Learn more about UC TechAlerts – Subscribe to categories and get notified of new UC technologies

Browse Category: Research Tools > Reagents

Categories

[Search within category]

Novel Assay Using Azide-Capture Agents

Prof. Min Xue from the University of California, Riverside and Prof. Wei Wei from the Institute for Systems Biology have developed materials and  methods to detect and measure FA uptake alone or simultaneously with protein detection in multiplex down to single-cell resolution. FA analogs with an azide functional group mimics natural FAs. Specially designed small polymers are used to efficiently assay the FA analogs and produce fluorescent or chemical signals upon binding. The technology is compatible with protein analysis and generally applicable to other metabolites and proteins. Fig 1: Schematic of the UCR-ISB method for detecting fatty acid uptake from single cells.  

Compositions and Methods for Modification of Cells

New chemistries are emerging for the direct attachment of complex molecules to cell surfaces. Chemistries that modify cells must perform under a narrow set of conditions in order to maintain cell viability. They must proceed in buffered aqueous media at the optimal physiological pH—typically pH 7.4—and within a temperature range of 4 – 37 ºC. Furthermore, these reactions must have sufficiently rapid kinetics to achieve high conversion even when confronted with the limits of surface diffusion characteristics. Due to these requirements, few chemistries exist that can attach molecules and proteins to live cells.  There is a need for improved methods of attaching proteins to living cells.   UC researchers have developed a convenient enzymatic strategy for the modification of cell surfaces for targeted immunotherapy applications.  

Peptides for Macromolecular Delivery

This invention includes: 1) Applying amphiphilic peptides (AP) (e.g. E5-TAT, INF7-TAT, or similar peptides) in novel scenarios for delivering CRISPR-Cas9 RNPs and performing genome editing in primary human cells of substantial clinical value (human pluripotent stem cells [HSPCs], T cells) and mouse neuronal progenitor cells in vitro or in vivo. These peptides have also been used for delivering plasmid DNA as a DNA vaccine into human cells in culture, and into mouse tissue in vivo to produce a robust immune response.  2) Novel peptide sequences (derivatives of E5-TAT or INF7-TAT) with improved properties and/or improved activity (relative to the founder peptides) in delivering cargo into target cells. The inventors have created a library of related sequences, all with distinct activity in delivering cargo to different cell types. These novel peptide sequences have been applied to the same scenarios as above, with improved outcomes compared to the parent peptides, E5-TAT or INF7-TAT, in genome editing, and may provide benefits in delivery of plasmid DNA as well. This is especially valuable when delivering to cell types that are notoriously difficult to transduce, such as HSPCs and T cells, leading to new therapeutic opportunities. Background Biological macromolecules offer great potential as therapeutics but the greatest hurdle that remains is their efficient cell entry into target cell types. Cell entry is limiting for two very promising technologies, DNA vaccines and genome editing, and this invention aims to address the unmet need. DNA vaccines would provide a rapid and inexpensive approach to vaccination for a wide range of viral pathogens, but have been hampered by poor delivery of the DNA into the target cells, resulting in an immune response not potent enough for effective vaccination. DNA vaccines involve delivering a plasmid which encodes a viral protein into the nucleus of human cells, where it can be transcribed and then expressed to be recognized by the immune system. In order to improve their efficacy, DNA vaccines have been delivered via in vivo electroporation, a painful process that requires specialized equipment and repeat dosing. Genome editing holds immense therapeutic promise for correcting the genetic mutations underlying disease, or for preventing or treating non-genetic disease. Delivery of the genome editing enzymes, such as CRISPR-Cas9, into the cytosol or nuclei of cells in need of manipulation remains the largest hurdle. Delivering CRISPR-Cas9 as a ribonucleoprotein (RNP) complex offers many advantages compared to other approaches (e.g. the use of viral vectors or lipid nanoparticles), but the RNP lacks an inherent method of cell entry. Amphiphilic peptides (AP) enable transduction of macromolecules into cells and therefore the inventors have aimed to apply APs to delivering cargo such as CRISPR-Cas9 RNPs as well as plasmid DNA into target cell types. The activity of a specific AP in delivering cargo into a the cytosol of a cell is often dependent on the specific cargo being delivered as well as the specific cell type. Therefore, the inventors have created libraries of related APs with diversity in their amino acid sequence in order to allow delivery of macromolecular cargo to a range of cell types, dependent on the specific application. A peptide sequence derived from influenza hemagluttinin sequence, HA2, has been previously described and applied as an endosomolytic peptide (ELP). When the HA2 sequence is appended with TAT, a positively charged cell penetrating peptide sequence derived from the HIV TAT protein, the fusion peptide “HA2-TAT” is able to deliver macromolecular cargo across the cell membrane and also act as an ELP to allow endosomal escape. Derivatives of the HA2-TAT sequence with changes in the amino acid sequence of the peptide, such as the peptide “E5-TAT,” has improved properties compared to HA2-TAT in solubility as well as delivering cargo into cells. The INF7-TAT peptide has similar properties, where INF7 is another glutamine-rich analog of HA2 with improved properties for endosomal escape. HA2 and its derivates (E5, INF7) with and without fusion to cell penetrating peptides (HA2-TAT, E5-TAT, INF7-TAT) have been applied to delivering macromolecular cargo such as proteins and nucleic acids into cells.

One-Pot Multienzyme Synthesis of Sialidase Reagents, Probes and Inhibitors

Researchers at the University of California, Davis, have developed an environmentally friendly one-pot multienzyme (OPME) method for synthesizing sialidase reagents, probes, and inhibitors.

(SD2020-306) Monitoring mRNA Translation by RNA Modifications -STAMP (Surveying Targets by APOBEC-Mediated Profiling)

RNA-binding proteins (RBPs) play essential roles in gene expression and other cellular functions. Thus their identification and the understanding of their mechanisms of action and regulation is key to unraveling physiology and disease. To measure translation efficiency and different steps of ribosome recruitment, the state of the art is ribosome profiling (or Ribo‐seq) and polysome profiling which uses millions of cells, sucrose gradients, centrifugation and often requires the removal of ribosomal RNA as part of the sequencing library preparation as it contaminates more than 50% of most ribosome/polysome libraries. Also, we cannot distinguish full length isoforms here, as the ribosome‐fragments are short.

Facile, Excitation-Based Spectral Microscopy For Fast Multicolor Imaging And Quantitative Biosensing

The number of color channels that can be concurrently probed in fluorescence microscopy is severely limited by the broad fluorescence spectral width. Spectral imaging offers potential solutions, yet typical approaches to disperse the local emission spectra notably impede the attainable throughput.    UC Berkeley researchers have discovered methods and systems for simultaneously imaging up to 6 subcellular targets, labeled by common fluorophores of substantial spectral overlap, in live cells at low (~1%) crosstalks and high temporal resolutions (down to ~10 ms), using a single, fixed fluorescence emission detection band. 

Improved Growth of Stem Cells in Culture

Prof. Talbot and her colleagues from the University of California, Riverside have developed a research tool to prolong the viability and pluripotency of stem cells in culture. The culture medium is supplemented with an additive that includes a source of acetate ions, a carboxylic acid, or a salt of the carboxylic acid, or a combination of these substances. Results have shown that this substrate medium allows for less stem cell death, faster colony growth, and causes cells to attach to and spread faster on the substrate. This provides tremendous advantages in stem cell colony morphology, growth, survival, maintenance of pluripotency, and dynamic behavior when compared to existing media.  Fig 1: Images of stem cells in culture before and after treatment  

Development of Methods and Assay for Measurement of Total Oxidized Phospholipid (OxPL)

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease in the United States. It can be broadly sub-classified into nonalcoholic fatty liver (NAFL), which is thought to have minimal risk of progression to cirrhosis, and nonalcoholic steatohepatitis (NASH), which is thought to have an increased risk of progression to cirrhosis. The current diagnostic gold standard for differentiating whether a patient with NAFLD has NAFL versus NASH is liver biopsy. However, liver biopsy is an invasive procedure, which is limited by sampling variability, cost, and may be complicated by morbidity and even death, although rare. Accurate, non-invasive, biomarkers for the detection of liver disease and liver disease progression e.g., progression to NASH, are currently also not available.

Monoclonal Antibodies Specific to Canine PD-1 and PD-L1

Researchers at the University of California, Davis have developed monoclonal antibodies with multiple applications relevant to canine PD-1 and PD-L1.

Kelch Like Family Member 11 (Klhl11) Autoantibodies As Markers Of Seminoma Associated Paraneoplastic Encephalitis In Men

Researchers at UCSF and Chan Zuckerberg Biohub have discovered a novel biomarker for an autoimmune disease that affects patients with testicular cancer.  The disease, known as “testicular cancer-associated paraneoplastic encephalitis,” can cause severe neurological symptoms.  The symptoms include loss of limb control, eye movement, and in some cases, speech.  The disease begins with testicular cancer, which in some cases causes the immune system to attack the brain.  Affected patients are often misdiagnosed and appropriate treatment is delayed. 

(SD2019-232) Technologies that can be Used to Selectively Bind Messenger RNA and Enhance Protein Translation

Control of gene expression is a general approach to treat diseases where there is too much or too little of a gene product. However, while there are many methods which are available to downregulate the expression of messenger RNA transcripts, very few strategies can upregulate the endogenous gene product. The vast majority of gene regulatory drugs which are commercially available or being developed are designed to knockdown gene expression (i.e. siRNAs, miRNAs, anti-sense, etc.). There exist some methods to enhance gene expression, such as the delivery of messenger RNAs; although, therapeutic delivery of such large and charged RNA molecules is technically challenging, inefficient, and may not be practical. There are also classical gene therapy approaches where a gene product is delivered as viral-encoded products (AAV or lentivirus-packaged). However, these methods suffer from not being able to accurately reproduce the correct alternatively spliced isoforms in the right ratios in cells.  

Hydrodealkenylative C(Sp3)–C(Sp2) Bond Scission

UCLA researchers in the Department of Chemistry and Biochemistry have developed a new chemical reaction that combines ozone, an iron salt, and a hydrogen atom donor to enable hydrodealkenylative cleavage of C(sp3)–C(sp2) bonds in a widely applicable manner.

Drug Repurposing To Explore Novel Treatment For Cushing Disease

UCLA researchers in the Department of Medicine and the Department of Molecular and Medicinal Pharmacology have identified several small molecule reagents to treat Cushing disease.

Development of Novel Fluorescent Puromycin Derivatives

Puromycin is an aminonucleoside antibiotic produced by the bacterium Streptomyces alboniger. Its mode of action is to inhibit protein synthesis by disrupting peptide transfer on ribosomes, leading to premature chain termination during protein translation. Puromycin blocks protein synthesis in both eukaryotes and prokaryotes and is routinely used as a research tool in cell culture. The native Puromycin is also used assays such as mRNA display. As such, derivatives have been synthesized in which the amino acid of the 3' end of adenosine based antibiotics is altered to change the compound's antibiotic activity. Other compounds have been synthesized with differing amino acids and functionalities to examine the effect it has on bacterial viability. The majority do not show useful absorption or emission profiles. What is needed is a method to track the compounds in biological systems.

Rapid, Sensitive Detection of Nucleic Acid Sequences in Environmental Samples

UCLA Researchers at the California NanoSystems Institute have developed a methodology that permits PCR-based detection of nucleic acid sequences in soil that does not require the isolation of DNA.

Reagent to Label Proteins via Lysine Isopeptide Bonds

Researchers in the UCLA Department of Chemistry and Biochemistry and the University of Texas-Medical Center, Houston Department of Microbiology and Molecular Genetics have modified the Corynebacterium diphtheriae (C. diphtheriae) sortase enzyme so that it can be used as a bioconjugation reagent in vitro.

Electrochemical Flash Fluorination and Radiofluorination

Researchers led by Saman Sadeghi from the Department of Molecular & Medical Pharmacology at UCLA have developed a new and simple process to make fluorinated organic compounds.

Protein-Coated Microparticles For Protein Standardization In Single-Cell Assays

Single-cell analysis offers powerful capabilities of identification of rare sub-populations of cells, understanding heterogeneity of cancerous tumors, and tracking cell differentiation and reprogramming. Despite great potentials for uncovering new biological systems and targeting diseases with precision medicine, single-cell approaches are composed of complex device processes that can cause bias in measurement.  In deep sequencing, technical variation in single cell expression data occurs during capture and pre-amplification steps. Similarly, in single-cell protein assays, technical variability can obscure functionally relevant variance.    To better control protein measurement quality in single-cell assays, researchers at the University of California, Berkeley developed a novel method to loading and release protein standard. This method utilizes the surface of modified and functionalized microparticles as vehicles to capture target proteins with desired concentrations. Chelation-assisted click chemistry is applied to demonstrate that protein standards with different molecular masses can be loaded and bounded in a single-cell protein assay. Microparticles are introduced into single-cell devices by either passive gravity, magnetic attraction, or other physicochemical forces. These protein standards from microparticles provide a reference to measure protein mass sizes from individual cells and a quality control for any biases in device fabrication, cell lysis, protein solubility, protein capture, and protein readouts (i.e. antibody probing).   

Scar Minimization Treatment: Fibrotic to Fat Cell Conversion

Clinical treatment for scar-less wound healing remains a highly desired, yet unmet need. UCI researchers have developed a method to minimize scarring during wound healing through cellular reprograming that encourages formation of new skin fat cells. This novel therapy is non-surgical and applicable to multiple types of scars and aging skin.

Methods for Enhancing Cell Populations for Articular Cartilage Repair

Cartilage lesion treatments require expanding cells from healthy donor cartilage which have limited availability and restricted potential to produce cartilage. This invention overcomes these challenges, presenting chemical and physical methods for enhancing cell populations capable of producing neocartilage. According to a 2015 global market report, tissue engineering technologies are expected to reach over 94B USD by 2022.

The Brightest, Red-Shifted Luciferase-Luciferin Bioluminescent Pairs

Researchers at the University of California, Riverside, have developed several new luciferase-luciferin pairs that have superior brightness and excellent performance in vitro and in vivo. Through directed evolution of the existing NanoLuc Luciferase and the use of diphenylterazine (DTZ) as a substrate, the emission extensity is more than doubled compared to NanoLuc-furimazine. Moreover, red-shifted emission of teLuc-DTZ makes it an excellent tool for in vivo imaging. teLuc-DTZ streamlines a variety of applications to afford high sensitivity and reproducibility. Furthermore, fusing teLuc to a fluorescent protein creates the Antares2-DTZ pair, with emissions further red-shifted to the > 600 nm range and 65 times more photons emitted above 600 nm than FLuc-D-Luciferin. Fig. 1 shows the relative emission intensity and the range of emitted wavelengths of light  

17S-FD-895: An Improved Synthetic Splice Modulator mirroring FD-895

The spliceosome, the cellular splicing machinery, regulates RNA splicing of messenger RNA precursors (pre-mRNAs) into maturation of protein coding RNAs. Recurrent mutations and copy number changes in genes encoding spliceosomal proteins and splicing regulatory factors have tumor promoting or suppressive functions in hematological malignancies, as well as some other cancers. Over the past 10 years, a list of natural products, including FD-895, pladienolide B, herboxidiene, and spliceostatin A, have been identified as spliceosome modulators. They have been shown to have anti-cancer effect in vitro and in vivo models. However, these compounds demonstrate poor metabolic stability and short half-lives in vivo, excluding them from entering clinical evaluation. This invention, 17S-FD-895, is an analog of FD-895 and was synthesized through the combination of total synthesis and synthetic methods, demonstrating improved stability and on-target effect. This new spliceosome targeting compound was evaluated in different secondary acute myeloid leukemia models and showed potent efficacy in inhibition of acute myeloid leukemia (AML) LSC and disruption of AML maintenance in vitro and in mouse xenograft models (Crews et al. 2016). The study by Crews et al. indicates the pivotal role of spliceosome in secondary acute myeloid leukemia and the therapeutic potential of targeting leukemia stem cells in this subtype of AML often unresponsive to current therapy.

Modulation Of p53 as a Cancer Therapeutic Target

Researchers at the University of California, Davis have designed peptides and oligonucleotide sequences to enhance p53 expression.

Biologically Applicable Water-Soluble Heterogeneous Catalysts For Parahydrogen-Induced Polarization

UCLA researchers in the Department of Chemistry and Biochemistry have developed a novel method of parahydrogen-induced polarization in water using heterogeneous catalysts.

Sieve Container For Contactless Media Exchange For Cell Growth

Media that contains nutrients and growth factors is necessary to grow all types of cells, a process that is widely used in many fields of research. Such media should be routinely changed either to different media or a fresh batch of the same media. This change currently involves either using a pipette to transfer cells from their current dish of media to a new dish, or aspirating the media out of the dish and replacing it with new media. Both methods have inherent risks to stressing and damaging the cells. Researchers at UCI have developed a unique dish for growing cells that allows for safer aspiration of the old media, which reduces stress and damage to the cells.

  • Go to Page: