Oligomerization reactions of deoxyribonucleotides on montmorillonite clay: The effect of mononucleotide structure,phosphate activation and montmorillonite composition on phosphodiester bond formation

2-d-5-GMP and 2-d-5-AMP bind 2 times more strongly to montmorillonite 22A than do 2-d-5-CMP and 5-TMP. The dinucleotide d(pG)₂ forms in 9.2% yield and the cyclic dinucleotide c(dpG)₂ in 5.4% yield in the reaction of 2-d-5-GMP with EDAC in the presence of montmorillonite 22A. The yield of d(pC)₂ (2.0%) is significantly lower but comparable to that obtained from 5-TMP. The yield of dimers which contain the phosphodiester bond decreases as the reaction medium is changed from 0.2 M NaCl to a mixture of 0.2 M NaCl and 0.075 M MgCl₂. A low yield of d(pA)₂ was observed in the condensation reaction of 5-ImdpA on montmorillonite 22A. The cyclic nucleotide (3,5-cdAMP) was obtained in 14% yield from 3-ImdpA. The yield of d(pA)₂ obtained when EDAC is used as the condensing agent increases with increasing iron content of the Na⁺-montmorillonite used as catalyst. Evidence is presented which shows that the acidity of the Na⁺-montmorillonite is a necessary but not sufficient factor for the montmorillonite catalysis of phosphodiester bond formation.     

Oligomerization reactions of ribonucleotides: The reaction of the 5′-phosphorimidazolide of nucleosides on montmorillonite and other minerals

The reaction of ImpA in the presence of Na⁺-montmorillonite 22A or Na⁺-Volclay in aqueous, pH 8 solution gives a 50–60% yield of dimers and trimers (pA)₂ and (pA)₃. The ratio of 3,5-phosphodiester bond formation is twice as great as 2,5-bond formation. The reaction requires the presence of Mg²⁺ and is inhibited by 0.4 M imidazole. N-methylimidazole enhances the rate of the reaction but does not cause major changes in yield or product composition. Higher yields were obtained when Li⁺- or Ca²⁺-montmorillonites were used in place of Na⁺-montmorillonite. Little or no phosphodiester bond formation was observed with Mg²⁺- or Al³⁺-montmorillonite. Montmorillonites other than 22A and Volclay exhibited little or no catalysis. In addition, little or no catalysis was exhibited in ferrugenous smectite, nontronite, allophane, imogolite or sepiolite. Oligomers were also formed by the reaction of ImpG, 2-methylImpG, ImpC and ImpU in the presence of Na⁺-montmorillonite. The pyrimidine nucleotides gave significantly lower yields of oligomers.     

Enhanced transfection efficiency of a systemically delivered tumor-targeting immunolipoplex by inclusion of a pH-sensitive histidylated oligolysine peptide

Successful cancer gene therapy depends on the development of non-toxic, efficient, tumor cell- specific systemic gene delivery systems. Our laboratory has developed a systemically administered, ligand–liposome complex that can effectively and preferentially deliver its therapeutic payload to both primary and metastatic tumors. To further improve the transfection efficiency of this targeting complex, a synthetic pH-sensitive histidylated oligolysine K[K(H)KKK]₅-K(H)KKC (HoKC), designed to aid in endosomal escape and condensation of DNA, was included in the complex. The presence of HoKC increased the in vitro transfection efficiency over that of the original complex. Moreover, no increase in cytotoxicity was observed due to the presence of the HoKC peptide. In a DU145 human prostate cancer xenograft tumor model in athymic nude mice, inclusion of the HoKC peptide did not interfere with the tumor targeting specificity of the i.v. administered ligand/liposome/DNA complex. Most importantly, the level of transgene expression was significantly elevated in the tumors, but not in the normal tissue in those animals receiving the complex incorporating HoKC. The in vivo enhancement of transfection efficiency by this modified gene delivery vehicle could lead to a reduction in the number of administrations required for antitumor efficacy.     

Mineral catalysis of the formation of dimers of 5′-AMP in aqueous solution: The possible role of montmorillonite clays in the prebiotic synthesis of RNA

The reaction of the 5-AMP with water soluble carbodiimide (EDAC) in the presence of Na⁺-montmorillonite 22A results in the formation of 2,5-(pA)₂ (18.9%), 3,5-(pA)₂ (11%), and AppA (4.8%). When poly(U) is used in place of the clay the product yields are 2,5-(pA)₂ (15.5%), 3,5-(pA)₂ (3.7%) and AppA (14.9%). The 3,5-cyclic dinucleotide, 3,5-c(pA)₂, is also formed when poly(U) is used. AppA is the principal reaction product when neither clay nor poly(U) is present in the reaction mixture. Products which contain the phophodiester bond are formed at different ionic strengths, pH and temperatures using Na⁺-montmorillonite. Phosphodiester bond formation was not observed when Cu²⁺-montmorillonite was used or when DISN was used in the place of EDAC. The extent catalysis of phophodiester bond formation varied with the particular clay mineral used. Those Na⁺-clays which bind 5-AMP more strongly are better catalysts. Cu²⁺-montmorillonite, which binds 5-AMP strongly, exhibits no catalytic activity.     

N–O bond cleavage mechanism(s) in nitrous oxide reductase

Quantum chemical calculations of active-site models of nitrous oxide reductase (N(2)OR) have been undertaken to elucidate the mechanism of N-O bond cleavage mediated by the supported tetranuclear Cu(4)S core (Cu(Z)) found in the enzymatic active site. Using either a minimal model previously employed by Gorelsky et al. (J. Am. Chem. Soc. 128:278-290, 2006) or a more extended model including key residue side chains in the active-site second shell, we found two distinct mechanisms. In the first model, N(2)O binds to the fully reduced Cu(Z) in a bent μ-(1,3)-O,N bridging fashion between the Cu(I) and Cu(IV) centers and subsequently extrudes N(2) while generating the corresponding bridged μ-oxo species. In the second model, substrate N(2)O binds loosely to one of the coppers of Cu(Z) in a terminal fashion, i.e., using only the oxygen atom; loss of N(2) generates the same μ-oxo copper core. The free energies of activation predicted for these two alternative pathways are sufficiently close to one another that theory does not provide decisive support for one over the other, posing an interesting problem with respect to experiments that might be designed to distinguish between the two. Effects of nearby residues and active-site water molecules are also explored.     

The binding and reactions of nucleotides and polynucleotides on iron oxide hydroxide polymorphs

The iron oxide hydroxide minerals goethite and akaganéite were likely constituents of the sediments present in, for instance, geothermal regions of the primitive earth. They may have adsorbed organics and catalyzed the condensation processes which led to the origins of life. The binding to and reactions of nucleotides and oligonucleotides with these minerals was investigated. The adsorption of adenosine, 5-AMP, 3-AMP, 5-UMP, and 5-CMP to these minerals was studied. Adenosine did not bind to goethite and akaganéite. The adsorption isotherms for the binding of the nucleotides revealed that they all had close to the same affinity for the mineral. Binding to goethite was about four times stronger than to akagan éite. There was little difference in the adsorption of each nucleotide suggesting the binding was between the negative charge on the phosphate group and the positive charges on the mineral surface. The absence of binding of adenosine is consistent with this explanation. Binding decreases as the pH increases due to the titration of the positive (acidic) centers on the minerals. Two times as many moles of polynucleotides were bound to these minerals as compared to the mononucleotides. Watson-Crick hydrogen bonding of adenosine and 5-AMP to poly(U) complexes with goethite and akaganéite was observed. There was no interaction of uridine with the poly(U)-goethite complex as expected if Watson-Crick hydrogen bonding is taking place. Neither goethite nor akaganéite catalyzed the oligomerization of the phosphorimidazolide of adenosine (ImpA). The template directed synthesis of oligomers of 5-GMP on the poly(C) bound to goethite was observed. Higher molecular weight oligomers were observed when the poly (C) was bound to goethite than was found in the absence of the mineral.     

Formation of RNA Oligomers on Montmorillonite: Site of Catalysis

Certain montmorillonites catalyze the self condensation of the 5-phosphorimidazolide of nucleosides in pH 8 aqueous electrolyte solutions at ambient temperatures leading to formation of RNA oligomers. In order to establish the nature of the sites on montmorillonite responsible for this catalytic activity, oligomerization reactions were run with montmorillonites which had been selectively modified (I) at the edges by (a) fluoride treatment, (b) silylation, (c) metaphosphate treatment of the anion exchange sites (II) in the interlayer by (a) saturation with quaternary alkylammonium ions of increasing size, (b) aluminum polyoxo cations. High pressure liquid chromatography, HPLC, analysis of condensation products for their chain lengths and yields indicated that modification at the edges did not affect the catalytic activity to a significant extent, while blocking the interlayer strongly inhibited product formation.