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Raman microscopic analysis of hepatitis delta virus (HDV) ribozyme crystals was performed to study the binding of [Mg.sup.2+] and Co(N[H.sub.3]).sub.6.sup.3+] to the HDV ribozyme. The data obtained supported two classes of inner sphere [Mg.sup.2+]-P[O.sub.2.sup.- binding sites, sites that Co[(N[H.sub.3]).sub.6.sup.3+] able to displace and others it failed to displace.

Several kinetic experiments are conducted to explain the impact of pH on the rate and metal binding properties of the different mutants of HDV ribozyme. The mechanisms involved in the interaction taking place between a catalytic metal ion and the cleavage site G.U wobble base pair are also discussed.

The vibrational signatures of RNA-bound magnesium hydrate in crystals of hepatitis delta virus (HDV) ribozyme are detected and the effects of magnesium hydrate binding to the nonbridging phosphate oxygens in the phosphodiester backbone are examined. Raman experiments have provided the potential to detect site-specific effects by using isotope and chemical substitutions.

Raman crystallography is employed to directly measure the value of p[K.sub.a] near neutrality for the catalytic cytosine in the genomic hepatitis delta virus (HDV) ribozyme. The results prove that such ribozymes can easily optimize the values of their side chains for protein transfer.

Divalent cations play critical structural and functional roles in many RNAs. While the hepatitis delta virus (HDV) ribozyme can undergo self-cleavage in the presence of molar concentrations of monovalent cations, divalent cations such as Mg2+ are required for efficient catalysis under physiological conditions. Moreover, the cleavage reaction can be inhibited with Co(NH3)63+, an analogue of Mg(H2O)62+. Here, the binding of Mg2+ and Co(NH3)63+ to the HDV ribozyme is studied by Raman microscopic analysis of crystals. Raman difference spectra acquired at different metal ion conditions reveal changes in the ribozyme. When Mg2+ alone is introduced to the ribozyme, inner sphere coordination of Mg(H2O)x2+ (x ⩽ 5) to nonbridging PO2- oxygen and changes in base stretches and phosphodiester group conformation are observed. In addition, binding of Mg2+ induces deprotonation of a cytosine assigned to the general acid C75, consistent with solution studies. When Co(NH3)63+ alone is introduced, deprotonation of C75 is again observed, as are distinctive changes in base vibrational ring modes and phosphodiester backbone conformation. In contrast to Mg2+ binding, Co(NH3)63+ binding does not perturb PO2- group vibrations, consistent with its ability to make only outer sphere contacts. Surprisingly, competitive binding studies reveal that Co(NH3)63+ ions displace some inner sphere-coordinated magnesium species, including ions coordinated to PO2- groups or the N7 of a guanine, likely G1 at the active site. These observations contrast with the tenet that Co(NH3)63+ ions displace only outer sphere magnesium ions. Overall, our data support two classes of inner sphere Mg2+-PO2- binding sites: sites that Co(NH3)63+ can displace and others it cannot. [ABSTRACT FROM AUTHOR]

Hybrid organic−inorganic polymer thin films of the form (−O−Zn−O−C2H4−)nhave been deposited from diethyl zinc and ethylene glycol using molecular layer deposition (MLD) over a range of substrate temperatures between 100 and 170 °C. Infrared transmission confirms that the films consist of Zn−O and ethylene-oxide units. In analogy with known alucone polymers of the form (−O−Al−O−R−)n, the zinc-based hybrid material is an example of a “zincone” polymer. In situ quartz crystal microbalance analysis indicated that the sequential surface reactions of diethyl zinc and ethylene glycol are sufficiently self-limiting and saturating to enable well-controlled MLD growth. Quantitative analysis of in situ quartz crystal microbalance and film thickness results indicate that ethylene glycol molecules can undergo a “double reaction” where the OH groups on both ends of the diol react with available Zn−C2H5surface sites to produce a relatively inert bridging alkane. The mass uptake per MLD cycle during Zn−hybrid film deposition decreases with increasing reaction temperature. Infrared transmission spectroscopy also shows that Zn−organic hybrid films are stable in dry air. However, the as-deposited ZnO−hybrid material could be hydrolyzed by H2O (for example, in ambient) resulting in films consisting of zinc oxide and zinc hydroxide with some carbon remnants. Spectroscopic ellipsometry indicates the thickness of hydrolyzed films increases linearly with reaction cycles, and scanning probe and transmission electron microscopy images show the hydrolyzed ZnO−hybrid film coating is uniform and conformal. The transmission electron micrographs also show the hydrolyzed Zn−hybrid films contain nanoscale porosity. These results suggest new pathways to fabricate organic−inorganic hybrid materials, including metal-organic framework structures. [ABSTRACT FROM AUTHOR]

The hepatitis delta virus (HDV) ribozyme uses a cytosine to facilitate general acid-base catalysis. Biochemical studies suggest that C75 has a PKa perturbed to near neutrality. To measure this pKa directly, Raman spectra were recorded on single ribozyme crystals using a Raman microscope. A spectral feature arising from a single neutral cytosine was identified at 1528 cm-1. At low pH, this mode was replaced with a new spectral feature. Monitoring these features as a function of pH revealed PKa values for the cytosine that couple anticooperatively with Mg2+ binding, with values of 6.15 and 6.40 in the presence of 20 and 2 mM Mg2+, respectively. These PKa values agree well with those obtained from ribozyme activity experiments in solution. To correlate the observed PKa with a specific nucleotide, crystals of C75U, which is catalytically inactive, were examined. The Raman difference spectra show that this mutation does not affect the conformation of the ribozyme. However, crystals of C75U did not produce a signal from a protonatable cytosine, providing strong evidence that protonation of C75 is being monitored in the wild-type ribozyme. These studies provide the first direct physical measurement of a PKa near neutrality for a catalytic residue in a ribozyme and show that ribozymes, like their protein enzyme counterparts, can optimize the pKa of their side chains for proton transfer. [ABSTRACT FROM AUTHOR]

The article demonstrates that Raman crystallography can be used to study metal ion interactions in RNA that can complement traditional but less direct crystallographic and biochemical methods. According to the authors, Raman crystallography permits both qualitative and semiquantitative dissection of metal ion binding and should prove useful in the analysis of other functional RNA and RNA-protein complexes.

The use of contact lenses for the early treatment of bacterial or fungal keratitis has become a new research focus. Two main requirements of the therapeutic contact lenses are antimicrobial ability and visible light transmittance. Silver nanoparticles (AgNPs), as a nonspecific antimicrobial component, have been loaded onto contact lenses for the treatment of bacterial and fungal keratitis. Recently, it was reported that, via a simple immersion method, AgNPs can be synthesized and fixed onto the surface of polydopamine (PDA)-coated materials. However, in this study, we found that the above traditional method has the disadvantages of poor AgNP loading and low visible light transmittance, which could be induced by a limited amount of phenolic hydroxyl groups on and second oxidation of the PDA coating, respectively. To overcome these disadvantages, in this paper, we provided a facile and novel method to robustly bind multilayer-AgNPs on contact lens surfaces by using dopamine as a reducing agent and bioglue. In comparing with the monolayer-AgNP-loaded contact lenses fabricated by the traditional method, the multilayer-AgNP-loaded contact lenses had excellent antimicrobial ability and better visible light transmittance. Moreover, the multilayer-AgNP-loaded contact lens had low cytotoxicity to human corneal epithelial cells and anti-inflammation properties. Furthermore, the shortcoming of decreasing visible light transmittance induced by excess adherence of AgNPs on the multilayer-AgNP-loaded contact lens was alleviated by decreasing the size of AgNPs through altering the concentration of dopamine and AgNO 3 . Contact lenses loaded with small AgNPs (Ag@PDA-2.5, diameter ≈ 25-50 nm) had approximately the same Ag + release and antimicrobial abilities, but significantly better visible light transmittance and anti-inflammatory properties than the contact lenses loaded with large AgNPs (Ag@PDA-5, diameter ≈ 50-75 nm). After that, in vivo testing indicated the promising therapeutic strategy of multilayer-AgNP-loaded contact lenses (Ag@PDA-2.5) for early bacterial keratitis and fungal keratitis. In addition, PDA coating could provide reactive sites to immobilize other biomolecules or drugs on this multilayer-AgNP-loaded contact lens for further combination therapies in treating bacterial or fungal keratitis. Finally, the stability of the visible light transmittance of the multilayer-AgNP-loaded contact lens was detected. The visible light transmittance of Ag@PDA-2.5 was weakened after being cultured with an extremely high concentration of bacteria, while it was stable in the moderate work environment. Though PDA coating had been wildly used to modify implantation devices, however, few studies about PDA coating modified contact lenses have been reported so far. Therefore, this research also provides an important basis for using PDA coating to modify a therapeutic contact lens.