An innovative sulfoxide-containing MS-cleavable cross-linker, DBrASO, specifically designed for cysteine residues and aimed at enhancing protein-protein interactions studies and protein complexes architecture analysis.

An innovative mass spectrometry platform that utilizes sulfoxide-containing MS-cleavable heterobifunctional photoactivated cross-linkers to enhance protein structural elucidation.

Researchers at UC Berkeley have developed methods to deliver biomolecules to plant cells using new plant-derived cell penetrating peptides (CPPs). Despite the revolution in DNA editing that the last decade has brought, plant genetic engineering has not been able to benefit to the same extent. This is due to certain challenges in plant physiology that limit the delivery of exogenous protein cargos, as required in the CRISPR-Cas9 system, primarily due to the plant cell wall. In mammalian cells, for instance, cargo delivery can be accomplished using cell-penetrating peptides (CPPs) which are short peptides that facilitate the transport of cargo molecules through the plasma membrane to the cytosol. While this technology has been optimized in mammalian cells, few have studied the delivery of CPPs in plants to verify whether the cell wall is permissible to these materials. Another barrier to the use of nanotechnologies for plant biomolecule delivery is the lack of quantitative validation of successful intracellular protein delivery. The near universal dependence on confocal microscopy to validate delivery of fluorescent proxy cargoes can be inappropriate for use in plants due to various physiological plant properties, for example intrinsic autofluorescence of plant tissues. Therefore, there exists an unmet need for new materials and methods to deliver biomolecules to plant cells and to confirm the delivery of proteins of varying sizes into walled plant tissues. Stage of Research The inventors have developed methods to deliver proteins into plant cells using cell penetrating peptides which are appropriate for use with CRISPR-Cas9 technology, siRNAs, zinc-finger nucleases, TALENs, and other DNA editing methods. They have also developed a biomolecule fluorophore-based assay to accurately quantitate protein delivery to plants cells.Stage of DevelopmentResearch - in vitro 

Many genomes encode Restriction-Modification systems (RMs) that act to protect the host cell from invading DNA by cutting at specific sites (frequently short 4-6 base reverse complement palindromes). RMs also protect host DNA from unfavorably being cut by modifying sites within the host DNA that could be targets by the host’s own surveillance enzymes. It is also not unusual to find that these enzymes are adjacent to each other in the host genome. Traditional approaches to understanding these sites involve finding a methylase that is typically adjacent to a restriction enzyme, and then extracting DNA, expressing protein and then testing DNA sequence for evidence of cutting. In certain laboratory research (e.g., programs that involve transforming DNA/RNA) it may be desirable to more comprehensively understand the sequences being surveilled by the host. Moreover, it may be desirable in certain laboratory research to know/predict which surveillance enzymes are present in a genome in order to affect cell transformation efficiency through evasion of those sequences.

See background of NCD 32985. 

The identifcation of high-affinity peptide epitopes displayed on MHC-I molecules is an important first step in understanding cell-mediated immune responses and in the development of targeted immunotherapies to treat infections or cancer. This task is typically addressed through the useof highly sensitive mass-spectroscopy approaches and machine learning algorithms. However, this approach is hampered by peptide loss during the upstream purification step. The approach is also hampered by a lack of specificity in purification.  This technology involves the use of peptide-receptive MHC-I molecules in complex made using the TAPBPR chaperone. The peptide receptive MHC-I can be immobilized on chromatography columns or magnetic beads. They can provide unprecedented levels of highly specific peptide recovery 

Biological fluids are complex, with compositions that vary constantly and evade molecular definition. Nevertheless, within these fluids proteins fluctuate, fold, function, and evolve as programmed. Synthetic heteropolymers capable of emulating such interactions would replicate how proteins behave in biological fluids, individually and collectively, leading the way toward synthetic biological fluids. However, while there exist known monomeric sequence requirements, the chemical and sequence characteristics of proteins at the segmental level, rather than the monomeric level, may be the key factor governing how proteins transiently interact with neighboring molecules (and how biological fluids collectively behave). To address this opportunity, UC Berkeley researchers have developed a new process of heteropolymer design for protein stabilization and synthetic mimics of biological fluids. The process leverages chemical characteristics and sequential arrangements along protein chains at the segmental level to design heteropolymer ensembles as mixtures of disordered, partially folded, and folded proteins. In studies, for each heteropolymer ensemble, the level of segmental similarity to that of natural proteins determines its ability to replicate many functions of biological fluids, including: assisting protein folding during translation; preserving the viability of fetal bovine serum without refrigeration; enhancing the thermal stability of proteins; and, behaving like synthetic cytosol under biologically relevant conditions. Molecular studies further translated protein sequence information at the segmental level into intermolecular interactions with a defined range, degree of diversity and temporal and spatial availability.

Single-cell analyses are revolutionizing biomedicine and biology, with genomics (DNA) and transcriptomics (RNA) tools leading the way. At the protein-level, single-cell analyses are limited to mass spectrometry and immunoassays. Neither assay provides comprehensive coverage of proteome for single cells, missing key protein forms (called isoforms).  UC Berkeley researchers have developed a hybrid droplet-electrophoresis device, termed “DropBlot”, to detect proteins from patient-derived tissue biospecimens relevant to clinical medicine and pathology. The DropBlot takes advantage of water-in-oil (W/O) droplets to encapsulate single cells derived from chemically fixed tissues, thus providing a picoliter-volume reaction chamber in which said cells are lysed and subjected to harsh lysis conditions (100ºC, 2 hours), as needed for fixed cells. We report an all-in-one microdevice to facilitate cell-laden droplet loading with >98% microwell occupancy. Droplets remain intact under the electric field and protein isoforms are shown to electromigrate out of the droplet and into a microfluidic separation channel where protein sizing takes place via the action of electrophoresis in a photoactive polyacrylamide (PA) gel. DropBlot has been successfully applied to live and fixed cancer cell lines and resolved proteins with high sensitivity.

Alternative splicing accounts for a considerable portion of transcriptomic diversity, as most protein-coding genes are spliced into multiple mRNA isoforms. However, errors in splicing patterns can give rise to mis-splicing with pathological consequences, such as the congenital diseases familial dysautonomia, Duchenne muscular dystrophy, and spinal muscular atrophy. Small nuclear RNA (snRNA) components of the U snRNP family have been proposed as a therapeutic modality for the treatment of mis-splicing. U1 snRNAs offer great promise, with prior studies demonstrating in vivo efficacy, suggesting additional preclinical development is merited. Improvements in enabling technologies, including screening methodologies, gene delivery vectors, and relevant considerations from gene editing approaches justify further advancement of U1 snRNA as a therapeutic and research tool.

Brief description not available

The main way to reduce the activity of RBPs in cells is through gene expression knockdown (i.e. siRNAs or antisense oligonucleotides). More recently, circular RNAs have been used as a competitive inhibitor of miRNA activity by capturing the Argonaute proteins – which already occurs naturally in cells. There are also no known small molecule inhibitors of RBPs.

Methyl-CpG-binding-protein 2 (MeCP2) is a nuclear protein expressed in all cell types, especially neurons. Mutations in the MECP2 gene cause Rett syndrome (RTT), an incurable neurological disorder that disproportionately affects young girls. Strategies to restore MeCP2 expression phenotypically reverse RTT-like symptoms in male and female MeCP2-deficient mice, suggesting that direct nuclear delivery of functional MeCP2 could restore MeCP2 activity.The inventors have discovered that ZF-tMeCP2, a conjugate of MeCP2(aa13-71, 313-484) and the cell-permeant mini-protein ZF5.3, binds DNA in a methylation-dependent manner and reaches the nucleus of model cell lines intact at concentrations above 700 nM. When delivered to live cells, ZF-tMeCP2 engages the NCoR/SMRT co-repressor complex and selectively represses transcription from methylated promoters. Efficient nuclear delivery of ZF-tMeCP2 relies on a unique endosomal escape portal provided by HOPS-dependent endosomal fusion.In a comparative evaluation, the inventors observed the Tat conjugate of MeCP2 (Tat-tMeCP2) (1) degrades within the nucleus, (2) is not selective for methylated promoters, and (3) traffics in a HOPS-independent manner. These results support the feasibility of a HOPS-dependent portal for delivering functional macromolecules to the cell interior using the cell-penetrant mini-protein ZF5.3. Such a strategy could broaden the impact of multiple families of protein-derived therapeutics.

Researchers at the University of California, Davis have developed a potential drug target for cancer and inflammation by studying the binding of integrins to P-selectin.

Researchers at the University of California, Davis have developed a potential therapeutic treatment for sepsis by modulating the interaction between integrins and Myeloid Differentiation factor 2 (MD-2).

Brief description not available

Researchers at the University of California, Davis have developed de novo positive allosteric modulators (PAMs) that bind to TRPV1 proteins involved with pain-sensing in order to provide analgesic effects.

Researchers at UCI have designed a high-throughput, cost-effective microfluidic platform as a research tool for performing genomic, proteomic, single-cell, pharmacological, and agricultural studies across multiple cell types.

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.

This invention illustrates that the secretome of hESCs, iPSCs and moreover, ALS patients’ iPSCs, robustly protect neuronal cells from apoptosis, diminish mislocalization of TDP43, and significantly improve the formation and maintenance of neurites of ALS-MNs. Such neuro-protection manifests in the genetic and in an acquired neuro-toxicity models. Importantly, administration of CM form ALS-iPSCs (ALS iPSC-CM) to transgenic mice that model human disease (SOD1G93A) prevented MN degeneration, maintained the innervation (neuro-muscular junctions), delayed onset of symptoms, and prolonged lifespan. Comparative proteomics and fractionation of conditioned medium outline specific proteins and fractions that are responsible for this neuroprotection. Translationally, this work suggests the rapid development of a new therapeutic for ALS.

This invention leverages the nuclease activity of CRISPR proteins for the direct, sensitive detection of specific nucleic acid sequences. This all-in-one detection modality includes an internal Nuclease Chain Reaction (NCR), which possesses an amplifying, feed-forward loop to generate an exponential signal upon detection of a target nucleic acid.Cas13 or Cas12 enzymes can be programmed with a guide RNA that recognizes a desired target sequence, activating a non-specific RNase or DNase activity. This can be used to release a detectable label. On its own, this approach is inherently limited in sensitivity and current methods require an amplification of genetic material before CRISPR-base detection. 

Viral infection is a multistep process involving complex interplay between viral life cycle and host immunity. One defense mechanism that hosts use to protect cells against the virus are nucleic-acid-mediated surveillance systems, such as RNA interference-driven gene silencing and CRISPR-Cas mediated gene editing. Another important stage for host cells to combat virus replication is translational regulation, which is particular important for the life cycle of RNA viruses, such as Hepatitis C virus and Coronavirus.  While efforts to characterize structural features of viral RNA have led to a better understanding of translational regulation, no systematical approaches to identify important host genes for controlling viral translation have been developed and little is known about how to regulate host-virus translational interaction to prevent and treat infections caused by RNA viruses.   UC Berkeley researchers have developed a high-throughput platform using CRISPR-based target interrogation to identify new therapeutics targets or repurposed drug targets for blocking viral RNA translation.  The new kits can also be used to identify important domains within target proteins that are required for regulating (viral RNA translation) and can inform drug design and development for treating RNA viruses.

UC Berkeley researchers have created a new technique that can rapidly “print” two-dimensional arrays of cells and proteins that mimic a wide variety of cellular environments in the body, be it the brain tissue surrounding a neural stem cell, the lining of the intestine or liver or the cellular configuration inside a tumor.  In the new technique, each cell or protein is tethered to a substrate with a short string of DNA. While similar methods have been developed that attach tethered cells or proteins one by one.  By repeating the process, up to 10 different kinds of cells or proteins can be tethered to the surface in an arbitrary pattern. This technique could help scientists develop a better understanding of the complex cell-to-cell messaging that dictates a cell’s final fate, from neural stem cell differentiating into a brain cell to a tumor cell with the potential to metastasize to an embryonic stem cell becoming an organ cell.

Mutated versions of Cas12a that remove its non-specific ssDNA cleavage activity without affecting site-specific double-stranded DNA cutting activity. These mutant proteins, in which a short amino acid sequence is deleted or changed, provide improved genome editing tools that will avoid potential off-target editing due to random ssDNA nicking.

This invention provides a means of selectively modulating individual Toll-like receptor (TLR) signaling pathways by introducing mutations in the trafficking protein Unc93b1. The desired cell population (e.g., T cells, dendritic cells) is removed from a patient and maintained in vitro, subjected to genomic editing at precise locations within the coding region of Unc93b1, and then reimplanted into the patient. Depending on the indication to be treated, a given TLR signaling pathway can either be enhanced or inhibited by changing the target sequence in Unc93b1. Uses for this invention include, but are not limited to, 1) boosting immune responses to pathogens or tumors, and 2) initiating tissue repair programs in immune cells.  It includes TLR3, 5, 7, 9.

Researchers in the UCLA Departments of Surgery and Molecular and Medical Pharmacology have developed a novel blood-based assay that can capture and characterize circulating tumor cells indicative of both early- and late-staged hepatocellular carcinoma (HCC).