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Results 1 through 20 out of 2039

in category biochemistry


1: Homology Directed Repair by Cas9:Donor Co-localization in Mammalian Cells

Philip JR Roche, Heidi Gytz et al.

6,397 downloads (posted 16 Jan 2018)

Homology directed repair (HDR) induced by site specific DNA double strand breaks (DSB) with CRISPR/Cas9 is a precision gene editing approach that occurs at low frequency in comparison to indel forming non homologous end joining (NHEJ). In order to obtain high HDR percentages in mammalian cells, we engineered Cas9 protein fused to a high-affinity monoavidin domain to deliver biotinylated donor DNA to a DSB site. In addition, we used the cationic polymer, polyethylenimine, to deliver Cas9 RNP-donor DNA complex into the cell. Combining these strategies improved HDR percentages of up to 90% in three tested loci (CXCR4, EMX1, and TLR) in standard HEK293 cells. Our approach offers a cost effective, simple and broadly applicable gene editing method, thereby expanding the CRISPR/Cas9 genome editing toolbox.


2: CRISPR/Cas9-APEX-mediated proximity labeling enables discovery of proteins associated with a predefined genomic locus in living cells

Samuel A. Myers, Jason Wright et al.

5,918 downloads (posted 04 Jul 2017)

The activation or repression of a gene's expression is primarily controlled by changes in the proteins that occupy its regulatory elements. The most common method to identify proteins associated with genomic loci is chromatin immunoprecipitation (ChIP). While having greatly advanced our understanding of gene expression regulation, ChIP requires specific, high quality, IP-competent antibodies against nominated proteins, which can limit its utility and scope for discovery. Thus, a method able to discover and identify prot...


3: Platform for rapid nanobody discovery in vitro

Conor McMahon, Alexander S Baier et al.

5,125 downloads (posted 16 Jun 2017)

Camelid single-domain antibody fragments ('nanobodies') provide the remarkable specificity of antibodies within a single immunoglobulin VHH domain. This unique feature enables applications ranging from their use as biochemical tools to therapeutic agents. Virtually all nanobodies reported to date have been obtained by animal immunization, a bottleneck restricting many applications of this technology. To solve this problem, we developed a fully in vitro platform for nanobody discovery based on yeast surface display of a ...


4: Enhanced proofreading governs CRISPR-Cas9 targeting accuracy

Janice S Chen, Yavuz S. Dagdas et al.

4,650 downloads (posted 06 Jul 2017)

The RNA-guided CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing. High-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) variants exhibit substantially reduced off-target cleavage in human cells, but the mechanism of target discrimination and the potential to further improve fidelity were unknown. Using single-molecule Förster resonance energy transfer (smFRET) experiments, we show that both SpCas9-HF1 and eSpCas9(1.1) are trapped in an inactive state whe...


5: CRISPR-Cas12a target binding unleashes single-stranded DNase activity

Janice S Chen, Enbo Ma et al.

4,435 downloads (posted 29 Nov 2017)

CRISPR-Cas12a (Cpf1) proteins are RNA-guided DNA targeting enzymes that bind and cut DNA as components of bacterial adaptive immune systems. Like CRISPR-Cas9, Cas12a can be used as a powerful genome editing tool based on its ability to induce genetic changes in cells at sites of double-stranded DNA (dsDNA) cuts. Here we show that RNA-guided DNA binding unleashes robust, non-specific single-stranded DNA (ssDNA) cleavage activity in Cas12a sufficient to completely degrade both linear and circular ssDNA molecules within mi...


6: DNA-dependent RNA cleavage by the Natronobacterium gregoryi Argonaute

Sunghyeok Ye, Taegeun Bae et al.

4,398 downloads (posted 20 Jan 2017)

We show here that, unlike most other prokaryotic Argonaute (Ago) proteins, which are DNA-guided endonucleases, the Natronobacterium gregoryi-derived Ago (NgAgo) can function as a DNA-guided endoribonuclease, cleaving RNA, rather than DNA, in a targeted manner. The NgAgo protein, in complex with 5′-hydroxylated or 5′-phosphrylated oligodeoxyribonucleotides (ODNs) of variable lengths, split RNA targets into two or more fragments in vitro, suggesting its physiological role in bacteria and demonstrating a potential for degr...


7: A chemical biology view of bioactive small molecules and a binder-based approach to connect biology to precision medicines

Stuart L. Schreiber

4,366 downloads (posted 07 Aug 2018)

Evidence is provided that the discovery of small-molecule binders can yield compounds that alter interactomes, protein modifications, cellular lifetimes, and ultimately the specific functions of proteins relevant to human health. These novel mechanisms of action are needed to accelerate the translation of insights from human biology into medicines.


8: Aequorea victoria’s secrets

Talley Lambert, Hadrien Depernet et al.

4,216 downloads (posted 19 Jun 2019)

Using mRNA-Seq and de novo transcriptome assembly, we identified, cloned and characterized nine previously undiscovered fluorescent protein (FP) homologs from Aequorea victoria and a related Aequorea species, with most sequences highly divergent from avGFP. Among these FPs are the brightest GFP homolog yet characterized and a reversibly photochromic FP that responds to UV and blue light. Beyond green emitters, Aequorea species express purple- and blue-pigmented chromoproteins (CPs) with absorbances ranging from green to...


9: Thermal Decomposition Of The Amino Acids Glycine, Cysteine, Aspartic Acid, Asparagine, Glutamic Acid, Glutamine, Arginine And Histidine

Ingrid M Weiss, Christina Muth et al.

4,149 downloads (posted 22 Mar 2017)

Calorimetry, thermogravimetry and mass spectrometry were used to follow the thermal decomposition of the eight amino acids G, C, D, N, E, Q, R and H between 185 °C and 280 °C. Endothermic heats of decomposition between 72 and 151 kJ/mol are needed to form 12 to 70 % volatile products. This process is neither melting nor sublimation. With exception of cysteine they emit mainly H2O, some NH3 and no CO2. Cysteine produces CO2 and little else. The reactions are described by polynomials, AA → a (NH3) + b (H2O) + c (CO2) + d ...


10: Doubling healthy lifespan using drug synergies

Tesfahun Dessale, Krishna Chaithanya Batchu et al.

3,986 downloads (posted 21 Jun 2017)

Pharmacological interventions that target human ageing would extend individual healthspan and result in dramatic economic benefits to rapidly ageing societies worldwide. For such interventions to be contemplated they need to comprise drugs that are efficacious when given to adults and for which extensive human safety data are available. Here we show that dramatic lifespan extension can be achieved in C.elegans by targeting multiple, evolutionarily conserved ageing pathways using drugs that are already in human use. By t...



Marin van Heel, Michael C. Schatz

3,709 downloads (posted 24 Nov 2017)

We are currently facing an avalanche of cryoEM (cryogenic Electron Microscopy) publications presenting beautiful structures at resolution levels of ~3 Angstrom: a true resolution revolution [Kuehlbrandt, Science 343(2014)1443,1444]. Impressive as these results may be, a fundamental statistical error has persisted in the literature that affects the numerical resolution values for practically all published structures. The error goes back to a misinterpretation of basic statistics and pervades virtually all popular cryo EM...


12: Cannabinoid glycosides: In vitro production of a new class of cannabinoids with improved physicochemical properties

Janee’ M. Hardman, Robert T. Brooke et al.

3,503 downloads (posted 30 Jan 2017)

The cannabinoid signaling system has recently garnered attention as a therapeutic target for numerous indications, and cannabinoids are now being pursued as new treatment options in diverse medical fields such as neurology, gastroenterology, pain management, and oncology. Cannabinoids are extremely hydrophobic and relatively unstable compounds, and as a result, formulation and delivery options are severely limited. Enzymatic glycosylation is a strategy to alter the physicochemical properties of small molecules, often im...


13: Modified TCA/acetone precipitation of plant proteins for proteomic analysis

Liangjie Niu, Hang Zhang et al.

3,441 downloads (posted 01 Aug 2018)

Protein extracts obtained from cells or tissues often require removal of interfering substances for the preparation of high-quality protein samples in proteomic analysis. A number of protein extraction methods have been applied to various biological samples. TCA/acetone precipitation and phenol extraction, a common method of protein extraction, is thought to minimize protein degradation and activity of proteases as well as reduce contaminants like salts and polyphenols. However, the TCA/acetone precipitation method reli...


14: A rationally designed and highly versatile epitope tag for nanobody-based purification, detection and manipulation of proteins

Hansjörg Götzke, Markus Kilisch et al.

3,363 downloads (posted 17 May 2019)

Specialized epitope tags are widely used for detecting, manipulating or purifying proteins, but often their versatility is limited. Here, we introduce the ALFA-tag, a novel, rationally designed epitope tag that serves an exceptionally broad spectrum of applications in life sciences while outperforming established tags like the HA, FLAG or myc tags. The ALFA-tag forms a small and stable α-helix that is functional irrespective of its position on the target protein in prokaryotic and eukaryotic hosts. We developed a nanobo...


15: Extracellular 2’3’-cGAMP is an immunotransmitter produced by cancer cells and regulated by ENPP1

Jacqueline A Carozza, Volker Böhnert et al.

3,283 downloads (posted 03 Feb 2019)

2’3’-cyclic GMP-AMP (cGAMP) is characterized as an intracellular second messenger that is synthesized in response to cytosolic dsDNA and activates the innate immune STING pathway. Our previous discovery of its extracellular hydrolase ENPP1 hinted at the existence of extracellular cGAMP. Here, using mass spectrometry, we detected that cGAMP is continuously exported as a soluble factor by an engineered cell line but then efficiently cleared by ENPP1, explaining why it has escaped detection until now. By developing potent,...


16: Structural basis for the RNA-guided ribonuclease activity of CRISPR-Cas13d

Cheng Zhang, Silvana Konermann et al.

3,213 downloads (posted 04 May 2018)

CRISPR-Cas endonucleases directed against foreign nucleic acids mediate prokaryotic adaptive immunity and have been tailored for broad genetic engineering applications. Type VI-D CRISPR systems contain the smallest known family of single effector Cas enzymes, and their signature Cas13d ribonuclease employs guide RNAs to cleave matching target RNAs. To understand the molecular basis for Cas13d function, we resolved cryo-electron microscopy structures of Cas13d-guide RNA binary complex and Cas13d-guide-target RNA ternary ...


17: T cell co-stimulatory receptor CD28 is a primary target for PD-1-mediated inhibition

Enfu Hui, Jeanne Cheung et al.

3,137 downloads (posted 09 Nov 2016)

Programmed death-1 (PD-1) is a co-inhibitory receptor that suppresses T cell activation and is an important cancer immunotherapy target. Upon activation by its ligand PD-L1, PD-1 is thought to suppress signaling through the T cell receptor (TCR). Here, by titrating the strength of PD-1 signaling in both biochemical reconstitution systems and in T cells, we demonstrate that the coreceptor CD28 is strongly preferred over the TCR as a target for dephosphorylation by PD-1- recruited Shp2 phosphatase. We also show that PD-1 ...


18: Live-cell mapping of organelle-associated RNAs via proximity biotinylation combined with protein-RNA crosslinking

Pornchai Kaewsapsak, David M Shechner et al.

2,716 downloads (posted 21 Jun 2017)

The spatial organization of RNA within cells is a crucial factor in a wide range of biological functions, spanning all kingdoms of life. However, a general understanding of RNA localization has been hindered by a lack of simple, high-throughput methods for mapping the transcriptomes of subcellular compartments. Here, we develop such a method, termed APEX-RIP, which combines peroxidase-catalyzed, spatially restricted in situ protein biotinylation with RNA-protein chemical crosslinking. We demonstrate that, using a single...


19: Electrophilic PROTACs that degrade nuclear proteins by engaging DCAF16

Xiaoyu Zhang, Vincent M Crowley et al.

2,664 downloads (posted 15 Oct 2018)

Ligand-dependent protein degradation has emerged as a compelling strategy to pharmacologically control the protein content of cells. So far, only a limited number of E3 ligases have been found to support this process. Here, we use a chemical proteomic strategy to discover that DCAF16 - a poorly characterized substrate recognition component of CUL4-DDB1 E3 ubiquitin ligases - promotes nuclear-restricted protein degradation upon modification by cysteine-directed heterobifunctional electrophilic compounds.


20: The relative values of the turnover number and the dissociation rate constant determine the definition of the Michaelis-constant

Gassan Nazzal

2,483 downloads (posted 11 May 2016)

In this work, we attempt to determine the assumptions of each case of the QSSA. We came to the conclusion that for an enzyme with average kinetics parameters the REA is a good approximation to derive the rate equation and the Km value tends to equal the dissociation constant Kd . The active site classifies the population of the substrate into two energy states, the ground state, and the transition state. The ratio Km/Kd is equal to the partition function of the assumed two-state-system. For the average enzyme , the part...