Nanopore Whole Transcriptome Analysis and Pathogen Surveillance by a Novel Solid-Phase Catalysis Approach.

The requirement of a large input amount (500 ng) for Nanopore direct RNA-seq presents a major challenge for low input transcriptomic analysis and early pathogen surveillance. The high RNA input requirement is attributed to significant sample loss associated with library preparation using solid-phase reversible immobilization (SPRI) beads. A novel solid-phase catalysis strategy for RNA library preparation to circumvent the need for SPRI bead purification to remove enzymes is reported here.

Direct Metatranscriptome RNA-seq and Multiplex RT-PCR Amplicon Sequencing on Nanopore MinION - Promising Strategies for Multiplex Identification of Viable Pathogens in Food.

Viable pathogenic bacteria are major biohazards that pose a significant threat to food safety. Despite the recent developments in detection platforms, multiplex identification of viable pathogens in food remains a major challenge. A novel strategy is developed through direct metatranscriptome RNA-seq and multiplex RT-PCR amplicon sequencing on Nanopore MinION to achieve real-time multiplex identification of viable pathogens in food.

Type II Restriction of Bacteriophage DNA With 5hmdU-Derived Base Modifications.

To counteract bacterial defense systems, bacteriophages (phages) make extensive base modifications (substitutions) to block endonuclease restriction. Here we evaluated Type II restriction of three thymidine (T or 5-methyldeoxyuridine, 5mdU) modified phage genomes: phage M6 with 5-(2-aminoethyl)deoxyuridine (5-edU), phage ViI (Vi1) with 5-(2-aminoethoxy)methyldeoxyuridine (5-emdU) and phage phi W-14 (a.k.

Solid-phase enzyme catalysis of DNA end repair and 3' A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA.

The use of next-generation sequencing (NGS) has been instrumental in advancing biological research and clinical diagnostics. To fully utilize the power of NGS, complete, uniform coverage of the entire genome is required. In this study, we identified the primary sources of bias observed in sequence coverage across AT-rich regions of the human genome with existing amplification-free DNA library preparation methods.

Enhancing Multistep DNA Processing by Solid-Phase Enzyme Catalysis on Polyethylene Glycol Coated Beads.

Covalent immobilization of enzymes on solid supports provides an alternative approach to homogeneous biocatalysis by adding the benefits of simple enzyme removal, improved stability, and adaptability to automation and high-throughput applications. Nevertheless, immobilized (IM) enzymes generally suffer from reduced activity compared to their soluble counterparts. The nature and hydrophobicity of the supporting material surface can introduce enzyme conformational change, spatial confinement, and limited substrate accessibility, all of which will result in loss of the immobilized enzyme activity.

Single-molecule visualization of a formin-capping protein 'decision complex' at the actin filament barbed end.

Precise control of actin filament length is essential to many cellular processes. Formins processively elongate filaments, whereas capping protein (CP) binds to barbed ends and arrests polymerization. While genetic and biochemical evidence has indicated that these two proteins function antagonistically, the mechanism underlying the antagonism has remained unresolved.

Probing homodimer formation of epidermal growth factor receptor by selective crosslinking.

Ligand binding promotes conformational rearrangement of the epidermal growth factor receptor (EGFR) leading to receptor autophosphorylation and downstream signaling. However, transient interactions between unstimulated EGFR molecules on the cell surface are not fully understood. In this report, we describe the investigation of homodimer formation of EGFR by means of an SNAP-tag based selective crosslinking approach (S-CROSS).

Alternative spliceosome assembly pathways revealed by single-molecule fluorescence microscopy.

Removal of introns from nascent transcripts (pre-mRNAs) by the spliceosome is an essential step in eukaryotic gene expression. Previous studies have suggested that the earliest steps in spliceosome assembly in yeast are highly ordered and the stable recruitment of U1 small nuclear ribonucleoprotein particle (snRNP) to the 5' splice site necessarily precedes recruitment of U2 snRNP to the branch site to form the "prespliceosome." Here, using colocalization single-molecule spectroscopy to follow initial spliceosome assembly on eight different S.

Three-color single molecule imaging shows WASP detachment from Arp2/3 complex triggers actin filament branch formation.

During cell locomotion and endocytosis, membrane-tethered WASP proteins stimulate actin filament nucleation by the Arp2/3 complex. This process generates highly branched arrays of filaments that grow toward the membrane to which they are tethered, a conflict that seemingly would restrict filament growth. Using three-color single-molecule imaging in vitro we revealed how the dynamic associations of Arp2/3 complex with mother filament and WASP are temporally coordinated with initiation of daughter filament growth.

The formin Daam1 and fascin directly collaborate to promote filopodia formation.

Filopodia are slender cellular protrusions that dynamically extend and retract to facilitate directional cell migration, pathogen sensing, and cell-cell adhesion. Each filopodium contains a rigid and organized bundle of parallel actin filaments, which are elongated at filopodial tips by formins and Ena/VASP proteins. However, relatively little is known about how the actin filaments in the filopodial shaft are spatially organized to form a bundle with appropriate dimensions and mechanical properties.

Substrates for improved live-cell fluorescence labeling of SNAP-tag.

The SNAP-tag labeling technology provides a simple, robust, and versatile approach to the imaging of fusion proteins for a wide range of experimental applications. Owing to the specific and covalent nature of the labeling reaction, SNAP-tag is well suited for the analysis and quantification of fused target protein using fluorescence microscopy techniques. In this report, we present our most recent findings on the labeling of SNAP-tag fusion proteins both in vitro and in cell culture with SNAP-tag substrates derived from single regioisomers of carboxyrhodamine dyes.

Near-infrared fluorescence imaging of mammalian cells and xenograft tumors with SNAP-tag.

Fluorescence in the near-infrared (NIR) spectral region is suitable for in vivo imaging due to its reduced background and high penetration capability compared to visible fluorescence. SNAP(f) is a fast-labeling variant of SNAP-tag that reacts with a fluorescent dye-conjugated benzylguanine (BG) substrate, leading to covalent attachment of the fluorescent dye to the SNAP(f). This property makes SNAP(f) a valuable tool for fluorescence imaging.

Two-color STED microscopy in living cells.

Diffraction-unlimited resolution provided by Stimulated Emission Depletion (STED) microscopy allows for imaging cellular processes in living cells that are not visible by conventional microscopy. However, it has so far not been possible to study dynamic nanoscale interactions because multicolor live cell STED microscopy has yet to be demonstrated and suitable labeling technologies and protocols are lacking. Here we report the first realization of two-color STED imaging in living cells.

Development of SNAP-tag fluorogenic probes for wash-free fluorescence imaging.

The ability to specifically attach chemical probes to individual proteins represents a powerful approach to the study and manipulation of protein function in living cells. It provides a simple, robust and versatile approach to the imaging of fusion proteins in a wide range of experimental settings. However, a potential drawback of detection using chemical probes is the fluorescence background from unreacted or nonspecifically bound probes.

Fluorescent site-specific labeling of Escherichia coli expressed proteins with Sfp phosphopantetheinyl transferase.

Fluorescent tagging of proteins has become a critical step in optical analysis of protein function in vitro and in living cells. Here we describe a two-tag system for expression and isolation of a protein of interest from Escherichia coli and subsequent site-specific fluorescent labeling with Sfp phosphopantetheinyl transferase (Sfp synthase). In the example presented, adenoviral protein E3-14.

Site-specific protein labeling by intein-mediated protein ligation.

Intein-mediated protein ligation (IPL) employs an intein to create a protein possessing a C-terminal thioester that can be ligated to a protein or peptide with an amino-terminal cysteine via a native peptide bond. Here we present a procedure to conduct isolation and labeling of recombinant proteins expressed in E. coli using synthetic short peptides possessing a fluorescent moiety.

Protein cyclization enhanced thermostability and exopeptidase-resistance of green fluorescent protein.

A mutant of green fluorescent protein (GFPmut3*) from the jellyfish Aequorea victoria was cyclized in vitro and in vivo by the use of a naturally split intein from the dnaE gene of Synechocystis species PCC6803 (Ssp). Cyclization of GFPmut3* was confirmed by amino acid sequencing and resulted in an increased electrophoretic mobility compared with the linear GFPmut3*. The circular GFPmut3* was 5 degrees C more thermostable than the linear form and significantly more resistant to proteolysis of exopeptidase.

Intein-mediated peptide arrays for epitope mapping and kinase/phosphatase assays.

Synthetic peptides are widely used for production and analysis of antibodies as well as in the study of protein modification enzymes. To circumvent the technical challenges of the existing techniques regarding peptide quantization and normalization, a new method of producing peptide arrays has been developed. This approach utilizes intein-mediated protein ligation that involves linkage of a carrier protein possessing a reactive carboxyl-terminal thioester to a peptide with an amino-terminal cysteine through a native peptide bond.

Purification of green fluorescent protein using a two-intein system.

A two-intein purification system was developed for the affinity purification of GFPmut3*, a mutant of green fluorescent protein. The GFPmut3* was sandwiched between two self-cleaving inteins. This approach avoided the loss of the target protein which may result from in vivo cleavage of a single intein tag.

Design, preparation and use of ligated phosphoproteins: a novel approach to study protein phosphatases by dot blot array, ELISA and Western blot assays.

The study of substrate specificity of protein phosphatases (PPs) is very challenging since it is difficult to prepare a suitable phosphorylated substrate. Phosphoproteins, phosphorylated by a protein kinase, or chemically synthesized phosphopeptides are commonly used substrates for PPs. Both types of these substrates have their advantages and limitations.

Use of intein-mediated phosphoprotein arrays to study substrate specificity of protein phosphatases.

Synthetic peptides incorporating various chemical moieties, for example, phosphate groups, are convenient tools for investigating protein modification enzymes, such as protein phosphatases (PPs). However, short peptides are sometimes poor substrates, and their binding to commonly used matrices is unpredictable and variable. In general, protein substrates for PPs are superior for enzymatic assays, binding to various matrices, and Western blot analysis.

Crystal structures of an intein from the split dnaE gene of Synechocystis sp. PCC6803 reveal the catalytic model without the penultimate histidine and the mechanism of zinc ion inhibition of protein splicing.

The first naturally occurring split intein was found in the dnaE gene of Synechocystis sp. PCC6803 and belongs to a subclass of inteins without a penultimate histidine residue. We describe two high-resolution crystal structures, one derived from an excised Ssp DnaE intein and the second from a splicing-deficient precursor protein.

Preparation of an immunoadsorbent coupled with a recombinant antigen to remove anti-acetylcholine receptor antibodies in abnormal serum.

An immunoadsorbent that removes anti-acetylcholine receptor antibodies (AChRAb) in abnormal serum of myasthenia gravis (MG) patient was efficiently prepared by an expression product, the functional fragment of AChR(alpha205) fused with maltose binding protein (MBP). The ligand can then covalently bind to amylose resin through MBP fusion protein. It was shown from the result of this study with anti-AChR mice sera that the removal rate of AChRAb on this immunoadsorbent reached 87+/-10% (mean value of 10 mice) and the maximally binding capacity of AChRAb was approximately 260 microg/g immunoadsorbent (wet weight).

Recent advances in protein splicing: manipulating proteins in vitro and in vivo.

Protein splicing is an intricate self-catalyzed protein rearrangement that converts an inactive protein precursor to biologically active proteins. In the past decade, mechanistic studies and extensive engineering of the naturally occurring protein splicing elements, termed inteins, has led to the development of numerous novel technologies. These intein-based methodologies permit in vitro and in vivo protein processing in ways previously not possible using traditional biochemical and genetic approaches.