Methyl green-pyronin; stoichiometry of reaction with nucleic acids

Study of the stoichiometry of the DNA-methyl green reaction by dialysis, precipitation of stain-nucleic acid mixtures, and the staining of nuclei of known DNA content, indicate that the compound consists of one dye molecule per 10 P. The significance of this result was discussed in the preceding paper (1). Histone and lanthanum (and probably other multivalent cations (3)) compete with the dye for the nucleic acid molecule, indicating a common site of attachment, presumably the phosphoric acid groups. With care in the avoidance of procedures which might depolymerize DNA, and the use of a buffer at about pH 4.1, a quantitative histochemical method for DNA by the use of methyl green is possible. Pyronin staining appears to be of qualitative significance only. Slight differences in degree of polymerization, as between the shad and mammalian DNA appear to have no effect on methyl green staining. It may be that a critical level of polymerization for DNA staining exists. This level must exceed 20 nucleotides to account for the 10 P to 1 dye molecule and the effect on the methyl green absorption spectrum; but it may be considerably greater. Beyond this critical level, whatever it may be, further polymerization probably has no influence on staining.     

Medicinal chemistry of plasmid DNA with peptide nucleic acids: A new strategy for gene therapy

In this chapter, we describe an approach using a peptide nucleic acid (PNA) clamp to directly and irreversibly modify plasmid DNA, without affecting either its supercoiled conformation or its ability to be efficiently transcribed. This strategy enables investigators to `functionalize' their gene of interest by direct coupling of ligands (fluorophores, peptide, proteins, sugars or oligonucleotides) to plasmid DNA. This approach provides versatile tools to study the mechanisms of gene delivery and to circumvent some of the main obstacles of synthetic gene delivery systems, such as specific targeting and efficient delivery. The proof-of-principal of PNA-dependent gene chemistry (PDGC) was demonstrated with a fluorescently labeled PNA that allowed generation of a highly fluorescent preparation of plasmid DNA that was functionally and conformationally intact. Fluorescent-PNA/DNA was used to identify critical parameters involved in naked DNA and non-viral gene delivery technology. The greatest potential of PDGC lies in the ability to attach specific ligands (e.g., peptides, proteins) to the plasmid DNA in order to overcome cellular barriers of non-viral gene delivery systems. In this regard, specific examples of ligands coupled to DNA are described and their effect on increasing the efficacy of gene therapy is presented.     

Medicinal chemistry of plasmid DNA with peptide nucleic acids: A new strategy for gene therapy

Summary In this chapter, we describe an approach using a peptide nucleic acid (PNA) clamp to directly and irreversibly modify plasmid DNA, without affecting either its supercoiled conformation or its ability to be efficiently transcribed. This strategy enables investigators to functionalize their gene of interest by direct coupling of ligands (fluorophores, peptide, proteins, sugars or oligonucleotides) to plasmid DNA. This approach provides versatile tools to study the mechanisms of gene delivery and to circumvent some of the main obstacles of synthetic gene delivery systems, such as specific targeting and efficient delivery. The proof-of-principal of PNA-dependent gene chemistry (PDGC) was demonstrated with a fluorescently labeled PNA that allowed generation of a highly fluorescent preparation of plasmid DNA that was functionally and conformationally intact. Fluorescent-PNA/DNA was used to identify critical parameters involved in naked DNA and non-viral gene delivery technology. The greatest potential of PDGC lies in the ability to attach specific ligands (e.g., peptides, proteins) to the plasmid DNA in order to overcome cellular barriers of non-viral gene delivery systems. In this regard, specific examples of ligands coupled to DNA are described and their effect on increasing the efficacy of gene therapy is presented     

Medicinal chemistry of plasmid DNA with peptide nucleic acids: A new strategy for gene therapy

Summary In this chapter, we describe an approach using a peptide nucleic acid (PNA) clamp to directly and irreversibly modify plasmid DNA, without affecting either its supercoiled conformation or its ability to be efficiently transcribed. This strategy enables investigators to 'functionalize' their gene of interest by direct coupling of ligands (fluorophores, peptide, proteins, sugars or oligonucleotides) to plasmid DNA. This approach provides versatile tools to study the mechanisms of gene delivery and to circumvent some of the main obstacles of synthetic gene delivery systems, such as specific targeting and efficient delivery. The proof-of-principal of PNA-dependent gene chemistry (PDGC) was demonstrated with a fluorescently labeled PNA that allowed generation of a highly fluorescent preparation of plasmid DNA that was functionally and conformationally intact. Fluorescent-PNA/DNA was used to identify critical parameters involved in naked DNA and non-viral gene delivery technology. The greatest potential of PDGC lies in the ability to attach specific ligands (e.g., peptides, proteins) to the plasmid DNA in order to overcome cellular barriers of non-viral gene delivery systems. In this regard, specific examples of ligands coupled to DNA are described and their effect on increasing the efficacy of gene therapy is presented.     

Electrochemical impedance spectroscopy sensor for ascorbic acid based on copper(I) catalyzed click chemistry

Copper(I) species can be acquired from the reduction of copper(II) by ascorbic acid (AA) in situ, and which in turn quantitative catalyze the azides and alkynes cycloaddition reaction. In this study, propargyl-functionalized ferrocene (propargyl-functionalized Fc) has been modified on the electrode through reacting with azide terminal modified Au electrode via copper(I) catalyzed azides and alkynes cycloaddition (CuAAC) reaction. The electrochemical impedance spectroscopy (EIS) measurement has been applied to test the electron transfer resistance of the Au electrode before and after click reaction. The changes of the fractional surface coverage (θ) with different AA concentrations are characterized. It is found that the θ value has a linear response to the logarithm of AA concentration in the range of 5.0 pmol/L to 1.0 nmol/L with the detection limits of 2.6 pmol/L. The sensor shows a good stability and selectivity. And it has been successfully applied to the AA detection in the real samples (urine) with satisfactory results.     

Borane-amine complexes--versatile reagents in the chemistry of nucleic acids and their analogs

A new method for synthesis of N-alkylated nucleosides was developed. Exceptionally mild and selective conversion of N-acyl to the corresponding N-alkyl nucleosides was achieved by reduction with borane-amine complexes. The borane-amine complexes were also used as efficient scavengers of a 4,4'-dimethoxytrityl (DMT) cation. Neutralization of the cation eliminated the boranophosphate group degradation during acidic DMT deprotection and allowed milder acidic conditions for the deprotection.     

Study of different copper (I) catalysts for the "click chemistry" approach to carbanucleosides

We compare herein the scope of three copper (I) catalysts on the synthesis of various 1,4-disubstitued-1,2,3-triazolo-carbanucleosides through a microwave (and thermic) assisted Huisgen 1,3-dipolar cycloaddition. The tetrakis(acetonitrile)copper hexafluorophosphate ([Cu(CH3CN)4]PF6), the imidazoline(mesythyl)copper bromide (Imes)CuBr, and the copper/copper sulfate Cu(0)/CuSO4 (II) mixture have been chosen for this study. Their influence in a catalytic amount will be analyzed according to the substituent of the alkyne, the solvent, or the heating method.     

Site-specific conjugation allows modulation of click reaction stoichiometry for pretargeted SPECT imaging

ABSTRACT

Antibody pretargeting is a promising strategy for improving molecular imaging, wherein the separation in time of antibody targeting and radiolabeling can lead to rapid attainment of high contrast, potentially increased sensitivity, and reduced patient radiation exposure. The inverse electron demand Diels-Alder ‘click’ reaction between trans-cyclooctene (TCO) conjugated antibodies and radiolabeled tetrazines presents an ideal platform for pretargeted imaging due to rapid reaction kinetics, bioorthogonality, and potential for optimization of both slow and fast clearing components. Herein, we evaluated a series of anti-human epidermal growth factor receptor 2 (HER2) pretargeting antibodies containing distinct molar ratios of site-specifically incorporated TCO. The effect of stoichiometry on tissue distribution was assessed for pretargeting TCO-modified antibodies (monitored by ¹²⁵I) and subsequent accumulation of an ¹¹¹In-labeled tetrazine in a therapeutically relevant HER2+tumor-bearing mouse model. Single photon emission computed tomography (SPECT) imaging was also employed to assess tumor imaging at various TCO-to-monoclonal antibody (mAb) ratios. Increasing TCO-to-mAb molar ratios correlated with increased in vivo click reaction efficiency evident by increased tumor distribution and systemic exposure of ¹¹¹In-labeled tetrazines. The pharmacokinetics of TCO-modified antibodies did not vary with stoichiometry. Pretargeted SPECT imaging of HER2-expressing tumors using ¹¹¹In-labeled tetrazine demonstrated robust click reaction with circulating antibody at ~2 hours and good tumor delineation for both the 2 and 6 TCO-to-mAb ratio variants at 24 hours, consistent with a limited cell-surface pool of pretargeted antibody and benefit from further distribution and internalization. To our knowledge, this represents the first reported systematic analysis of how pretargeted imaging is affected solely by variation in click reaction stoichiometry through site-specific conjugation chemistry.     

Radiation chemistry of nucleic acids: identification of the major hydroperoxy thymine

The major product from γ-radiation of thymine in aerated aqueous solution is shown to be $\text{cis}$-5-hydroxy-6-hydroperoxy-5,6-dihydrothymine by considering its uv, ir, nmr spectral data, the nmr deshielding effect on the OOH proton, and its reduction to $\text{cis}$-thymine glycol. These suggest that the earlier structural assignment [Cadet and Teoule, Biochem. Biophys. Acta., $\text{238}$, 8 (1971)] was in error. Furthermore, HOOH oxidation of either $\text{cis}$- or $\text{trans}$-thymine glycol in acidic condition gives this hydro-peroxide in yields >90%. This result again directly contradicts that reported in the previous paper. Our present findings are readily explained by considering the chemistry of thymine glycols and the reaction of OH radicals with thymine. Also, contrary to the earlier notion that this peroxide should be unstable, we find that it is sufficiently stable for studying the thymine peroxide interaction with chromosomes, bacterial cells and nucleic acid components.     

Interaction of nucleic acids. VI. Interaction of purine with nucleic acids

The interaction of purine with DNA, tRNA, poly A, poly C, and poly A. poly U complex was investigated. In the presence of purine, the nucleic acids in coil form (such as denatured DNA, poly A and poly C in neutral solutions, or tRNA) have lower optical rotations. In addition, hydrodynamic studies indicate that in purine solutions the denatured DNA has a higher viscosity and a decreased sedimentation coefficient. These findings indicate that through interaction with purine, the bases along the poly-nucleotide chain are unstacked and are separated farther from each other, resulting in increased assymmetry (and possibly volume) of the whole polymer. Thus, the de-naturation effect of purine reported previously can be explained by this preferential interaction of purine with the bases of nucleic acids in coil form through a hydrophobic-costacking mechanism. Results from studies on optical rotation and helix-coil transition show that the interaction of purine is greater with poly A than with poly C. The influence of temperature, Mg⁺⁺ concentration, ionic strength, and purine concentration on the effect of purine on nucleic acid conformation has also been investigated. In all these situations the unraveling of nucleic acid conformation occurs at much lower temperatures (20–40°C lower) in the presence of purine (0.2–0.6M).     

Lysozyme association with nucleic acids

Lysozyme is well known for the ability to hydrolyze the cell wall of bacteria. Based on the similarity of structure between lysozyme and histones as seen from the results of X-ray crystal structure determinations, we have postulated that binding to nucleic acids may be another biological function of lysozyme. We have therefore begun a systematic study of the interactions of lysozyme and related molecules with nucleic acids, and present here a preliminary report. Binding to DNA and RNA has been demonstrated from gel electrophoresis, enzyme activity, and coprecipitation studies. We suggest that this function of lysozyme will provide an explanation why Lee-Huang et al. (1999) [Proc. Natl. Acad. Sci. USA 96, 2678-2681] were able to call lysozyme a "killer protein" against the AIDS virus, and may provide a new avenue of research on AIDS therapy.     

Detection of transglutaminase activity using click chemistry

Transglutaminase 2 (TG2) is a Ca(2+)-dependent enzyme able to catalyze the formation of ε(γ-glutamyl)-lysine crosslinks between polypeptides, resulting in high molecular mass multimers. We have developed a bioorthogonal chemical method for the labeling of TG2 glutamine-donor proteins. As amine-donor substrates we used a set of azide- and alkyne-containing primary alkylamines that allow, after being crosslinked to glutamine-donor proteins, specific labeling of these proteins via the azide-alkyne cycloaddition. We demonstrate that these azide- and alkyne-functionalized TG2 substrates are cell permeable and suitable for specific labeling of TG2 glutamine-donor substrates in HeLa and Movas cells. Both the Cu(I)-catalyzed and strain promoted azide-alkyne cycloaddition proved applicable for subsequent derivatization of the TG2 substrate proteins with the desired probe. This new method for labeling TG2 substrate proteins introduces flexibility in the detection and/or purification of crosslinked proteins, allowing differential labeling of cellular proteins.     

Interactions of porphyrins with nucleic acids

The interactions of nucleic acids with water-soluble porphyrins and metalloporphyrins have been investigated by stopped-flow and temperature-jump techniques. Both natural DNA (calf thymus) and synthetic homopolymers [poly(dG-dC) and poly(dA-dT)] have been employed. The porphyrins studied belong to the tetrakis(4-N-methylpyridyl)porphine (H2TMpyP-4) series and can be divided into two groups: (i) those which have no axial ligands when bound to nucleic acids [e.g., Ni(II), Cu(II), and the nonmetallic derivatives] and (ii) those which maintain axial ligands upon binding [e.g., Mn(III), Fe(III), Co(III), and Zn(II) derivatives]. The reaction of both axially and nonaxially liganded porphyrins at AT sites is too rapid to be measured by the kinetic methods utilized, whereas at GC sites the interaction of the nonaxially liganded porphyrins is in the millisecond time range and can be monitored by both stopped-flow and temperature-jump techniques. These results corroborate previous static studies, utilizing visible spectroscopy and circular dichroism, which indicate that the formation of an intercalated complex occurs only at GC base pair sites with porphyrins which do not possess axial ligands. With all the porphyrins investigated, the complexes formed at AT sites are envisioned as being of an "external" type involving some degree of overlap between the porphyrin and the bases of the duplex. In relaxation experiments of poly-(dG-dC) with H2TMpyP-4, a large, reproducible effect is observed which can be analyzed as a single exponential. Rate constants for association and dissociation of the H2TMpyP-4/poly(dG-dC) complex are 3.7 X 10(5) M-1 s-1 and 1.8 s-1, respectively. Relaxation studies of mixtures of poly(dA-dT) and poly(dG-dC) with H2TMpyP-4 indicate that the transfer of the porphyrin from one homopolymer to another occurs via a mechanism involving dissociation rather than direct transfer.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA-templated click chemistry for creation of novel DNA binding molecules

We have developed a new methodology for producing new molecules that bind to dsDNA using DNA-templated click chemistry. The click reactions between the minor groove binding peptide and acridine intercalators were accelerated by the addition of dsDNA. Furthermore, the resulting peptide–acridine conjugate showed a slightly stronger binding to dsDNA. These results indicate that the DNA-templated click chemistry is applicable for screening new binding molecules.