Department of Pharmaceutical Sciences, UCI

Many strategies have been implemented to enrich an RNA population for a selectable function, but demarcation of the optimal functional motifs or minimal structures within longer libraries remains a lengthy and tedious process. To overcome this problem, we have developed a technique that isolates minimal active segments from complex heterogeneous pools of RNAs. This method allows for truncations to occur at both 5 and 3 ends of functional domains and introduces independent primer-binding sequences, thereby removing sequence and structure bias introduced by constant-sequence regions. We show examples of minimization for genomic and synthetic aptamers and demonstrate that the method can directly reveal an active RNA assembled from multiple strands, facilitating the development of heterodimeric structures used in cellular sensors. This approach provides a pipeline to experimentally define the boundaries of active domains and accelerate the discovery of functional RNAs.

High shear spinning top (ST) typhoon-like fluid flow in a rapidly rotating inclined tube within a vortex fluidic device (VFD) approaches homochirality throughout the liquid with toroids of bundled single-walled carbon nanotubes (SWCNTs) twisted into stable chiral lemniscates (in the shape of Figure 8s), predominantly as the R-or S-structures, for the tube rotating clockwise (CW) or counterclockwise (CCW). However, this is impacted by the Earth's magnetic field (B_E). Theory predicts 1-20 MPa pressure for their formation, with their absolute chirality determined from scanning electron microscopy (SEM) and atomic force microscopy (AFM) images. Thus, the resultant lemniscate structures establish the absolute chirality of the inner and outer components of the ST flow. These chiral flows and lemniscates can be flipped to the opposite chirality by changing the orientation of the tube relative to the inclination angle of B_E, by moving the geographical location. Special conditions prevail where the tangential angle of the outer and inner flow of the ST becomes periodically aligned with B_E, which respectively dramatically reduce the formation of toroids (and thus lemniscates) and formation of lemniscates from the toroids formed by the double-helical (DH) flow generated by side wall Coriolis forces and Faraday waves.

Use of one addictive drug typically influences the behavioral response to other drugs, either administered at the same time or a subsequent time point. The nature of the drugs being used, as well as the timing and dosing, also influence how these drugs interact. Here, we tested the effects of adolescent THC exposure on the development of morphine-induced behavioral adaptations following repeated morphine exposure during adulthood. We found that adolescent THC administration paradoxically prevented the development of anxiety-related behaviors that emerge during a forced abstinence period following morphine administration but facilitated reinstatement of morphine CPP. Following forced abstinence, we then mapped the whole-brain response to a moderate dose of morphine and found that adolescent THC administration led to an overall increase in brain-wide neuronal activity and increased the functional connectivity between frontal cortical regions and the ventral tegmental area. Last, we show using rabies virus-based circuit mapping that adolescent THC exposure triggers a long-lasting elevation in connectivity from the frontal cortex regions onto ventral tegmental dopamine cells. Our study adds to the rich literature on the interaction between drugs, including THC and opioids, and provides potential neural substates by which adolescent THC exposure influences responses to morphine later in life.

The human genome contains thousands of potentially coding short open reading frames (sORFs). While a growing set of microproteins translated from these sORFs have been demonstrated to mediate important cellular functions, the majority remains uncharacterized. In our study, we performed a high-throughput CRISPR-Cas9 knock-out screen targeting 11,776 sORFs to identify microproteins essential for cancer cell line growth. We show that the CENPBD2P gene encodes a translated sORF and promotes cell fitness. We selected five additional candidate sORFs encoding microproteins between 11 and 63 amino acids in length for further functional assessment. Green fluorescent protein fusion constructs of these microproteins localized to distinct subcellular compartments, and the majority showed reproducible biochemical interaction partners. Studying the fitness and transcriptome of sORF knock-outs and complementation with the corresponding microprotein, we identify rescuable phenotypes while also illustrating the limitations and caveats of our pipeline for sORF functional screening and characterization.

Alchemical free energy calculations are becoming an increasingly prevalent tool in drug discovery efforts. Over the past decade, significant progress has been made in automating various aspects of this technique. However, one aspect hampering wider application is the construction of perturbation networks to connect ligands of interest. More specifically, ligand pairs with large dissimilarities should be avoided since they can lower convergence and decrease accuracy. Here, we propose a technique for automatic generation of intermediate molecules to break up problematic edges─calculations connecting two different ligands or molecules─into smaller perturbations. To this end, a modular tool was developed that generates intermediates for a molecule pair by enumerating R-group combinations called IMERGE-FEP (Intermediate MolEculaR GEnerator for Free Energy Perturbation). Intermediate enumeration of multiple, representative congeneric series showed that intermediates increase similarity regarding shared substructures, geometry, and LOMAP scores. Taken together, this tool eases integration of intermediate steps into free energy calculation protocols.

A structurally novel metabolite, fatuamide A (1), was discovered from a laboratory cultured strain of the marine cyanobacterium Leptolyngbya sp., collected from Faga'itua Bay, American Samoa. A bioassay-guided approach using NCI-H460 human lung cancer cells directed the isolation of fatuamide A, which was obtained from the most cytotoxic fraction. The planar structure of fatuamide A was elucidated by integrated NMR and MS/MS analysis, and a combination of bioinformatic and computational approaches was used to deduce the absolute configuration at its eight stereocenters. A putative hybrid PKS/NRPS biosynthetic gene cluster responsible for fatuamide A production was identified from the sequenced genomic DNA of the cultured cyanobacterium. The biosynthetic gene cluster possessed elements that suggested fatuamide A binds metals, and this metallophore property was demonstrated by native metabolomics and indicated a preference for binding copper. The producing strain was found to be highly resistant to toxicity from elevated copper concentrations in culture media.

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.

Across cell types and organisms, thousands of RNAs display asymmetric subcellular distributions. Studying this process requires quantifying abundances of specific RNAs at precise subcellular locations. To analyze subcellular transcriptomes, multiple proximity-based techniques have been developed in which RNAs near a localized bait protein are specifically labeled, facilitating their biotinylation and purification. However, these complex methods are often laborious and require expensive enrichment reagents. To streamline the analysis of localized RNA populations, we developed Oxidation-Induced Nucleotide Conversion sequencing (OINC-seq). In OINC-seq, RNAs near a genetically encoded, localized bait protein are specifically oxidized in a photo-controllable manner. These oxidation events are then directly detected and quantified using high-throughput sequencing and our software package, PIGPEN, without the need for biotin-mediated enrichment. We demonstrate that OINC-seq can induce and quantify RNA oxidation with high specificity in a dose- and light-dependent manner. We further show the spatial specificity of OINC-seq by using it to quantify subcellular transcriptomes associated with the cytoplasm, ER, nucleus, and the inner and outer membranes of mitochondria. Finally, using transgenic zebrafish, we demonstrate that OINC-seq allows proximity-mediated RNA labeling in live animals. In sum, OINC-seq together with PIGPEN provide an accessible workflow for analyzing localized RNAs across different biological systems.

Vancomycin continues to be a widely used antibiotic of last resort in treating drug-resistant pathogens despite the emergence of vancomycin-resistant strains such as vancomycin-resistant Enterococci (VRE). This communication reports that conjugation of vancomycin to a second antibiotic that targets a different region of lipid II enhances and rescues its antibiotic activity. Conjugation of vancomycin to a minimal teixobactin pharmacophore in which residues 1-6 are replaced with an aromatic amide results in substantial enhancement in activity over the individual components or mixtures thereof. Three conjugates with minimum inhibitory concentrations (MICs) of 0.5 μg/mL against methicillin-resistant Staphylococcus aureus (MRSA) and 0.063-0.125 μg/mL against methicillin-susceptible Staphylococcus aureus (MSSA) were identified. Each of these conjugates is also active against VRE, even though the individual components are inactive, with the most active conjugate (Cbp-Lys10-teixo7-11-vanco) having an MIC of 2-4 μg/mL. These findings demonstrate that conjugation of vancomycin to a minimal teixobactin pharmacophore is an effective strategy for enhancing the activity of vancomycin against important Gram-positive pathogens.