Combination of aptamer and drug for reversible anticoagulation in cardiopulmonary bypass

Content introduction:

• Combination of aptamer and drug for reversible anticoagulation in cardiopulmonary bypass


• De novo DNA synthesis using polymerase-nucleotide conjugates


• Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos


• Precise, automated control of conditions for high-throughput growth of yeast and bacteria with eVOLVER


• Encoding human serine phosphopeptides in bacteria for proteome-wide identification of phosphorylation-dependent interactions

1. Combination of aptamer and drug for reversible anticoagulation in cardiopulmonary bypass
Unfractionated heparin (UFH), the standard anticoagulant for cardiopulmonary bypass (CPB) surgery, carries a risk of post-operative bleeding and is potentially harmful in patients with heparin-induced thrombocytopenia–associated antibodies. To improve the activity of an alternative anticoagulant, the RNA aptamer 11F7t, Ruwan Gunaratne at Duke University in Durham, North Carolina, USA and his colleagues solved X-ray crystal structures of the aptamer bound to factor Xa (FXa). The finding that 11F7t did not bind the catalytic site suggested that it could complement small-molecule FXa inhibitors. They demonstrate that combinations of 11F7t and catalytic-site FXa inhibitors enhance anticoagulation in purified reaction mixtures and plasma. Aptamer–drug combinations prevented clot formation as effectively as UFH in human blood circulated in an extracorporeal oxygenator circuit that mimicked CPB, while avoiding side effects of UFH. An antidote could promptly neutralize the anticoagulant effects of both FXa inhibitors. Their results suggest that drugs and aptamers with shared targets can be combined to exert more specific and potent effects than either agent alone.



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2. De novo DNA synthesis using polymerase-nucleotide conjugates
Oligonucleotides are almost exclusively synthesized using the nucleoside phosphoramidite method, even though it is limited to the direct synthesis of ∼200 mers and produces hazardous waste. Here, Sebastian Palluk at Joint BioEnergy Institute in California, USA and his colleagues describe an oligonucleotide synthesis strategy that uses the template-independent polymerase terminal deoxynucleotidyl transferase (TdT). Each TdT molecule is conjugated to a single deoxyribonucleoside triphosphate (dNTP) molecule that it can incorporate into a primer. After incorporation of the tethered dNTP, the 3′ end of the primer remains covalently bound to TdT and is inaccessible to other TdT–dNTP molecules. Cleaving the linkage between TdT and the incorporated nucleotide releases the primer and allows subsequent extension. They demonstrate that TdT–dNTP conjugates can quantitatively extend a primer by a single nucleotide in 10–20 s, and that the scheme can be iterated to write a defined sequence. This approach may form the basis of an enzymatic oligonucleotide synthesizer.

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3. Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos
Rapid, efficient generation of knock-in mice with targeted large insertions remains a major hurdle in mouse genetics. Here, Bin Gu at Hospital for Sick Children in Toronto, Ontario, Canada and his colleagues describe two-cell homologous recombination (2C-HR)-CRISPR, a highly efficient gene-editing method based on introducing CRISPR reagents into embryos at the two-cell stage, which takes advantage of the open chromatin structure and the likely increase in homologous-recombination efficiency during the long G2 phase. Combining 2C-HR-CRISPR with a modified biotin–streptavidin approach to localize repair templates to target sites, they achieved a more-than-tenfold increase (up to 95%) in knock-in efficiency over standard methods. They targeted 20 endogenous genes expressed in blastocysts with fluorescent reporters and generated reporter mouse lines. They also generated triple-color blastocysts with all three lineages differentially labeled, as well as embryos carrying the two-component auxin-inducible degradation system for probing protein function. They suggest that 2C-HR-CRISPR is superior to random transgenesis or standard genome-editing protocols, because it ensures highly efficient insertions at endogenous loci and defined 'safe harbor' sites.

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4. Precise, automated control of conditions for high-throughput growth of yeast and bacteria with eVOLVER
Precise control over microbial cell growth conditions could enable detection of minute phenotypic changes, which would improve our understanding of how genotypes are shaped by adaptive selection. Although automated cell-culture systems such as bioreactors offer strict control over liquid culture conditions, they often do not scale to high-throughput or require cumbersome redesign to alter growth conditions. Brandon G Wong at Boston University in Boston, Massachusetts, USA and his colleagues report the design and validation of eVOLVER, a scalable do-it-yourself (DIY) framework, which can be configured to carry out high-throughput growth experiments in molecular evolution, systems biology, and microbiology. High-throughput evolution of yeast populations grown at different densities reveals that eVOLVER can be applied to characterize adaptive niches. Growth selection on a genome-wide yeast knockout library, using temperatures varied over different timescales, finds strains sensitive to temperature changes or frequency of temperature change. Inspired by large-scale integration of electronics and microfluidics, they also demonstrate millifluidic multiplexing modules that enable multiplexed media routing, cleaning, vial-to-vial transfers and automated yeast mating.

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5. Encoding human serine phosphopeptides in bacteria for proteome-wide identification of phosphorylation-dependent interactions
Post-translational phosphorylation is essential to human cellular processes, but the transient, heterogeneous nature of this modification complicates its study in native systems. Karl W Barber at Yale University in New Haven, Connecticut, USA and his colleagues developed an approach to interrogate phosphorylation and its role in protein-protein interactions on a proteome-wide scale. They genetically encoded phosphoserine in recoded E. coli and generated a peptide-based heterologous representation of the human serine phosphoproteome. They designed a single-plasmid library encoding >100,000 human phosphopeptides and confirmed the site-specific incorporation of phosphoserine in >36,000 of these peptides. They then integrated their phosphopeptide library into an approach known as Hi-P to enable proteome-level screens for serine-phosphorylation-dependent human protein interactions. Using Hi-P, they found hundreds of known and potentially new phosphoserine-dependent interactors with 14-3-3 proteins and WW domains. These phosphosites retained important binding characteristics of the native human phosphoproteome, as determined by motif analysis and pull-downs using full-length phosphoproteins. This technology can be used to interrogate user-defined phosphoproteomes in any organism, tissue, or disease of interest.

Read more, please click https://www.nature.com/articles/nbt.4150