A Self‐immolative Molecular Beacon for Amplified Nucleic Acid Detection

Fluorogenic hybridization probes allow the detection of RNA and DNA sequences in homogeneous solution. Typically, one target molecule activates the fluorescence of a single probe molecule. This limits the sensitivity of nucleic acid detection. Herein, we report a self‐immolative molecular beacon (iMB) that escapes the one‐target/one‐probe paradigm. The iMB probe includes a photoreductively cleavable N‐alkyl‐picolinium (NAP) linkage within the loop region. A fluorophore at the 5’‐end serves, on the one hand, as a reporter group and, on the other hand, as a photosensitizer of a NAP‐linker cleavage reaction. In the absence of target, the iMB adopts a hairpin shape. Quencher groups prevent photo‐induced cleavage. The iMB opens upon hybridization with a target, and both fluorescent emission as well as photo‐reductive cleavage of the NAP linker can occur. In contrast to previous chemical amplification reactions, iMBs are unimolecular probes that undergo cleavage leading to products that have lower target affinity than the probes before reaction. Aided by catalysis, the method allowed the detection of 5 pm RNA target within 100 min.h2020 european research council http://dx.doi.org/10.13039/100010663Peer Reviewe

Toward conditional control of Smac mimetic activity by RNA‐templated reduction of azidopeptides on PNA or 2′‐OMe‐RNA

Oligonucleotide templated reactions can be used to control the activity of functional molecules based on the presence of a specific trigger sequence. We report an RNA‐controlled reaction system to conditionally restore the N‐terminal amino group and thus binding affinity of azide‐modified Smac mimetic compounds (SMCs) for their target protein X‐linked Inhibitor of Apoptosis Protein (XIAP). Two templated reactions were compared: Staudinger reduction with phosphines and a photocatalytic reaction with Ru(bpy)2(mcbpy). The latter proved faster and more efficient, especially for the activation of a bivalent SMC, which requires two consecutive reduction steps. The templated reaction proceeds with turnover when 2′‐OMe‐RNA probes are used, but is significantly more efficient with PNA, catalyzing a reaction in the presence of low, substoichiometric amounts (1%‐3%, 10 nM) of target RNA.European Research Council, Horizon 2020 ProgrammePeer Reviewe

Dissecting the role of protein phosphorylation: a chemical biology toolbox

Protein phosphorylation is a crucial regulator of protein and cellular function, yet, despite identifying an enormous number of phosphorylation sites, the role of most is still unclear. Each phosphoform, the particular combination of phosphorylations, of a protein has distinct and diverse biological consequences. Aberrant phosphorylation is implicated in the development of many diseases. To investigate their function, access to defined protein phosphoforms is essential. Materials obtained from cells often are complex mixtures. Recombinant methods can provide access to defined phosphoforms if site-specifically acting kinases are known, but the methods fail to provide homogenous material when several amino acid side chains compete for phosphorylation. Chemical and chemoenzymatic synthesis has provided an invaluable toolbox to enable access to previously unreachable phosphoforms of proteins. In this review, we selected important tools that enable access to homogeneously phosphorylated protein and discuss examples that demonstrate how they can be applied. Firstly, we discuss the synthesis of phosphopeptides and proteins through chemical and enzymatic means and their advantages and limitations. Secondly, we showcase illustrative examples that applied these tools to answer biological questions pertaining to proteins involved in signal transduction, control of transcription, neurodegenerative diseases and aggregation, apoptosis and autophagy, and transmembrane proteins. We discuss the opportunities and challenges in the field.Peer Reviewe

El fútbol como negocio: una introducción a la gestión deportiva en Europa y Brasil

Uno de los aportes de este libro es el énfasis que los autores ponen en el carácter global del negocio del fútbol y en el análisis de cada uno de los actores que están implicados en este tipo de empresa: desde los dirigentes y gerentes de los clubes, hasta los consumidores a nivel local, pasando por los jugadores, las empresas de telecomunicaciones y de informática, y las marcas deportivas. Además, como lo señala Rory Miller, en el negocio globalizado del fútbol existen perdedores y ganadores. Revertir la primera de estas posiciones requiere de algo más que cambiar las formas de propiedad, como explican los capítulos de Carles Murillo y Oliver Seitz, autores que han ocupado puestos en la gerencia de los clubes que analizan: el FC Barcelona y el Coritiba Foot Ball Club, respectivamente

The integrated 3-point correlation function of cosmic shear

We present the integrated 3-point shear correlation function $i\zeta_{\pm}$ -- a higher-order statistic of the cosmic shear field -- which can be directly estimated in wide-area weak lensing surveys without measuring the full 3-point shear correlation function, making this a practical and complementary tool to 2-point statistics for weak lensing cosmology. We define it as the 1-point aperture mass statistic $M_{\mathrm{ap}}$ measured at different locations on the shear field correlated with the corresponding local 2-point shear correlation function $\xi_{\pm}$. Building upon existing work on the integrated bispectrum of the weak lensing convergence field, we present a theoretical framework for computing the integrated 3-point function in real space for any projected field within the flat-sky approximation and apply it to cosmic shear. Using analytical formulae for the non-linear matter power spectrum and bispectrum, we model $i\zeta_{\pm}$ and validate it on N-body simulations within the uncertainties expected from the sixth year cosmic shear data of the Dark Energy Survey. We also explore the Fisher information content of $i\zeta_{\pm}$ and perform a joint analysis with $\xi_{\pm}$ for two tomographic source redshift bins with realistic shape-noise to analyse its power in constraining cosmological parameters. We find that the joint analysis of $\xi_{\pm}$ and $i\zeta_{\pm}$ has the potential to considerably improve parameter constraints from $\xi_{\pm}$ alone, and can be particularly useful in improving the figure of merit of the dynamical dark energy equation of state parameters from cosmic shear data.Comment: Accepted for publication in MNRAS; v2 matches the accepted manuscript; 18 pages + appendi

Towards a templated reaction that translates RNA in cells into a proaptotic peptide‐PNA conjugate

Nucleic acid-templated chemistry opens the intriguing prospect of triggering the synthesis of drugs only in diseased cells. Herein, we explore the feasibility of using RNA-templated chemical reactions for the activation of a known Smac peptidomimetic compound (SMC), which has proapoptotic activity. Two peptide nucleic acid (PNA) conjugates were used to enable conditional activation of a masked SMC by reduction of an azide either by Staudinger reduction or catalytic photoreduction using a ruthenium complex. The latter provided ~135 nM SMC-PNA on as little as 10 nM (0.01 eq.) template. For the evaluation of the templated azido-SMC reduction system in cellulo, a stable HEK 293 cell line was generated, which overexpressed a truncated, non-functional form of the XIAP mRNA target. We furthermore describe the development of electroporation protocols that enable a robust delivery of PNA conjugates into HEK 293 cells. The action of the reactive PNA conjugates was evaluated by viability and flow cytometric apoptosis assays. In addition, electroporated probes were re-isolated and analyzed by ultra-high performance liquid chromatography (UPLC). Unfortunately, the ruthenium-PNA conjugate proved phototoxic, and treatment of cells with PNA-linked reducing agent and the azido-masked SMC conjugate did not result in a greater viability loss than treatment with scrambled sequence controls. Intracellular product formation was not detectable. A control experiment in total cellular RNA isolate indicated that the templated reaction can in principle proceed in a complex system. The results of this first-of-its-kind study reveal the numerous hurdles that must be overcome if RNA molecules are to trigger the synthesis of pro-apoptotic drugs inside cells.European Research Council's Horizon 2020Peer Reviewe

Cosmology from the integrated shear 3-point correlation function: simulated likelihood analyses with machine-learning emulators

The integrated shear 3-point correlation function $\zeta_{\pm}$ measures the correlation between the local shear 2-point function $\xi_{\pm}$ and the 1-point shear aperture mass in patches of the sky. Unlike other higher-order statistics, $\zeta_{\pm}$ can be efficiently measured from cosmic shear data, and it admits accurate theory predictions on a wide range of scales as a function of cosmological and baryonic feedback parameters. Here, we develop and test a likelihood analysis pipeline for cosmological constraints using $\zeta_{\pm}$. We incorporate treatment of systematic effects from photometric redshift uncertainties, shear calibration bias and galaxy intrinsic alignments. We also develop an accurate neural-network emulator for fast theory predictions in MCMC parameter inference analyses. We test our pipeline using realistic cosmic shear maps based on $N$-body simulations with a DES Y3-like footprint, mask and source tomographic bins, finding unbiased parameter constraints. Relative to $\xi_{\pm}$-only, adding $\zeta_{\pm}$ can lead to $\approx 10-25\%$ improvements on the constraints of parameters like $A_s$ (or $\sigma_8$) and $w_0$. We find no evidence in $\xi_{\pm} + \zeta_{\pm}$ constraints of a significant mitigation of the impact of systematics. We also investigate the impact of the size of the apertures where $\zeta_{\pm}$ is measured, and of the strategy to estimate the covariance matrix ($N$-body vs. lognormal). Our analysis solidifies the strong potential of the $\zeta_{\pm}$ statistic and puts forward a pipeline that can be readily used to improve cosmological constraints using real cosmic shear data.Comment: 21 pages, 11 figures, 3 tables. Comments welcom

Enhancing Auxiliary-Mediated Native Chemical Ligation at Challenging Junctions with Pyridine Scaffolds

To expand the scope of native chemical ligation (NCL) beyond reactions at cysteine, ligation auxiliaries are appended to the peptide N-terminus. After the introduction of a pyridine-containing auxiliary, which provided access to challenging junctions (proline or β-branched amino acids), we herein probe the role of the pyridine-ring nitrogen. We observed side reactions leading to preliminary auxiliary loss. We describe a new easy to attach β-mercapto-β-(4-methoxy-2-pyridinyl)-ethyl (MMPyE) auxiliary, which 1) has increased stability; 2) enables NCL at sterically encumbered junctions (e. g., Leu-Val); and 3) allows removal under mildly basic (pH 8.5) conditions was introduced. The synthesis of a 120 aa long peptide containing eight MUC5AC tandem repeats via ligation of two 60mers demonstrates the usefulness. Making use of hitherto unexplored NCL to tyrosine, the MMPyE auxiliary provided access to a head-to-tail-cyclized 21-mer peptide and a His6-tagged hexaphosphorylated peptide comprising 6 heptapeptide repeats of the RNA polymerase II C-terminal domain.Peer Reviewe