Department of Pharmaceutical Sciences, UCI

Antisense oligonucleotides (ASOs) and small interfering RNA (siRNA) therapeutics highlight the power of oligonucleotides in silencing disease-causing messenger RNAs (mRNAs). Another promising class of gene-silencing oligonucleotides is RNA-cleaving nucleic acid enzymes, which offer the potential for allele-specific RNA inhibition with greater precision than ASOs and siRNAs. Herein, we chemically evolved the nucleolytic DNA enzyme (DNAzyme) 10-23, by incorporating the modifications that are essential to the success of ASO drugs, including 2'-fluoro, 2'-O-methyl, and 2'-O-methoxyethyl RNA analogues, and backbone phosphorothioate, to enhance catalytic efficiency by promoting RNA substrate binding and preventing dimerization of 10-23. These ASO-like DNAzymes cleaved structured RNA targets in long transcripts, showed prolonged intracellular stability, and downregulated mRNA and protein levels of both exogenously transfected eGFP and endogenously elevated oncogenic c-MYC. In colon cancer HCT116 cells, the downregulation of oncogenic c-MYC RNA resulted in cell cycle arrest, reduced proliferation, and increased apoptosis. RACE (rapid amplification of cDNA ends) polymerase chain reaction and Sanger sequencing confirmed precise, site-specific mRNA transcript cleavage with minimal RNase H activation in cells. By merging ASO structural and pharmacokinetic advantages with DNAzyme catalytic versatility, these ASO-like 10-23 variants offer a promising new class of potent gene-silencing agents, representing a significant step toward therapeutic DNAzyme development.

High-damaging Candida albicans strains tend to form hyphae and exacerbate intestinal inflammation in ulcerative colitis patients through IL-1β-dependent mechanisms. Fungal agglutinin-like sequence (Als) proteins worsen DSS-induced colitis in mouse models. FADD and caspase-8 are important regulators of gut homeostasis and inflammation. However, whether they link directly to fungal proteins is not fully understood. Here, we report that Als proteins induce IL-1β release in immune cells. We show that hyphal Als3 is internalized in macrophages and interacts with caspase-8 and the inflammasome adaptor apoptosis-associated speck-like protein containing a CARD (ASC). Caspase-8 is essential for Als3-mediated ASC oligomerization and IL-1β processing. In non-immune cells, Als3 is associated with cell death core components FADD and caspase-8. N-terminal Als3 (N-Als3) expressed in Jurkat cells partially inhibits apoptosis. Mechanistically, N-Als3 promotes oligomerization of FADD and caspase-8 through their death effector domains (DEDs). N-Als3 variants with a mutation in the peptide-binding cavity or amyloid-forming region are impaired in DED oligomerization. Together, these results demonstrate that DEDs are intracellular sensors of Als3. This study identifies additional potential targets to control hypha-induced inflammation.

This paper reports highly active analogues of clovibactin in which the rare, noncanonical amino acid d-hydroxyasparagine is replaced with the commercially available amino acid d-threonine. Sequential mutation of leucines 2, 7, and 8 to the more hydrophobic homologue cyclohexylalanine dramatically increases the antibiotic activity of d-Thr₅-clovibactin. The resulting analogues (d-Cha₂,d-Thr₅-clovibactin, Cha₇,d-Thr₅-clovibactin, and Cha₈,d-Thr₅-clovibactin) are readily prepared by standard peptide synthesis techniques and exhibit excellent activity (≤1 μg/mL) against the Gram-positive, drug-resistant pathogens MRSA and VRE.

Growth in the development of engineered polymerases for synthetic biology has led to renewed interest in assays that can measure the fidelity of polymerases that are capable of synthesizing artificial genetic polymers (XNAs). Conventional approaches require purifying the XNA intermediate of a replication cycle (DNA → XNA → DNA) by denaturing polyacrylamide gel electrophoresis, which is a slow, costly, and inefficient process that requires a large-scale transcription reaction and careful extraction of the XNA strand from the gel slice. In an effort to streamline the assay, we developed a purification-free approach in which the XNA transcription and reverse transcription steps occur inside the matrix of a hydrogel-coated magnetic particle. Accordingly, a DNA primer cross-linked throughout the gel matrix is annealed to a template of defined sequence and extended with XNA. Following removal of the DNA template, the XNA product strand is copied back into DNA, recovered, amplified, cloned, and sequenced. Performing the replication cycle in the hydrogel format drastically reduces the time and reaction scales required to measure the fidelity of an XNA polymerase, making it easier to evaluate the properties of a range of candidate XNA polymerases.

The architecture of cells and the tissue they form within multicellular organisms are highly complex and dynamic. Cells optimize their function within tissue microenvironments by expressing specific subsets of RNAs. Advances in cell tagging methods enable spatial understanding of RNA expression when merged with transcriptomics. However, these techniques are currently limited by the spatial resolution of the tagging, the number of RNAs that can be sequenced, and multiplexing to isolate spatially-distinct cells within the same tissue landscape. To address these limitations, we developed CrossSeq, which employs photocrosslinking fluorescent probes and confocal microscopy activation to demarcate user-defined regions of interest on fixed cells for multiplexed spatial transcriptomic analysis. We investigate phenyl azide and diazirine crosslinking scaffolds and define their photoactivity profiles. We then deploy the aryl azide scaffold with three fluorophores for multiplexing on glyoxal fixed cells and analyze the defined populations using flow cytometry. Finally, we apply CrossSeq to investigate an in vitro MDA-MB-231-LM2 metastatic cancer migration model to evaluate changes in gene expression at the migratory cell front versus the exterior population. We anticipate this new technology will be a valuable tool addition as it will enable easier access to spatial transcriptomic analysis for the scientific community using conventional microscopy and analysis techniques.

Loss-of-function sequence variants in KCNA1, which encodes the voltage-gated potassium channel Kv1.1, cause Episodic Ataxia Type 1 (EA1) and epilepsy. Due to a paucity of drugs that directly rescue mutant Kv1.1 channel function, current therapeutic strategies for KCNA1-linked disorders involve indirect modulation of neuronal excitability. Native Americans have traditionally used conifer extracts to treat paralysis, weakness, and pain, all of which may involve altered electrical activity and/or Kv1.1 dysfunction specifically. Here, screening conifer extracts, we found that Chamaecyparis pisifera increases wild-type (WT) Kv1.1 activity, as does its prominent metabolite, the abietane diterpenoid pisiferic acid. Uniquely, pisiferic acid also restored function in 12/12 EA1-linked mutant Kv1.1 channels tested in vitro. Crucially, pisiferic acid (1 mg/kg) restored WT function in Kv1.1^E283K/+ mice, a model of human EA1. Experimentally validated all-atom molecular dynamics simulations in a neuron-like membrane revealed that the Kv1.1 voltage-sensing domain (VSD) also acts as a ligand-binding domain akin to those of classic ligand-gated channels; binding of pisiferic acid induces a conformational shift in the VSD that ligand-dependently opens the pore. Conifer metabolite pisiferic acid is a promising and versatile therapeutic lead for EA1 and other Kv1.1-linked disorders.

Huntingtons disease (HD) is caused by a CAG repeat expansion in the HTT gene, leading to altered gene expression. However, the mechanisms leading to disrupted RNA processing in HD remain unclear. Here we identify TDP-43 and the N6-methyladenosine (m6A) writer protein METTL3 to be upstream regulators of exon skipping in multiple HD systems. Disrupted nuclear localization of TDP-43 and cytoplasmic accumulation of phosphorylated TDP-43 occurs in HD mouse and human brains, with TDP-43 also co-localizing with HTT nuclear aggregate-like bodies distinct from mutant HTT inclusions. The binding of TDP-43 onto RNAs encoding HD-associated differentially expressed and aberrantly spliced genes is decreased. Finally, m6A RNA modification is reduced on RNAs abnormally expressed in the striatum of HD R6/2 mouse brain, including at clustered sites adjacent to TDP-43 binding sites. Our evidence supports TDP-43 loss of function coupled with altered m6A modification as a mechanism underlying alternative splicing in HD.

Specialized or secondary metabolites are small molecules of biological origin, often showing potent biological activities with applications in agriculture, engineering and medicine. Usually, the biosynthesis of these natural products is governed by sets of co-regulated and physically clustered genes known as biosynthetic gene clusters (BGCs). To share information about BGCs in a standardized and machine-readable way, the Minimum Information about a Biosynthetic Gene cluster (MIBiG) data standard and repository was initiated in 2015. Since its conception, MIBiG has been regularly updated to expand data coverage and remain up to date with innovations in natural product research. Here, we describe MIBiG version 4.0, an extensive update to the data repository and the underlying data standard. In a massive community annotation effort, 267 contributors performed 8304 edits, creating 557 new entries and modifying 590 existing entries, resulting in a new total of 3059 curated entries in MIBiG. Particular attention was paid to ensuring high data quality, with automated data validation using a newly developed custom submission portal prototype, paired with a novel peer-reviewing model. MIBiG 4.0 also takes steps towards a rolling release model and a broader involvement of the scientific community. MIBiG 4.0 is accessible online at https://mibig.secondarymetabolites.org/.

Degrons are short amino acid sequences that can facilitate the degradation of protein substrates. They can be classified as either ubiquitin-dependent or -independent based on their interactions with the ubiquitin proteasome system (UPS). These amino acid sequences are often found in exposed regions of proteins serving as either a tethering point for an interaction with an E3 ligase or initiating signaling for the direct degradation of the protein. Recent advancements in the protein degradation field have shown the therapeutic potential of both classes of degrons through leveraging their degradative effects to engage specific protein targets. This review explores what targeted protein degradation applications degrons can be used in and how they have inspired new degrader technology to target a wide variety of protein substrates.