biological molecules

Quantum dots (QDs) have a number of unique optical properties that are advantageous in the development of bioanalyses based on fluorescence resonance energy transfer (FRET). Researchers have used QDs as energy donors in FRET schemes for the analysis of nucleic acids, proteins, proteases, haptens, and other small molecules. This paper reviews these applications of QDs. Existing FRET technologies can potentially be improved by using QDs as energy donors instead of conventional fluorophores. Superior brightness, resistance to photobleaching, greater optimization of FRET efficiency, and/or simplified multiplexing are possible with QD donors. The applicability of the Förster formalism to QDs and the feasibility of using QDs as energy acceptors are also reviewed.

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Figure A ligand capped core/shell quantum dot acting as energy donor in a FRET process with aconjugated Cy3 labeled oligonucleotide