An engineered polymerase enabling the synthesis of threose nucleic acid (TNA) for advanced therapeutic applications.

This technology introduces a novel class of synthetic genetic polymers, capable of enhancing protein target binding and mimicking antibodies, for therapeutic and diagnostic applications.

Researchers from UC San Diego developed a new technology that facilitates pooling of nuclear expressed antisense RNAs (NEARs) to identify consequential RNA processing events such as alternative or constitutive RNA splicing or polyadenylation.This technology will identify a phenotype of interest and/or a group of RNA processing events (for example RNA splicing sites of interest or alternatively spliced exons), and transduce cells with a library of NEARs targeting these events. Applications include: Normal 0 false false false EN-US X-NONE X-NONE MicrosoftInternetExplorer4 /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-pagination:widow-orphan; font-size:10.0pt; font-family:"Times New Roman",serif;} Research tool. As screens to identify exons of phenotypic relevance in a high throughput manner.Therapeutic target identification. To identify therapeutic targets of cancer cell suppression, such as poison exons in cancer specific transcripts.Therapeutic discovery. As a therapeutic agent to identify therapeutic NEARs for splicing related disorders.  

Existing RNA-targeting tools for sequence-specific manipulation include anti-sense oligos (ASOs), designer PUF proteins and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas systems. However, there are significant limitations to each of the current tools. ASOs are usually not available for most RNA manipulations other than gene silencing. Designer proteins, such as PUF (Pumilio and FBF homology protein), possess low RNA recognition efficiency and it remains challenging to target RNA sequences >8-nucleotides (nt) in length. The bulky Cas protein (Cas13d: average 930 amino acids) leads to complication for transgene delivery and concerns of its immunogenicity due to its bacterial origin. Mutants of zinc finger(ZnF) proteins in ZRANB2 recognize a single-strand RNA containing a novel GGG motif with micromolar affinity, compared to the original motif GGU. These mutants serve as a foundation for RNA-binding ZnF designer protein engineering for in vivo RNA sequence-specific targeting.ZnFs are generally compact domains (~3kDa each) that have been successfully engineered for DNA recognition as modular arrays. A ZnF-based system has unique advantages, especially in a therapeutic context: (1) Broad application with the possibility to fuse with other effector domains; (2) High efficiency of RNA recognition (3 RNA bases recognized per 30-amino-acid ZnF) with a small size of protein. Only 4 ZnFs (~100 aa) is required for specific targeting in the transcriptome. (3) Humanized components without immunogenic concern.By engineering new sequence specificity of the ZRANB2 ZnF1, researchers from UC San Diego identified 13 mutants that altered their preferred RNA binding motif from GGU to GGG. They are N24R, N24H, N14D/N24R, N14D/N24H, N14R/N24R, N14R/N24H, N14H/N24R, N14H/N24H, N14Q/N24R, N14Q/N24H, N14E/N24R, N14S/N24R, N14E/N24H.

Hybridization capture approaches allow targeted high-throughput sequencing analysis at reduced costs compared to shotgun sequencing. Hybridization capture is particularly useful in analyses of genomic data from ancient, environmental, and forensic samples, where target content is low, DNA is fragmented and multiplex PCR or other targeted approaches often fail. Hybridization capture involves the use of "bait" nucleotides that capture genomic sequences that are of particular interest for the researcher. Current bait synthesis methods require large-scale oligonucleotide chemical synthesis and/or in vitro transcription. Both RNA and DNA bait generation requires synthesizing template oligonucleotides using phosphoramidite chemistry. Microarray-based synthesis generates oligonucleotides in femtomole scales with high chemical coupling error rates. Templates synthesized at small-scale require enzymatic amplification before use in hybridization capture.The solution proposed here involves a simple and highly efficient method to generate target probes using isothermal amplification. Target sequences are circularized and then amplified by rolling circle amplification. This method generates concatemers comprising thousands of copies of the target seqeuence. Restriction digestion of the amplified product then produces probes to use in target enrichment applications. 

Researchers at the University of California, Davis have identified DNA methylation biomarkers in placenta, as well as maternal and newborn blood, allowing early autism diagnosis and risk assessment.

A novel bioluminescent platform for in vivo tracking and visualization of RNA dynamics without the need for excitation light.

Professor Hideaki Tsutsui and colleagues from the University of California, Riverside have developed a portable handheld device for nucleic acid extraction. With its high-speed motor, knurled lysis chamber for rapid sample lysis, and quick nucleic acid extraction using paper disks, this device can yield ready-to-use extracts in just 12 minutes, significantly reducing the time required for sample preparation. This technology is advantageous over current methods as it can be expedited without the need for cumbersome specimen collection, packaging, and submission, shortening the turnaround time.  

Researchers at the University of California, Davis have developed a method and composition for modifying genetic sequences using Adenosine deaminases that act on RNA (ADARs).

Although viral delivery of CRISPR genome editors is the most widely used method for in vivo cell editing, viral vectors can be immunogenic, carry the risk of vector genome integration and can induce off-target DNA damage due to continuous genome editor expression. Lipid-nanoparticle (LNP):mRNA complexes are non-virally derived vehicles for in vivo delivery that have provided for genome editing in the liver. However, developing LNP:mRNA complexes that can edit non-liver tissues remains a challenge.  UCB researchers have created new LNP compositions and methods for delivery that have increased efficiency for delivering a molecular payload such as CRISPR-Cas effector proteins, guide RNAs, and/ nucleic acids encoding same. 

Optimizing the editing efficiency of CRISPR-mediated enzymes is still needed.  This is especially true in therapeutic use cases, when it would be ideal to attain high rates of editing via a low, transient dose of the enzyme in the ribonucleoprotein (RNP) format used for multiple ex vivo clinical trials. Because many CRISPR enzymes are of bacterial origin, fusion to NLS motifs can greatly enhance editing efficiency. However, CRISPR protein yields can decrease – sometimes dramatically – if the construct bears toomany NLSs. UC Berkeley researchers have developed CRISPR proteins with enhanced editing efficiencies by introducing multiple nuclear localization signal (NLS) fused at rationally selected sites within the backbone of CRISPR-Cas9. These Cas9 variants showed they can improve editing efficiency in T cells compared to constructs with terminally-fused NLS sequences and can be produced with high purity and yield.  

Understanding the function of RNAs requires visualizing their location and dynamics in live cells. However, direct labeling and imaging individual endogenous RNAs in living cells is still needed. UC Berkeley researchers have developed a method to directly resolve individual endogenous RNA transcripts in living cells using programmable RNA-guided and RNA-targeting CRISPR-Csm complexes coupled with a variety of crRNAs that collectively span along the transcripts of interest.  The researchers demonstrated robust labeling of MAP1B and NOTCH2 mRNAs in several cell lines. We tracked NOTCH2 and MAP1B transcript transient dynamics in living cells, captured distinct mobilities of individual transcripts in different subcellular compartments, and detected translation dependent and independent RNA motions.  

Rapid virus evolution generates proteins essential to infectivity and replication but with unknown function due to extreme sequence divergence. Using a database of 67,715 newly predicted protein structures from 4,463 eukaryotic viral species, it was found that 62% of viral proteins are structurally distinct and lack homologs in the Alphafold database. Structural comparisons suggested putative functions for >25% of unannotated viral proteins.  UC Berkeley researcher have created new single stranded DNA (ssDNA) bindingproteins and double stranded (dsDNA) binding proteins, and methods and compositions for using them, such as binding to target DNA.   

Before recombinant antibody expression plasmids can be designed, sequncing of the antibody light and heavy chain variable regions is necessary. Several other methods of sequencing antibody variable regions are available. Some involve high throughput RNA sequencing. These techniques are unavailable to many labs; they require the preparation of RNA-seq libraries, and computational analysis. As a result, the cost of performing such techniques is substantial and with sequencing cores being oversubscribed, turnaround can be as long as weeks to months. Other methods involve PCR and Sanger sequencing. However PCR amplification of variable regions results from difficulties in generating universal primers that can amplify any given variable region - particularly given the inherent low sequence identity in the 5' leader sequence of antibody light chains and heavy chains upstream of the variable regions. Sometimes degenerate primers can be used, but amplification success rate is only 80-90% due to non-specific priming and/or failure to prime at all. In addition, there is a significant risk that the variable regions of the parental myeloma line can amplify using the degenerate primers. 5' RACE (rapid amplification of 5' cDNA ends) can also be used, but mRNA degradation, cDNA purification and poly-A addition between reverse transcription and PCR, makes the technique long and difficult to perform. Non degenerate primers can be used, but each variable region requires multiple amplification attempts with different primer sets as well as sequence validation using mass spectrometry. And with both of these methods, primer derived mutations can be introduced. Mass spectrometry can be used to determine antibody variable regions, but these can result in ambiguous sequences because of isobaric residues such as isoleucine and leucine. But this method is time consuming, requires huge amounts of purified monoclonal antibody, is expensive and is inaccessible to most researchers. This technology involves a template switch reverse transcription of hybridoma RNA with at least three chain specific RT primers - one for the kappa chain, one for the lambda chain, and at least one for the heavy chain (for efficiency, this can be limited to IgG in a first pass). These are amplified in three separate PCR reactions and sequenced using Sanger sequencing.

mRNA-based cancer therapies include vaccination via mRNA delivery of tumor neoantigens, delivery of mRNA encoding for immune checkpoint and other protein therapeutics, and induced expression of anticancer surface proteins such as CAR expression in T cells. Success requires transfection of a critical number of immune cells together with appropriate immune-stimulation to effectively drive anti-tumor responses. UC Berkeley researchers have developed an adjuvant-assisted mRNA LNP delivery method that uses mRNA LNP and adjuvant to enhance delivery of nucleic acids to immune cells in vivo and stimulate immune cells. They demonstrated the use of this system to reduce mRNA reporter protein expression in the liver and enhance protein expression in the spleen in mice and also demonstrated this system can be used to genetically engineer T cells by delivering a Cre-recombinase mRNA construct- transfection and editing of approximately 4% of T cells is achieved in vivo. The immune response is superior in our system compared to current, commercial lipid nanoparticle delivery technologies.

TnpB protein has generated interest as a potential compact genome-editing tool, due to the short amino acid sequence (408 AAs for ISDra2 TnpB), which overlaps with the wRNA sequence in their genomes of origin. There is a need for compositions and methods that provide more efficient TnpB systems. UC Berkeley researchers have created variant TnpB proteins and variant wRNAs that increase cleavage activity and/or DNA binding activity (e.g., revealed as endonuclease activity such as on-target endonuclease activity). These variant TnpB proteins include an amino acid sequence having one or more amino acid substitutions relative to a corresponding wild type TnpB protein. Also provided are variant TnpB wRNAs that can form a complex with a TnpB protein and a second nucleotide sequence that can hybridize to a target sequence of a target nucleic acid, thereby guiding the complex to the target sequence.

Brief description not available

Various ubiquitin plasmids from Michael Rape lab, including but not limited to: pCS2-no his-ubiquitin wtpCS2-no his-ubiquitin all RpET30a-ubiquitin (no tag)pET30a-ubiquitin K0 (no tag) 

When model organisms are exposed to chemicals, resulting mutagenesis can provide insights on the chemical’s genotoxicity, which is an indicator of the chemical’s potential to cause cancer or birth defects. In fact, direct mutagenesis assays in bacteria are one of the three assays required by regulatory agencies for demonstrating the safety of potential clinical compounds. Mutagenesis assays are also used to study various DNA processes, such as replication, repair, damage tolerization, and homeostasis.

Researchers at the University of California, Davis have identified a short peptide which rapidly promotes protein degradation in cancerous enzymes and induces cell differentiation to kill lymphomas.

Researchers at the University of California, Davis have developed a virally derived homolog to increase the inflammatory response desirable in cancer immunotherapy.

Researchers at the University of California, Davis have developed a viral peptide therapeutic that targets MYC-based cancerous tumors.

Researchers at the University of California, Davis (“UC Davis”) have developed fusion proteins effective in inhibiting the replication of diverse groups of viruses that can be useful in controlling vector-borne virus transmission as well as reducing vector populations.

Presently, antiviral strategies are mostly focused on targeting viral proteins. However, the high mutation rates of RNA viruses, such as SARS-CoV-2, make the development of effective antiviral drugs very challenging. Disrupting viral-host interactions such as by targeting pro-viral, non-essential human genes will more likely prove effective against new variants or future coronavirus outbreaks.

Researchers from UC San Diego designed a new means to facilitate the enzymatic insertion of a variety of functionalized PreQ1 derivatives into a 17 nucleotide DNA hairpin which can then be appended to DNAs of interest for a variety of applications.Background: While harnessing the programmable power of nucleic acids is no new revelation for science, new innovative applications that realize this power have been crucial to scientific advancements of late. These innovative strategies often rely heavily on nucleic acid modifications.For many technical applications, precision is the key to its success, and it is necessary to have the means to carry out an efficient, site-specific, modification of the nucleic acid substrate. While a variety of site-specific enzymatic RNA modification strategies have been well established, the same is not true for DNA modifications, particularly single stranded DNA (ssDNA). Currently enzymatic modification of ssDNA is limited to 3’ insertion of modified nucleobases and the 5’ insertion of modified phosphate groups. Consequently, there is a need for higher precision methods and compositions for enzymatic modification of ssDNA.