Comparison of femtosecond laser and continuous wave UV sources for protein-nucleic acid crosslinking

Crosslinking proteins to the nucleic acids they bind affords stable access to otherwise transient regulatory interactions. Photochemical crosslinking provides an attractive alternative to formaldehyde-based protocols, but irradiation with conventional UV sources typically yields inadequate product amounts. Crosslinking with pulsed UV lasers has been heralded as a revolutionary technique to increase photochemical yield, but this method had only been tested on a few protein-nucleic acid complexes. To test the generality of the yield enhancement, we have investigated the benefits of using approximately 150 fs UV pulses to crosslink TATA-binding protein, glucocorticoid receptor and heat shock factor to oligonucleotides in vitro. For these proteins, we find that the quantum yields (and saturating yields) for forming crosslinks using the high-peak intensity femtosecond laser do not improve on those obtained with low-intensity continuous wave (CW) UV sources. The photodamage to the oligonucleotides and proteins also has comparable quantum yields. Measurements of the photochemical reaction yields of several small molecules selected to model the crosslinking reactions also exhibit nearly linear dependences on UV intensity instead of the previously predicted quadratic dependence. Unfortunately, these results disprove earlier assertions that femtosecond pulsed laser sources provide significant advantages over CW radiation for protein-nucleic acid crosslinking.     

Galactose-derived phosphonate analogues as potential inhibitors of phosphatidylinositol biosynthesis in mycobacteria

Galactose-based phosphonate analogues of myo-inositol-1-phosphate and phosphatidylinositol have been synthesized from methyl beta-d-galactopyranoside. Michaelis-Arbuzov reaction of isopropyl diphenyl phosphite or triisopropyl phosphite with a 6-iodo-3,4-isopropylidene galactoside afforded the corresponding phosphonates. Deprotection of the diphenyl phosphonate afforded methyl beta-d-galactoside 6-phosphonate, an analogue of myo-inositol-1-phosphate. The diisopropyl esters of the diisopropyl phosphonate were selectively deprotected and the corresponding anion was coupled with 1,2-dipalmitoyl-sn-glycerol using dicyclohexylcarbodiimide. Deprotection afforded a methyl beta-d-galactoside-derived analogue of phosphatidylinositol. The galactose-derived analogues of phosphatidylinositol and myo-inositol-1-phosphate were not substrates for mycobacterial mannosyltransferases (at concentrations up to 1 mM) involved in phosphatidylinositol mannoside biosynthesis in a cell-free extract of Mycobacterium smegmatis. The galactose-derived phosphonate analogue of phosphatidylinositol was shown to be an inhibitor at 0.01 mM of PimA mannosyltransferase involved in the synthesis of phosphatidylinositol mannoside from phosphatidylinositol, and a weaker inhibitor of the next mannosyltransferase(s), which catalyzes the mannosylation of phosphatidylinositol mannoside.     

Synthesis, improved antisense activity and structural rationale for the divergent RNA affinities of 3'-fluoro hexitol nucleic acid (FHNA and Ara-FHNA) modified oligonucleotides

The synthesis, biophysical, structural, and biological properties of both isomers of 3'-fluoro hexitol nucleic acid (FHNA and Ara-FHNA) modified oligonucleotides are reported. Synthesis of the FHNA and Ara-FHNA thymine phosphoramidites was efficiently accomplished starting from known sugar precursors. Optimal RNA affinities were observed with a 3'-fluorine atom and nucleobase in a trans-diaxial orientation. The Ara-FHNA analog with an equatorial fluorine was found to be destabilizing. However, the magnitude of destabilization was sequence-dependent. Thus, the loss of stability is sharply reduced when Ara-FHNA residues were inserted at pyrimidine-purine (Py-Pu) steps compared to placement within a stretch of pyrimidines (Py-Py). Crystal structures of A-type DNA duplexes modified with either monomer provide a rationalization for the opposing stability effects and point to a steric origin of the destabilization caused by the Ara-FHNA analog. The sequence dependent effect can be explained by the formation of an internucleotide C-F···H-C pseudo hydrogen bond between F3' of Ara-FHNA and C8-H of the nucleobase from the 3'-adjacent adenosine that is absent at Py-Py steps. In animal experiments, FHNA-modified antisense oligonucleotides formulated in saline showed a potent downregulation of gene expression in liver tissue without producing hepatotoxicity. Our data establish FHNA as a useful modification for antisense therapeutics and also confirm the stabilizing influence of F(Py)···H-C(Pu) pseudo hydrogen bonds in nucleic acid structures.     

Grating of single Lu@C(82) molecules using supramolecular network

A supramolecular grating of single Lu@C(82) molecules was obtained by depositing Lu@C(82) molecules onto a room temperature PTCDI-melamine network.     

Suicide inhibition of alpha-oxamine synthases: structures of the covalent adducts of 8-amino-7-oxononanoate synthase with trifluoroalanine

The irreversible inhibition of 8-amino-7-oxononanoate synthase by trifluoroalanine involves decarboxylative defluorination of the inhibitor-PLP aldimine followed by attack of the conjugated imine by the amino group of the active site lysine to afford a covalently bound difluorinated intermediate which can subsequently undergo further HF losses and hydrolysis to afford a 2-(pyridoximine phosphate) acetoyl protein adduct.     

Use of a lensed fiber for a large-field-of-view, high-resolution, fiber-scanning microendoscope

We report the application of a lensed fiber to a miniaturized fiber raster scanner in order to reduce the fiber's output beam size, thereby allowing for a compact and flexible endoscope capable of a large field of view (FOV) and high spatial resolution. For a proof of principle, the fabricated lensed fiber scanner is paired with a miniaturized gradient-index assembly to achieve a one-photon lateral resolution of 1.1 μm with a FOV that has a diameter of 440 μm.     

Manganese Triacetate Oxidation of 3β, 3aβ, 6-Trimethyl-3a, 7aβ-dihydro-2(3H), 5(4H)-benzofurandione

The manganese triacetate oxidation of hindered α¹-positions in a model enone, 3β, 3aβ, 6-trimethyl-3a, 7aβ-dihydro-2(3H), 5(4H)-benzofurandione, provided a procedure for the introduction of the α′-hydroxyenone functionality characteristic of the A ring of certain quassinoids.     

Arsenic speciation analysis

Nearly two dozen arsenic species are present in the environmental and biological systems. Differences in their toxicity, biochemical and environmental behaviors require the determination of these individual arsenic species. Considerable analytical progresses have been made toward arsenic speciation analysis over the last decade. Hyphenated techniques involving a highly efficient separation and a highly sensitive detection have become the techniques of choice. Methods based on high-performance liquid chromatography separation with inductively coupled plasma mass spectrometry, hydride generation atomic spectrometry, and electrospray mass spectrometry detection have been shown most useful for arsenic speciation in environmental and biological matrices. These hyphenated techniques have resulted in the determination of new arsenic species, contributing to a better understanding of arsenic metabolism and biogeochemical cycling. Methods for extracting arsenic species from solid samples and for stabilizing arsenic species in solutions are required for obtaining reliable arsenic speciation information.