X-ray crystallographic analysis of the hydration of A- and B-form DNA at atomic resolution

We have determined single crystal structures of an A-DNA decamer and a B-DNA dodecamer at 0.83 and 0.95 A, respectively. The resolution of the former is the highest reported thus far for any right-handed nucleic acid duplex and the quality of the diffraction data allowed determination of the structure with direct methods. The structures reveal unprecedented details of DNA fine structure and hydration; in particular, we have reexamined the overall hydration of A- and B-form DNA, the distribution of water around phosphate groups, and features of the water structure that may underlie the B to A transition.     

Synthesis and photometric properties of efficient white-emitting phosphor of M-AMG transition metal complexes for OLED applications

Progression in lighting sources mainly depended on new, robust energy-efficient diodes due to their advanced photometric properties. All organic light-emitting sources are constant energy-efficient devices and will be the light of the future. We explore the potential of transition metal complexes by focusing on cobalt(II), nickel(II), and copper (II) with aminoguanidine naphthoate as white phosphors in organic light-emitting diodes (OLEDs). The phosphors synthesized at optimized temperature were characterized structurally and thermally by spectral, thermal, and diffraction techniques. The photophysical studies of the target compound in several organic solvents having divergent polarity were also studied, and the results were exhibited. Photometric properties of the complexes were studied using photoluminescence, CIE (Commission internationale de l'éclairage) chromaticity coordinates, correlated color temperature, color purity, Duv, and TLCI (Television Lighting Consistency Index) to verify the applicability of complexes as phosphors. Excellent luminescence property with a high coloring index for (Cu(2NA-AMG-2H₂O)) opens the advanced avenue for light sources and serves as vital constituents for light-emitting diodes.     

Basigin (CD147) is the target for organomercurial inhibition of monocarboxylate transporter isoforms 1 and 4: the ancillary protein for the insensitive MCT2 is EMBIGIN (gp70)

Translocation of monocarboxylate transporters MCT1 and MCT4 to the plasma membrane requires CD147 (basigin) with which they remain tightly associated. However, the importance of CD147 for MCT activity is unclear. MCT1 and MCT4 are both inhibited by the cell-impermeant organomercurial reagent p-chloromercuribenzene sulfonate (pCMBS). Here we demonstrate by site-directed mutagenesis that removal of all accessible cysteine residues on MCT4 does not prevent this inhibition. pCMBS treatment of cells abolished co-immunoprecipitation of MCT1 and MCT4 with CD147 and enhanced labeling of CD147 with a biotinylated-thiol reagent. This suggested that CD147 might be the target of pCMBS, and further evidence for this was obtained by treatment of cells with the bifunctional organomercurial reagent fluorescein dimercury acetate that caused oligomerization of CD147. Site-directed mutagenesis of CD147 implicated the disulfide bridge in the Ig-like C2 domain of CD147 as the target of pCMBS attack. MCT2, which is pCMBS-insensitive, was found to co-immunoprecipitate with gp70 rather than CD147. The interaction between gp70 and MCT2 was confirmed using fluorescence resonance energy transfer between the cyan fluorescent protein- and yellow fluorescent protein-tagged MCT2 and gp70. pCMBS strongly inhibited lactate transport into rabbit erythrocytes, where MCT1 interacts with CD147, but not into rat erythrocytes where it interacts with gp70. These data imply that inhibition of MCT1 and MCT4 activity by pCMBS is mediated through its binding to CD147, whereas MCT2, which associates with gp70, is insensitive to pCMBS. We conclude that ancillary proteins are required to maintain the catalytic activity of MCTs as well as for their translocation to the plasma membrane.     

Probing the influence of stereoelectronic effects on the biophysical properties of oligonucleotides: comprehensive analysis of the RNA affinity, nuclease resistance, and crystal structure of ten 2'-O-ribonucleic acid modifications

The syntheses of 10 new RNA 2'-O-modifications, their incorporation into oligonucleotides, and an evaluation of their properties such as RNA affinity and nuclease resistance relevant to antisense activity are presented. All modifications combined with the natural phosphate backbone lead to significant gains in terms of the stability of hybridization to RNA relative to the first-generation DNA phosphorothioates (PS-DNA). The nuclease resistance afforded in particular by the 2'-O-modifications carrying a positive charge surpasses that of PS-DNA. However, small electronegative 2'-O-substituents, while enhancing the RNA affinity, do not sufficiently protect against degradation by nucleases. Similarly, oligonucleotides containing 3'-terminal residues modified with the relatively large 2'-O-[2-(benzyloxy)ethyl] substituent are rapidly degraded by exonucleases, proving wrong the assumption that steric bulk will generally improve protection against nuclease digestion. To analyze the factors that contribute to the enhanced RNA affinity and nuclease resistance we determined crystal structures of self-complementary A-form DNA decamer duplexes containing single 2'-O-modified thymidines per strand. Conformational preorganization of substituents, favorable electrostatic interactions between substituent and sugar-phosphate backbone, and a stable water structure in the vicinity of the 2'-O-modification all appear to contribute to the improved RNA affinity. Close association of positively charged substituents and phosphate groups was observed in the structures with modifications that protect most effectively against nucleases. The promising properties exhibited by some of the analyzed 2'-O-modifications may warrant a more detailed evaluation of their potential for in vivo antisense applications. Chemical modification of RNA can also be expected to significantly improve the efficacy of small interfering RNAs (siRNA). Therefore, the 2'-O-modifications introduced here may benefit the development of RNAi therapeutics.     

RNA interference and chemically modified small interfering RNAs

RNA interference (RNAi) is a powerful biological process for specific silencing of gene expression in diversified eukaryotic cells and has tremendous potential for functional genomics, drug discovery through in vivo target validation, and development of novel gene-specific medicine. The future success of this technology relies on identifying appropriate chemical modifications to improve stability, potency and in vivo cellular delivery. The present review summarizes the role of the chemist's toolbox in this emerging technology.     

Detection of alkali metal ions in DNA crystals using state-of-the-art X-ray diffraction experiments

The observation of light metal ions in nucleic acids crystals is generally a fortuitous event. Sodium ions in particular are notoriously difficult to detect because their X-ray scattering contributions are virtually identical to those of water and Na^+…O distances are only slightly shorter than strong hydrogen bonds between well-ordered water molecules. We demonstrate here that replacement of Na⁺ by K⁺, Rb⁺ or Cs⁺ and precise measurements of anomalous differences in intensities provide a particularly sensitive method for detecting alkali metal ion-binding sites in nucleic acid crystals. Not only can alkali metal ions be readily located in such structures, but the presence of Rb⁺ or Cs⁺ also allows structure determination by the single wavelength anomalous diffraction technique. Besides allowing identification of high occupancy binding sites, the combination of high resolution and anomalous diffraction data established here can also pinpoint binding sites that feature only partial occupancy. Conversely, high resolution of the data alone does not necessarily allow differentiation between water and partially ordered metal ions, as demonstrated with the crystal structure of a DNA duplex determined to a resolution of 0.6 Å.     

Inhibition of (cytosine C5)-methyltransferase by oligonucleotides containing flexible (cyclopentane) and conformationally constrained (bicyclo[3.1.0]hexane) abasic sites

Pseudorotationally locked sugar analogues based on bicyclo[3.1.0]-hexane templates were placed in DNA duplexes as abasic target sites in the M. HhaI recognition sequence. The binding affinity of the enzyme increases when the abasic site is constrained to the South conformation and decreases when it is constrained to the North conformation. A structural understanding of these differences is provided.     

Investigation of the proposed interdomain ribose zipper in hairpin ribozyme cleavage using 2'-modified nucleosides

The hairpin ribozyme achieves catalytic cleavage through interaction of essential nucleotides located in two distinct helical domains that include internal loops. Initial docking of the two domains is ion dependent and appears to be followed by a structural rearrangement that allows the ribozyme to achieve a catalytically active state that can undergo cleavage. The proposed structural rearrangement may also be ion dependent and is now of increased importance due to recent evidence that docking is not rate limiting and that metal ions are unlikely to be involved in the chemical cleavage step. An initial structural model of the docked hairpin ribozyme included a proposal for a ribose zipper motif that involves two pairs of hydroxyl groups at A(10) and G(11) in domain A pairing with C(25) and A(24) in domain B, respectively. We have used a chemical functional group substitution technique to study whether this proposed ribose zipper is likely to be present in the active, conformationally rearranged ribozyme that is fit for cleavage. We have chemically synthesized a series of individually modified hairpin ribozymes containing 2'-analogues of nucleosides, that include 2'-deoxy and 2'-deoxy-2'-fluoro at each of the four nucleoside positions, 2'-amino-2'-deoxy, 2'-deoxy-2'-thio, and 2'-arabino at position C(25), and 2'-oxyamino at position A(10), as well as some double substitutions, and we studied their cleavage rates under both single- and multiple-turnover conditions. We conclude that at least some of the hydrogen-bonding interactions in the ribose zipper motif, either as originally proposed or in a recently suggested structural variation, are unlikely to be present in the active rearranged form of the ribozyme that undergoes cleavage. Instead, we provide strong evidence for a very precise conformational positioning for the residue C(25) in the active hairpin. A precise conformational requirement would be expected for C(25) if it rearranges to form a base-triple with A(9) and the essential residue neighboring the cleavage site G(+1), as recently proposed by another laboratory. Our results provide further support for conformational rearrangement as an important step in hairpin ribozyme cleavage.     

Beta-carotene studies in Spirulina

The increasing importance of natural beta-carotene in fighting xerophthalmia and cancer has given special importance to algal sources of beta-carotene. The susceptibility to quick degradation of this valuable nutrient in oxygen atmosphere, light or heat calls for specific attention to processing and storage practices. In the case of Spirulina it was found that initial losses of beta-carotene on spray drying were between 7 and 10%. On storage in coloured bottles containing air, more than 50% was lost in less than 45 days. The particle size of the dried material seems to have an influence. Flakes (about 20 mesh+) retained 52% of the original beta-carotene level while the spray-dried fine powder (100 mesh-), retained only 34% of the original level. This is explainable in terms of surface area available for active reaction which is higher in the powder than in flakes. This questions the suitability of using spray drying for Spirulina drying. In this paper, data will be presented to substantiate the behaviour of beta-carotene on drying and storage by various methods.