Branched chemically modified poly(A) tails enhance the translation capacity of mRNA.

Although messenger RNA (mRNA) has proved effective as a vaccine, its potential as a general therapeutic modality is limited by its instability and low translation capacity. To increase the duration and level of protein expression from mRNA, we designed and synthesized topologically and chemically modified mRNAs with multiple synthetic poly(A) tails. Here we demonstrate that the optimized multitailed mRNA yielded ~4.7-19.5-fold higher luminescence signals than the control mRNA from 24 to 72 h post transfection in cellulo and 14 days detectable signal versus <7 days signal from the control in vivo. We further achieve efficient multiplexed genome editing of the clinically relevant genes Pcsk9 and Angptl3 in mouse liver at a minimal mRNA dosage. Taken together, these results provide a generalizable approach to synthesize capped branched mRNA with markedly enhanced translation capacity.

Spatial atlas of the mouse central nervous system at molecular resolution.

Spatially charting molecular cell types at single-cell resolution across the 3D volume is critical for illustrating the molecular basis of brain anatomy and functions. Single-cell RNA sequencing has profiled molecular cell types in the mouse brain1,2, but cannot capture their spatial organization. Here we used an in situ sequencing method, STARmap PLUS3,4, to profile 1,022 genes in 3D at a voxel size of 194 × 194 × 345 nm3, mapping 1.09 million high-quality cells across the adult mouse brain and spinal cord. We developed computational pipelines to segment, cluster and annotate 230 molecular cell types by single-cell gene expression and 106 molecular tissue regions by spatial niche gene expression. Joint analysis of molecular cell types and molecular tissue regions enabled a systematic molecular spatial cell-type nomenclature and identification of tissue architectures that were undefined in established brain anatomy. To create a transcriptome-wide spatial atlas, we integrated STARmap PLUS measurements with a published single-cell RNA-sequencing atlas1, imputing single-cell expression profiles of 11,844 genes. Finally, we delineated viral tropisms of a brain-wide transgene delivery tool, AAV-PHP.eB5,6. Together, this annotated dataset provides a single-cell resource that integrates the molecular spatial atlas, brain anatomy and the accessibility to genetic manipulation of the mammalian central nervous system.

Exosomal lipid PI4P regulates small extracellular vesicle secretion by modulating intraluminal vesicle formation.

Membrane lipids play vital roles in small extracellular vesicle (sEV) biogenesis. However, the function of various lipids in the biogenesis of sEVs is still poorly understood. Phosphoinositolphosphates (PIPs), a group of the most critical lipids in vesicle transport, can undergo rapid conversion in response to a variety of cell signals, which in turn influence the generation of vesicles. Due to the challenge in detecting the low amount of PIP content in biological samples, the function of PIPs in sEVs has been insufficiently investigated. Here, we employed an LC-MS/MS method to detect the levels of PIPs in sEVs. We revealed phosphatidylinositol-4-phosphate (PI4P) was the main PI-monophosphate in macrophage-derived sEVs. The release of sEVs was regulated in a time-dependent manner and correlated with the PI4P level during the lipopolysaccharide (LPS) stimulation. In terms of mechanism, within 10 h of LPS treatment, the LPS-induced production of type I interferon inhibited the expression of PIP-5-kinase-1-gamma, which increased the PI4P content on multivesicular bodies (MVBs) and recruited RAB10, member RAS oncogene family, to promote sEV generation. When LPS stimulation was extended to 24 h, the heat shock protein family A member 5 (HSPA5) expression level was elevated. PI4P interacted with HSPA5 on the Golgi or endoplasmic reticulum away from MVBs, which disrupted the continuous fast sEV release. In conclusion, the present study demonstrated an inducible sEV release model response to LPS treatment. The inducible release may be due to PI4P regulating the generation of intraluminal vesicles secreted as sEVs.

Photoactivation of Innate Immunity Receptor TLR8 in Live Mammalian Cells by Genetic Encoding of Photocaged Tyrosine.

The effectiveness of innate immune responses relies on an intricate balance between activation and regulation. TLR8, a member of the Toll-like receptor (TLR) family, plays a fundamental role in host defense by sensing viral single-stranded RNAs (ssRNAs). However, the molecular recognition and regulatory mechanism of TLR8 is not fully understood, especially in a whole-cell environment. Here, we engineer the first light-controllable TLR8 model by genetically encoding a photocaged tyrosine, NBY, into specific sites of TLR8. In the caged forms, the activity of TLR8 is masked but can be restored upon decaging by exposure to UV light. To explain the mechanism clearly, we divide the sites with light responsiveness into three groups. They can separately block the ligands that bind to the pockets of TLR8, change the interaction modes between two TLR8 protomers, and interfere with the interactions between TLR8 cytosolic domains with its downstream adaptor. Specifically, we use this chemical caging strategy to probe and evaluate the function of several tyrosine sites located at the interface of TLR8 homodimers with a previously unknown regulatory mode, which may provide a new strategy for TLR8 modulator development. Effects on downstream signaling pathways are monitored at the transcriptional and translational levels in various cell lines. By photoactivating specific cells within a larger population, this powerful tool can provide novel mechanistic insights, with potential in biotechnological and pharmaceutical applications.

Orthosteric-allosteric dual inhibitors of PfHT1 as selective antimalarial agents.

Artemisinin-resistant malaria parasites have emerged and have been spreading, posing a significant public health challenge. Antimalarial drugs with novel mechanisms of action are therefore urgently needed. In this report, we exploit a "selective starvation" strategy by inhibiting Plasmodium falciparum hexose transporter 1 (PfHT1), the sole hexose transporter in P. falciparum, over human glucose transporter 1 (hGLUT1), providing an alternative approach to fight against multidrug-resistant malaria parasites. The crystal structure of hGLUT3, which shares 80% sequence similarity with hGLUT1, was resolved in complex with C3361, a moderate PfHT1-specific inhibitor, at 2.3-Å resolution. Structural comparison between the present hGLUT3-C3361 and our previously reported PfHT1-C3361 confirmed the unique inhibitor binding-induced pocket in PfHT1. We then designed small molecules to simultaneously block the orthosteric and allosteric pockets of PfHT1. Through extensive structure-activity relationship studies, the TH-PF series was identified to selectively inhibit PfHT1 over hGLUT1 and potent against multiple strains of the blood-stage P. falciparum Our findings shed light on the next-generation chemotherapeutics with a paradigm-shifting structure-based design strategy to simultaneously target the orthosteric and allosteric sites of a transporter.

Structural Basis for Blocking Sugar Uptake into the Malaria Parasite Plasmodium falciparum.

Plasmodium species, the causative agent of malaria, rely on glucose for energy supply during blood stage. Inhibition of glucose uptake thus represents a potential strategy for the development of antimalarial drugs. Here, we present the crystal structures of PfHT1, the sole hexose transporter in the genome of Plasmodium species, at resolutions of 2.6 Å in complex with D-glucose and 3.7 Å with a moderately selective inhibitor, C3361. Although both structures exhibit occluded conformations, binding of C3361 induces marked rearrangements that result in an additional pocket. This inhibitor-binding-induced pocket presents an opportunity for the rational design of PfHT1-specific inhibitors. Among our designed C3361 derivatives, several exhibited improved inhibition of PfHT1 and cellular potency against P. falciparum, with excellent selectivity to human GLUT1. These findings serve as a proof of concept for the development of the next-generation antimalarial chemotherapeutics by simultaneously targeting the orthosteric and allosteric sites of PfHT1.