Higher-order structure influences critical functions in nearly all RNAs. Most single-nucleotide resolution RNA structure determination technologies cannot be used to analyze RNA from scarce biological samples, like viral genomes. To make quantitative RNA structure analysis applicable to a much wider array of RNA structure-function problems, the Weeks and Allbritton laboratories collaborated to develop a high-sensitivity selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) approach for structural analysis of low abundance RNAs. Their findings are published in the Journal of the American Chemical Society.
SHAPE characterization of the RNA genome of the recently discovered XMRV virus – inside virion particles – revealed functional determinants critical for virus packaging. High sensitivity SHAPE technologies are poised to accelerate functional and structural characterization of many low abundance RNAs, including viral genomes and rare biological mutants.
Members of the Johnson Group describe in Organic Letters, how the three-component coupling of Mg acetylides, silyl glyoxylates, and nitroalkenes results in a highly diastereoselective Kuwajima–Reich/vinylogous Michael cascade that provides tetrasubstituted silyloxyallene products.
The regio- and diastereoselectivity were studied using DFT calculations. These silyloxyallenes were then converted to cyclopentenols and cyclopentitols via a unique Lewis acid assisted Henry cyclization. The alkene functionality present in the cyclopentanol products can be elaborated using diastereoselective ketohydroxylation reactions.
The pregnane X receptor (PXR), a member of the nuclear receptor superfamily, regulates the expression of drug-metabolizing enzymes in a ligand-dependent manner. The conventional view of nuclear receptor action is that ligand binding enhances the receptor's affinity for coactivator proteins, while decreasing its affinity for corepressors. To date, however, no known rigorous biophysical studies have been conducted to investigate the interaction among PXR, its coregulators, and ligands. In a collaborative work published in Biochemistry, researchers in the Thompson and Redinbo groups used steady-state total internal reflection fluorescence microscopy (TIRFM) and total internal reflection with fluorescence recovery after photobleaching to measure the thermodynamics and kinetics of the interaction between the PXR ligand binding domain and a peptide fragment of the steroid receptor coactivator-1 (SRC-1) in the presence and absence of the established PXR agonist, rifampicin.
Equilibrium dissociation and dissociation rate constants of 5 μM and 2 s-1, respectively, were obtained in the presence and absence of rifampicin, indicating that the ligand does not enhance the affinity of the PXR and SRC-1 fragments. Additionally, TIRFM was used to examine the interaction between PXR and a peptide fragment of the corepressor protein, the silencing mediator for retinoid and thyroid receptors (SMRT). An equilibrium dissociation constant of 70 μM was obtained for SMRT in the presence and absence of rifampicin. These results strongly suggest that the mechanism of ligand-dependent activation in PXR differs significantly from that seen in many other nuclear receptors.
Polymer solar cells have some noteworthy advantages over mainstream inorganic-based solar cells, such as significantly reduced material/fabrication costs, flexible substrates, and low weight of finished solar cells. Thus polymer-based solar cells have become a very intensely researched field, interfacing chemistry, physics, and engineering. Rapid progress has been made with, for example, reports of power-conversion efficiency as high as 10%.
The central question — how to rationally design polymers to reach higher efficiency — has remained at the top of research priorities. As leaders in the design and synthesis of conjugated polymers for solar cells, researchers in the You Group attempt to answer this core question in a Perspective published as a cover article in the journal Macromolecules. From their unique vantage point, Huaxing Zhou, Liqiang Yang, and Wei You comprehensively review the progress in the polymer materials design for solar cells in the past decade and a half. Additionally, they offer inspiring recommendations in the section of "Outlook and Challenges," hoping to stimulate the field to come up with new ideas to push the efficiency even higher, to 15% and beyond.
Mesoporous silica nanospheres (MSNs) are a promising material for magnetic resonance imaging (MRI) contrast enhancement, as they can carry high loadings of Gd(III) complexes. MSN-based MRI contrast agents can circumvent many of the limitations of small molecule contrast agents such as low contrast enhancement efficiency, potential toxicity, and the inability to specifically target disease tissues. Nanoparticle-based MRI contrast agents must be cleared in a timely fashion to avoid the long-term toxicity.
Researchers in the Lin Group, as published in the journal Small, report the incorporation of a cleavable Gd(III) chelate into the MSN material such that the chelate is rapidly cleared after injection. The material was further functionalized with poly(ethylene glycol) and a targeting ligand to impart biocompatibility and target specificity. The effectiveness of this material as a MRI contrast agent was effectively demonstrated in vivo with human colon and pancreatic adenocarcinoma cells; the chelate was successfully cleaved and cleared via the renal excretion pathway.
Protein arginine methyltransferase 10 (PRMT10) is a type I arginine methyltransferase that is essential for regulating flowering time in Arabidopsis thaliana. Scientists in the Redinbo Group present in the Journal of Molecular Biology, a 2.6 Å resolution crystal structure of A. thaliana PRMT 10 (AtPRMT10) in complex with a reaction product, S-adenosylhomocysteine. The structure reveals a dimerization arm that is 12–20 residues longer than PRMT structures elucidated previously; as a result, the essential AtPRMT10 dimer exhibits a large central cavity and a distinctly accessible active site.
The researchers employed molecular dynamics to examine how dimerization facilitates AtPRMT10 motions necessary for activity, and they show that these motions are conserved in other PRMT enzymes. Finally, functional data reveal that the 10 N-terminal residues of AtPRMT10 influence substrate specificity, and that enzyme activity is dependent on substrate protein sequences distal from the methylation site. Taken together, these data provide insights into the molecular mechanism of AtPRMT10, as well as other members of the PRMT family of enzymes. They highlight differences between AtPRMT10 and other PRMTs but also indicate that motions are a conserved element of PRMT function.
Researchers in the Berkowitz Group have calculated the Potential of Mean Force (PMF) for the interaction between a model zwitterionic bilayer and a model charged bilayer. To understand the role of water, Changsun Eun and Max Berkowitz, as described in the Journal of Chemical Physics, separated the PMF into two components: one due to direct interaction and the other due to water-mediated interaction.
In their calculations, they observed that water-mediated interaction is attractive at larger distances and repulsive at shorter. The calculation of the entropic and enthalpic contributions to the solvent-mediated components of the PMF showed that attraction is entropically dominant, while repulsion is dominated by the enthalpy.
α-Oxyketones are important structural motifs commonly found in biologically active small molecules and natural products, and are versatile intermediates in organic synthesis. Typical approaches to α-oxyketone synthesis require pre-existing carbonyl groups that can be either oxidized or reduced. An attractive alternative strategy is the direct synthesis of α-oxyketones from alkenes via a multiple-electron oxidation, ketooxygenation. All prior ketooxygenations require the use of expensive and/or toxic transition-metal catalysts, such as Ru or Os, and are performed under harsh oxidizing conditions not readily amenable to complex molecular settings.
As reported in Chemical Science, the Alexanian Group has developed a metal-free ketooxygenation of alkenes using hydroxamic acids and O2. This process delivers α-oxyketones directly from simple alkenes with high levels of regio- and stereocontrol under mild conditions. This aerobic process is the fourth example published from the Alexanian Lab that highlights the unique reactivity of hydroxamic acids in intra- and intermolecular alkene addition reactions.
Alternative ion activation methods in a quadrupole ion trap mass spectrometer (QITMS) have been studied by researchers in the Glish Group, to determine their utility for iTRAQ. The collisional activation methods, thermally assisted CID (TA-CID) and high amplitude short time excitation (HASTE) CID, allow the low-mass cut-off (LMCO) to be reduced to ∼10% of the parent ion mass-to-charge ratio allowing the iTRAQ reporter ions to be trapped and detected.
An alternative to CID for ion activation/dissociation in a QITMS is infrared multiphoton photodissociation (IRMPD), which can be performed at LMCOs of <10% of the parent ion mass-to-charge ratio. Published in the International Journal of Mass Spectrometry are experiments comparing the efficiency of these methods for relative quantification using iTRAQ. All methods generated the reporter ions but there were differences in overall MS/MS efficiency and the conversion efficiency to the iTRAQ reporter ions.
Mammalian mitochondrial translational initiation factor 3 (IF3mt) binds to the small subunit of the ribosome displacing the large subunit during the initiation of protein biosynthesis. About half of the proteins in mitochondrial ribosomes have homologs in bacteria while the remainder are unique to the mitochondrion. To obtain information on the ribosomal proteins located near the IF3mt binding site, researchers in the Spremulli Group, in collaboration with colleagues at the Department of Biochemistry and Molecular Biology, Pennsylvania State University, performed cross-linking studies, followed by identification of the cross-linked proteins by mass spectrometry. Their results are published in Biochimica et Biophysica ACTA (BBA) - Proteins & Proteomics.
IF3mt cross-links to mammalian mitochondrial homologs of the bacterial ribosomal proteins S5, S9, S10, and S18-2 and to unique mitochondrial ribosomal proteins MRPS29, MRPS32, MRPS36 and PTCD3 (Pet309) which has now been identified as a small subunit ribosomal protein. IF3mt has extensions on both the N- and C-termini compared to the bacterial factors. Cross-linking of a truncated derivative lacking these extensions gives the same hits as the full length IF3mt except that no cross-links were observed to MRPS36. IF3 consists of two domains separated by a flexible linker. Cross-linking of the isolated N- and C-domains was observed to a range of ribosomal proteins particularly with the C-domain carrying the linker which showed significant cross-linking to several ribosomal proteins not found in prokaryotes.
Sample transport and electrokinetic injection bias are well characterized in capillary electrophoresis and simple microchips, but a thorough understanding of sample transport on devices combining electroosmosis, electrophoresis, and pressure-driven flow is lacking. In work published in Electrophoresis, researchers from the Allbritton Group evaluate the effects of electric fields from 0 to 300 V/cm, electrophoretic mobilities from 10-4 to 10-6 cm2/Vs, and pressure-driven fluid velocities from 50 to 250 µm/s on sample injection in a microfluidic chemical cytometry device. By studying a continuous sample stream, they found that increasing electric field strength and electrophoretic mobility result in improved injection and that COMSOL simulations accurately predict sample transport.
The effects of pressure-driven fluid velocity on injection are complex, and relative concentration values lie on a surface defined by pressure-driven flow rates. For high-mobility analytes, this surface is flat, and sample injection is robust despite fluctuations in flow rate. For lower mobility analytes, the surface becomes steeper, and injection depends strongly on pressure-driven flow. These results indicate generally that device design must account for analyte characteristics and specifically that this device is suited to high-mobility analytes. The team demonstrates that for a suitable pair of peptides fluctuations in injection volume are correlated; electrokinetic injection bias is minimized; and electrophoretic separation is achieved.
Nanoscale coordination polymers (NCPs) have been demonstrated as an interesting platform for the delivery of methotrexate (MTX), an antifolate cancer drug, as they possess many potential advantages over small-molecule chemotherapeutics such as high payloads, lower systemic toxicity, tunability, and enhanced tumor uptake. NCPs also overcome the limitations of existing nanoparticle formulations that have very low drug loadings.
Researchers in the Lin Group, published in Chemical Science, report the incorporation of MTX as a building block in an NCP formulation with exceptionally high drug loadings (up to 79.1 wt%) and the selective delivery of the NCP to cancer cells. Encapsulation of the NCP in a functionalized lipid bilayer allows for targeted delivery and controlled release to cancer cells. A phosphor can be doped into the NCPs for monitoring particle uptake by optical imaging. The lipid-coated and anisamide-targeted NCPs have superior in vitro efficacy against acute lymphoblastic leukemia cells when compared to the free drug.