Site-specific terminal and internal labeling of RNA by poly(A) polymerase tailing and copper-catalyzed or copper-free strain-promoted click chemistry

The modification of RNA with fluorophores, affinity tags and reactive moieties is of enormous utility for studying RNA localization, structure and dynamics as well as diverse biological phenomena involving RNA as an interacting partner. Here we report a labeling approach in which the RNA of interest--of either synthetic or biological origin--is modified at its 3'-end by a poly(A) polymerase with an azido-derivatized nucleotide. The azide is later on conjugated via copper-catalyzed or strain-promoted azide-alkyne click reaction. Under optimized conditions, a single modified nucleotide of choice (A, C, G, U) containing an azide at the 2'-position can be incorporated site-specifically. We have identified ligases that tolerate the presence of a 2'-azido group at the ligation site. This azide is subsequently reacted with a fluorophore alkyne. With this stepwise approach, we are able to achieve site-specific, internal backbone-labeling of de novo synthesized RNA molecules.     

Autophosphorylation of gatekeeper tyrosine by symbiosis receptor kinase

Plant receptor-like kinases (RLKs) share their evolutionary origin with animal interleukin-1 receptor-associated kinase (IRAK)/Pelle family of soluble kinases and are distinguished by having tyrosine as ‘gatekeeper’. This position is adjacent to the hinge region and is hidden in a hydrophobic pocket of the catalytic cleft of protein kinases and is therefore least probable to be a target for any modification. This communication illustrates the accessibility of the gatekeeper site (Y670) towards both autophosphorylation and dephosphorylation in the recombinant cytoplasmic domain of symbiosis receptor kinase from Arachis hypogaea (AhSYMRK). Autophosphorylation on gatekeeper tyrosine was detected prior to extraction but never under in vitro conditions. We hypothesize gatekeeper phosphorylation to be associated with synthesis/maturation of AhSYMRK and this phenomenon may be prevalent among RLKs.     

Development of selectable marker free,insect resistant,transgenic mustard (Brassica juncea) plants using Cre/loxmediated recombination

β-Fructofuranosidases (or invertases) catalyse the commercially-important biotransformation of sucrose into short-chain fructooligosaccharides with wide-scale application as a prebiotic in the functional foods and pharmaceutical industries. We identified a β-fructofuranosidase gene (CmINV) from a Ceratocystis moniliformis genome sequence using protein homology and phylogenetic analysis. The predicted 615 amino acid protein, CmINV, grouped with an existing clade within the glycoside hydrolase (GH) family 32 and showed typical conserved motifs of this enzyme family. Heterologous expression of the CmINV gene in Saccharomyces cerevisiae BY4742∆suc2 provided further evidence that CmINV indeed functions as a β-fructofuranosidase. Firstly, expression of the CmINV gene complemented the inability of the ∆suc2 deletion mutant strain of S. cerevisiae to grow on sucrose as sole carbohydrate source. Secondly, the recombinant protein was capable of producing short-chain fructooligosaccharides (scFOS) when incubated in the presence of 10% sucrose. Purified deglycosylated CmINV protein showed a molecular weight of ca. 66 kDa and a Km and Vmax on sucrose of 7.50 mM and 986 μmol/min/mg protein, respectively. Its optimal pH and temperature conditions were determined to be 6.0 and 62.5°C, respectively. The addition of 50 mM LiCl led to a 186% increase in CmINV activity. Another striking feature was the relatively high volumetric production of this protein in S. cerevisiae as one mL of supernatant was calculated to contain 197 ± 6 International Units of enzyme. The properties of the CmINV enzyme make it an attractive alternative to other invertases being used in industry.     

Why are Hot Holes Easier to Extract than Hot Electrons from Methylammonium Lead Iodide Perovskite?

Charge‐carriers photoexcited above a semiconductor's bandgap rapidly thermalize to the band‐edge. The cooling of these difficult to collect “hot” carriers caps the available photon energy that solar cells–including efficient perovskite solar cells–may utilize. Here, the dynamics and efficiency of hot carrier extraction from MAPbI₃ (MA = methylammonium) perovskite by spiro‐OMeTAD (a hole‐transporting layer) and TiO₂ (an electron‐transporting layer) are investigated and explained using both ultrafast electronic spectroscopy and theoretical modeling. Time‐resolved spectroscopy reveals a quasi‐equilibrium distribution of hot carriers forming upon excess‐energy excitation of the perovskite–a distribution largely unaffected by the presence of TiO₂. In contrast, the quasi‐equilibrium distribution of hot carriers is virtually nonexistent when spiro‐OMeTAD is present, which is indicative of efficient hot hole extraction at the interface of MAPbI₃. Density functional theory calculations predict that deep energy‐levels of MAPbI₃ exhibit electronically delocalized character, with significant overlap with the localized valence band charge of the spiro‐OMeTAD molecules lying on the surface of MAPbI₃. Consequently, hot holes are easily extracted from the deep energy‐levels of MAPbI₃ by spiro‐OMeTAD. These findings uncover the origins of efficient hot hole extraction in perovskites and offer a practical blueprint for optimizing solar cell interlayers to enable hot carrier utilization.     

Synthetic studies on dendritic glycoclusters: a convergent palladium-catalyzed strategy.

A facile Pd-catalyzed strategy by which multiantennary glycoclusters and sugar dendrons can be readily assembled in one-step is described.     

An investigation into possible quantum chaos in the H<Subscript>2</Subscript> molecule under intense laser fields via Ehrenfest phase space (EPS) trajectories

By employing the Ehrenfest "phase space" trajectory method for studying quantum chaos, developed in our laboratory, the present study reveals that the H₂ molecule under intense laser fields of three different intensities, I?=?1?×?10¹⁴ W/cm², 5?×?10¹⁴ W/cm², and 1?×?10¹⁵ W/cm², does not show quantum chaos. A similar conclusion is also reached through the Loschmidt echo (also called quantum fidelity) calculations reported here for the first time for a real molecule under intense laser fields. Thus, a long-standing conjecture about the possible existence of quantum chaos in atoms and molecules under intense laser fields has finally been tested and not found to be valid in the present case.     

Polymorphism of fecundity genes (FecB, FecX, and FecG) in the Indian Bonpala sheep

The present study was designed for screening polymorphism of known fecundity genes in prolific Indian Bonpala sheep. Employing tetra-primer amplification refractory mutation system PCR, 11-point mutations of BMP1B, BMP15, and GDF9 genes of 97 Bonpala ewes were genotyped. The FecB locus of the BMPR1B gene and two loci (G1 and G4) of GDF9 gene were found to be polymorphic. In FecB locus, three genotypes, namely, wild type (Fec++, 0.02), heterozygous (FecB+, 0.23), and mutant (FecBB, 0.75) were detected. At G1 locus of GDF9 gene, three genotypes, namely, wild type (GG, 0.89), heterozygous (GA, 0.10), and mutant (AA, 0.01) were detected. At G4 locus of GDF9 gene, three genotypes, namely, wild type (AA, 0.01), heterozygous (AG, 0.14), and mutant (GG, 0.85) were detected. Statistically no significant correlation of polymorphism of FecB, G1, and G4 loci and litter size was found in this breed. All five loci of BMP15 and three loci of GDF 9 genes were monomorphic. This study reports Bonpala sheep as the first sheep breed where concurrent polymorphism at three important loci (FecB, G1, and G4) of two different fecundity genes (BMPR1B and GDF9) has been found.