Biology subjects

Lian, Y.

Publications and source records attributed to Lian, Y..

At least 19 recordsLinked to original sources

bioRxiv2026

Receptor-like kinase RLK15 binds a fungal RNase effector to enhance autophosphorylation and activate host defense

Fungal pathogens secrete diverse effectors to suppress host immunity, yet the plant receptors that perceive these virulence factors often remain elusive. Here, we identify Ss1624, a secreted RNase from the sugarcane smut pathogen Sporisorium scitamineum, and show that it is recognized by the plasma-membrane-localized CrRLK1L-family receptor kinase RLK15. This recognition triggers canonical immune outputs, including reactive oxygen species burst, callose deposition, defense gene activation, and induces hypersensitive cell death. Mechanistically, Ss1624 physically interacts with the receptor-like kinase RLK15, which is required for Ss1624-triggered cell death. Functional assays indicate that this interaction enhances RLK15 autophosphorylation in a kinase activity-dependent manner, suggesting that Ss1624 can act as an elicitor. Transcript profiling further reveals coordinated expression patterns of Ss1624 and RLK15 during infection, and perturbation of Ss1624 expression alters RLK15 transcript levels and pathogenic outcomes. Extending these findings to rice, the ortholog OsRLK15 contributes positively to resistance to bacterial and fungal diseases without yield penalty in rice. Our results support a model in which a secreted RNase effector is directly sensed by a receptor kinase to initiate broad-spectrum immune responses, providing insights into host-pathogen interactions and identifying RLK15 as a candidate for crop improvement.

microbiology↗

bioRxiv2026

Intranuclear polyglycine aggregation drives neurodegeneration through epigenetic repression of chromatin accessibility and transcription

Polyglycine (polyG) proteins translated from expanded GGC trinucleotide repeats are implicated in a growing group of neuromuscular degenerative disorders characterized by intranuclear inclusions, yet the pathogenic importance of aggregate localization and the mechanisms underlying polyG-induced neurodegeneration remain unclear. Here we show that intranuclear polyG aggregates are markedly more pathogenic than cytoplasmic aggregates in cellular and mouse models. Intranuclear aggregation causes greater cell death, more severe behavioral deficits and neuropathology, and earlier mortality. Mechanistically, intranuclear polyG aggregates impair nascent RNA synthesis and are associated with a transcriptionally repressive chromatin state marked by globally reduced chromatin accessibility, decreased H3K27 acetylation, and increased HDAC3 expression across cellular, mouse, and human disease tissue. Using a light-inducible system, we further show that this transcriptional impairment depends on insoluble intranuclear aggregate formation rather than diffuse polyG alone. Pharmacological HDAC inhibition partially restores histone acetylation and transcriptional output and ameliorates behavioral and pathological abnormalities. Together, these findings identify intranuclear polyG aggregates as the more pathologically relevant species and uncover epigenetic repression of chromatin accessibility and transcription as a potentially common mechanism underlying polyG diseases.

neuroscience↗

bioRxiv2026

Machine learning-guided spatial omics for tissue-scale discovery of cell-type-specific architectures

Multiplexed protein imaging enables spatially resolved analysis of molecular organization in tissues, but existing spatial proteomics platforms remain constrained in scalability, throughput, and integration with RNA measurements and interpretable computational analysis. Here, we present an integrated spatial omics framework that combines highly multiplexed protein and RNA imaging with explainable machine learning to map cell-type-specific molecular and structural architectures at tissue scale. Using this platform, we simultaneously profiled up to 46 proteins and 79 RNA species across [~]370,000 cells in intact mouse brain tissue at diffraction-limited subcellular resolution ([~]260 nm). We developed a scalable, open-source computational pipeline for large-scale image processing and analysis, and show that nuclear protein and chromatin features alone are sufficient to accurately classify brain cell types and their spatial organization. Incorporation of explainable deep learning further enabled identification of human-interpretable, cell-type-specific subnuclear structural features directly from imaging data, with independent quantitative validation. Together, this integrated experimental and computational framework enables tissue-scale spatial proteomics-based cell-type classification and structural feature discovery, providing a broadly applicable platform for mechanistic studies, high-content screening, and translational applications.

systems biology↗

bioRxiv2025

A new dual-affinity peptide nucleic acid for targeting miRNA-21 precursor rescues tumor repressor PTEN expression

MicroRNAs (miRNAs) have multiple functions in cells and are related to many diseases including cancer by regulating posttranscriptional gene expression. The microRNA precursors (pre-miRs), which can be cleaved by Dicer endonuclease to produce mature miRNAs, often have a cleavage site consisting of both double-stranded (ds) and single-stranded (ss) RNA structures. Peptide nucleic acid (PNA) is a kind of analogue of DNA which can hybridize to DNA or RNA through Watson-Crick and Hoogsteen pairing. We previously reported a novel dual-affinity PNA (daPNA) platform that can simultaneously form a duplex and a triplex with a target RNAs ssRNA-dsRNA junction region. The PNA-RNA complex structure is stabilized by forming antisense PNA (asPNA)-ssRNA duplex immediately adjacent to a chemically modified dsRNA-binding PNA (dbPNA)-dsRNA triplex. In this study, we further explored the application of the daPNA platform to target the precursor of miR-21, which is considered an oncogene. We have designed a set of PNAs including asPNAs, dbPNAs, and daPNAs. The nondenaturing polyacrylamide gel electrophoresis (PAGE) and biolayer interferometry (BLI) data reveal that daPNA-21-10 can strongly bind to pre-miR-21 with high specificity. The data show that pre-miR-21 is easily targeted by traditional antimir, asPNA or dbPNA and optimization of the dsRNA-ssRNA junction position is needed for identifying a tightly binding daPNA. Furthermore, daPNA-21-10 not only inhibits the Dicer activity on pre-miR-21 in cell-free assays, but also down-regulates the expression of miR-21, rescuing PTEN protein expression in cells. Taken together, the biofunction and programmability of daPNA make it a promising platform for probing and regulating miRNA biogenesis and many other RNA-involved biological processes. HighlightsO_LISimultaneous recognition of dsRNA and ssRNA regions C_LIO_LIRNA targeting by dividing and conquering through a dsRNA-ssRNA junction construction C_LIO_LISubstrate-specific inhibition of Dicer acting on pre-miR-21 C_LIO_LIDerepressing PTEN expression by inhibiting miR-21 maturation C_LI O_FIG O_LINKSMALLFIG WIDTH=170 HEIGHT=200 SRC="FIGDIR/small/668835v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@c8af64org.highwire.dtl.DTLVardef@172d2bdorg.highwire.dtl.DTLVardef@1698ea1org.highwire.dtl.DTLVardef@112fa16_HPS_FORMAT_FIGEXP M_FIG C_FIG eToc BlurbLian et al. show that the miR-21 precursor structure containing the Dicer cleavage site can be targeted by a new type of dual-affinity peptide nucleic acids (daPNAs) through the creation of a dsRNA-ssRNA junction, energetically optimized for the simultaneous duplex and triplex formation. Pre-miR-21 structure, which is hardly accessible to traditional antisense strategies and triplex formation alone, can be targeted by a daPNA with high sequence/structure specificity and strong binding affinity. daPNA platform has a great potential in probing many other RNA structures and broad therapeutic applications.

molecular biology↗

bioRxiv2025

Lactucopicrin: A sesquiterpene lactone with anti-inflammatory activity modulates the crosstalk between NF-kB and AHR pathways

Chronic inflammatory diseases, including inflammatory bowel diseases and cardiovascular diseases, share inflammation as a central pathological feature. The transcription factors NF-kB and AHR are key regulators of inflammatory responses, yet their interplay remains underexplored in the context of natural anti-inflammatory compounds. This study investigates the anti-inflammatory properties of sesquiterpene lactones derived from Cichorium intybus L. (chicory), focusing on their modulation of NF-kB and AHR signaling pathways. Using a TNF-induced inflammation model in macrophages, endothelial, and intestinal epithelial cells, we identified lactucopicrin as a potent NF-kB antagonist. Notably, in silico docking and functional assays revealed lactucopicrin as a novel AHR modulator. Crucially, silencing AHR expression attenuated lactucopicrin-mediated NF-kB inhibition, uncovering a previously unrecognized AHR-NF-kB crosstalk mechanism. These findings not only position lactucopicrin as a dual-pathway modulator but also highlight the therapeutic potential of chicory-derived sesquiterpene lactones in treating inflammation-driven diseases and opening new avenues for leveraging natural products in targeted anti-inflammatory strategies.

molecular biology↗

bioRxiv2025

Towards the Development of Isoenergetic Peptide Nucleic Acid Based Probes Targeting Double-Stranded RNAs Through Enhancing Sequence-Specific Stacking Interactions

Peptide nucleic acid (PNA), a synthetic nucleic acid analog, exhibits substantial potential in biotechnology and therapeutic applications due to its high binding affinity and nuclease/protease resistance. Chemically modified PNAs capable of forming stable triplex structures with double-stranded RNAs (dsRNAs) under near-physiological conditions further expand their utility by enabling sequence-specific precise targeting and probing of functional RNA structural motifs. However, the presence of inverted Watson-Crick pairs (C-G and U-A) may significantly weaken the triplex formation of the dsRNA-binding PNAs (dbPNAs). Our previous work demonstrated that dbPNA P3 (composed of L, T, and Q monomers for the recognition of G-C, A-U, and C-G base pairs, respectively) can stimulate ribosomal frameshifting by binding to rHP2, a model RNA hairpin structure in an mRNA, albeit with suboptimal efficiency, due to its significantly weakened Q*C-G triple formation. We hypothesize that incorporating s2U adjacent to Q may offer unique stacking and hydrogen bonding interactions facilitating the development of isoenergetic dbPNA-based probes binding toward dsRNAs with varied sequences. In this study, we investigate how incorporating s2U adjacent to Q residues in P3 influences its binding to rHP2. Bio-layer interferometry (BLI) and non-denaturing polyacrylamide gel electrophoresis (PAGE) analyses demonstrate that substituting T with s2U at the N-terminal position adjacent to Q (P3-2QT) enhances binding affinity by [~]10-fold compared to unmodified P3, whereas C-terminal substitution (P3-TQ2) yields only a 2-fold improvement. Consistent with these findings, a cell-free dual-luciferase reporter assay reveals that P3-2QT significantly increases ribosomal frameshifting efficiency compared to P3 and P3-TQ2. Molecular dynamics simulations further indicate that P3-2QT maintains enhanced PNA-PNA stacking stability, particularly between s2U3 and Q4, suggesting a structural basis for its superior activity. Intriguingly, analogous s2U substitution in the P5 oligomer with the Q replaced by L does not confer a comparable enhancement in binding to the target RNA (rHP1) and frameshifting stimulation, highlighting the context-dependent nature of this modification. To assess the broader applicability of the s2U-Q motif, we examined its effect in dbPNAs targeting the RNA panhandle structure of influenza virus A and precursor microRNA-21, respectively. Both PAGE and BLI data confirm that s2U incorporation improves binding affinity, reinforcing the generality of this strategy. These findings underscore the potential of sequence-dependent uracil thiolation in optimizing triplex-forming dsRNA-binding PNAs, warranting further exploration of modified nucleobase designs to enhance their binding and functional properties. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=159 SRC="FIGDIR/small/662161v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@15d86aborg.highwire.dtl.DTLVardef@1347369org.highwire.dtl.DTLVardef@852040org.highwire.dtl.DTLVardef@84b0a_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIThiolation of uracil upstream of Q base in PNA markedly enhances triplex binding affinity and enables isoenergetic and orthogonal targeting/activation. C_LIO_LIN-terminal s2U modification adjacent to Q residues increases PNA-dsRNA binding by [~]10-fold and significantly boosts ribosomal frameshifting efficiency. C_LIO_LIBiophysical assays and molecular dynamics simulations reveal that thiolation improves PNA stacking stability and slows dissociation, thereby enhancing functional activity toward structured RNA targets. C_LIO_LIThe general applicability of s2U modification is demonstrated with influenza A virus RNA panhandle structure and precursor micoRNA-21 targets, underscoring its broad potential for optimizing PNA-based therapeutics and biotechnological tools. C_LI

biochemistry↗

bioRxiv2025

Single oocyte full-length isoform sequencing unveils the impact of transposable elements on RNA diversity and stability during oocyte maturation

The oocyte-specific isoforms play crucial roles in oocyte maturation, while current understanding of the oocyte transcriptome is mainly focused on gene level. Here, we utilized single cell full-length isoform sequencing based on third generation sequencing to detect entire transcripts in human and mouse oocytes. Isoform diversity during oocyte maturation was systematically profiled, including 6,736 and 4,902 putative novel human and mouse transcripts, respectively. More than half of novel isoforms were categorized as the novel-not-in-catalog (NNC) and may serve specific functions in oocytes, including novel isoforms of ARHGAP18, colocalized with microtubules and targeted knockdown disrupting oocyte maturation. Moreover, over 25% of NNC isoforms were derived from transposable elements (TEs), and their incorporation within transcripts could enhance isoform stability during oocyte maturation. Altogether, our findings represent a valuable resource showcasing the complexity and diversity of RNA isoforms in oocytes, as well as TE co-option for novel isoform generation and isoform stability enhancement.

developmental biology↗

bioRxiv2025

Cell type distribution of intrathecal antisense oligonucleotide activity in deep brain regions of non-human primates

Intrathecally administered RNase H1-active gapmer antisense oligonucleotides (ASOs) are promising therapeutics for brain diseases where lowering the expression of one target gene is expected to be therapeutically beneficial. Such ASOs are active, to varying degrees, across most or all cell types in the cortex and cerebellum of mouse and non-human primate (NHP), brain regions with substantial drug accumulation. Intrathecally delivered ASOs, however, exhibit a gradient of exposure across the brain, with more limited drug accumulation and weaker target engagement in deep brain regions of NHP. Here, we profiled the activity of a tool ASO against Malat1 in three deep brain regions of NHP: thalamus, caudate, and putamen. All neuronal subtypes exhibited knockdown similar to, or deeper than, the bulk tissue. Among non-neuronal cells, knockdown was deepest in microglia and weakest in endothelial stalk. Overall, we observed broad target engagement across all cell types detected, supporting the relevance of intrathecal ASOs to diseases with deep brain involvement.

neuroscience↗

bioRxiv2025

Zinc Finger Repressors mediate widespread PRNP lowering in the nonhuman primate brain and profoundly extend survival in prion disease mice

Prion disease is a rapidly progressing and invariably fatal neurodegenerative disorder with no approved treatment. The disease is caused by the self-templated misfolding of the prion protein (PrP) into toxic species, ultimately leading to neurodegeneration and death. We evaluated a novel epigenetic regulation approach using Zinc Finger Repressors (ZFRs) to ablate PrP expression at the transcriptional level. When delivered using adeno-associated virus (AAV), ZFRs potently and specifically reduced prion mRNA expression by >95% in vitro and to near undetectable levels within single neurons in vivo. In wildtype mice, ZFRs stably lowered neuronal PrP expression throughout the central nervous system for at least 17 months. In mice inoculated with misfolded PrP, AAV-ZFRs given at either early or late disease stages profoundly extended lifespan, significantly reduced PrP in the brain, and improved an array of molecular, histological, biomarker, and behavioral readouts. Finally, we delivered a ZFR targeting the human prion gene (PRNP) to cynomolgus monkeys using a novel blood-brain-barrier penetrant AAV capsid. Extensive bulk and single-cell assessments revealed widespread ZFR expression and PRNP repression in all 35 brain regions assessed, providing the first demonstration of epigenetic regulation across the nonhuman primate neuraxis following a single intravenous (IV) dose. These results highlight the potential of a one-time IV administered ZFR treatment for prion disease and other neurological disorders.

neuroscience↗

bioRxiv2024

Emergence of Sparse Coding, Balance and Decorrelation from a Biologically-Grounded Spiking Neural Network Model of Learning in the Primary Visual Cortex

1Many computational studies attempt to address the question of information representation in biological neural networks using an explicit optimization based on an objective function. These approaches begin with principles of information representation that are expected to be found in the network and from which learning rules can be derived. This study approaches the question from the opposite direction; beginning with a model built upon the experimentally observed properties of neural responses, homeostasis, and synaptic plasticity. The known properties of information representation are then expected to emerge from this substrate. A spiking neural model of the primary visual cortex (V1) was investigated. Populations of both inhibitory and excitatory leaky integrate-and-fire neurons with recurrent connections were provided with spiking input from simulated ON and OFF neurons of the lateral geniculate nucleus. This network was provided with natural image stimuli as input. All synapses underwent learning using spike-timing-dependent plasticity learning rules. A homeostatic rule adjusted the weights and thresholds of each neuron based on target homeostatic spiking rates and mean synaptic input values. These experimentally grounded rules resulted in a number of the expected properties of information representation. The network showed a temporally sparse spike response to inputs and this was associated with a sparse code with Gabor-like receptive fields. The network was balanced at both slow and fast time scales; increased excitatory input was balanced by increased inhibition. This balance was associated with decorrelated firing that was observed as population sparseness. This population sparseness was both the cause and result of the decorrelation of receptive fields. These observed emergent properties (balance, temporal sparseness, population sparseness, and decorrelation) indicate that the network is implementing expected principles of information processing: efficient coding, information maximization (infomax), and a lateral or single-layer form of predictive coding. These emergent features of the network were shown to be robust to randomized jitter of the values of key simulation parameters.

neuroscience↗

bioRxiv2024

Divalent siRNA for prion disease

Prion protein (PrP) lowering is effective in animal models of prion disease and is being tested clinically in prion disease patients, but there remains a need for more potent PrP-lowering drug candidates. Inspired by the reported potency and duration of action of divalent short interfering RNA (siRNA), a new oligonucleotide drug modality for the central nervous system, we sought to discover and develop a new PrP-lowering drug candidate. Herein we identify a mouse Prnp-targeting divalent siRNA molecule, 1682-s4, that lowers PrP to 49% residual brain expression in wild-type mice, and, in the context of intracerebral infection with Rocky Mountain Laboratory (RML) prions, achieves a 2.7-fold increase in survival time with pre-symptomatic chronic treatment and 64% increase in survival time with a single dose after symptom onset. We describe the generation of two transgenic mouse lines, Tg25109 and Tg26372, expressing the full human PRNP gene and its non-coding sequence, and demonstrate their utility for in vivo discovery of potent human PRNP-targeting oligonucleotides. We discover siRNA sequence 2439 against human PRNP and compare its potency in different divalent siRNA chemical scaffolds. We determine that both the fixed UU tail and extended nucleic acid linkages of scaffold s4 contribute to superior potency compared to other scaffolds tested, offering 9.4 and 15.9 percentage points respectively of additional PrP knockdown. A single dose of 348 {micro}g of 2439-s4 lowered whole brain hemisphere human PrP in transgenic mice to 17% residual after 30 days, while 52 {micro}g lowered PrP to 49% residual. 1-2% of the dose of 2439-s4 delivered into cerebrospinal fluid is retained in the brain, and the median effective tissue concentration is estimated at 1.2 micrograms per gram of tissue. Good Laboratory Practices toxicology studies identified no significant liabilities, and the U.S. FDA has cleared an Investigational New Drug application to bring 2439-s4 into clinical trials.

neuroscience↗

bioRxiv2024

PrP turnover in vivo and the time to effect of prion disease therapeutics

PrP lowering is effective against prion disease in animal models and is being tested clinically. Therapies in the current pipeline lower PrP production, leaving pre-existing PrP to be cleared according to its own half-life. We hypothesized that PrPs half-life may be a rate-limiting factor for the time to effect of PrP-lowering drugs, and one reason why late treatment of prion-infected mice is not as effective as early treatment. Using isotopically labeled diet with targeted mass spectrometry, as well as antisense oligonucleotide treatment followed by timed PrP measurement, we estimate a half-life of 5-6 days for PrP in the brain. PrP turnover is not affected by over-or under-expression. Mouse PrP and human PrP have similar turnover rates measured in wild-type or humanized knock-in mice. CSF PrP appears to mirror brain PrP in real time in rats. PrP in the colon is readily quantifiable and has a half-life just slightly shorter than in brain. An under-expressed pathogenic mutant PrP, corresponding to D178N in humans, exhibits an accelerated turnover rate. Our data may inform the design of both preclinical and clinical studies of PrP-lowering drugs. Author SummaryPrion disease is a fatal brain disease caused by misfolding of the prion protein (PrP). Emerging therapies for prion disease seek to reduce the amount of PrP produced in the brain in order to delay onset of disease or slow progression. Mouse studies have shown that if these therapies are initiated too late, their benefit is limited or they may not help at all. Here we measure the half-life of PrP in the mouse brain, and find that it is about 5 days. When drugs are used to lower PrP by cutting up the RNA that encodes PrP, the RNA drops rapidly while the protein lags behind, and does not reach its minimum level until 4 weeks after the drug is dosed. This half-life is about the same regardless of the species of PrP (mouse or human) and whether or not the brain is infected with prions. Cerebrospinal fluid appears to reflect the real-time levels of brain PrP with no appreciable lag. PrP can be measured in colon, which may be useful in animal studies of systemic drugs to lower PrP. PrP turns over more quickly in the presence of a pathogenic genetic variant, the equivalent of the human D178N variant. These findings suggest that clinical trials can monitor PrP in cerebrospinal fluid to look at drug activity, but should plan timepoints far enough post-dose to account for PrPs rate of turnover, and should focus on patients who will survive long enough to benefit from the drug.

neuroscience↗

bioRxiv2024

Distinct cortical spatial representations learned along disparate visual pathways

Recent experimental studies have discovered diverse spatial properties, such as head direction tuning and egocentric tuning, of neurons in the postrhinal cortex (POR) and revealed how the POR spatial representation is distinct from the retrosplenial cortex (RSC). However, how these spatial properties of POR neurons emerge is unknown, and the cause of distinct cortical spatial representations is also unclear. Here, we build a learning model of POR based on the pathway from the superior colliculus (SC) that has been shown to have motion processing within the visual input. Our designed SC-POR model demonstrates that diverse spatial properties of POR neurons can emerge from a learning process based on visual input that incorporates motion processing. Moreover, combining SC-POR model with our previously proposed V1-RSC model, we show that distinct cortical spatial representations in POR and RSC can be learnt along disparate visual pathways (originating in SC and V1), suggesting that the varying features encoded in different visual pathways contribute to the distinct spatial properties in downstream cortical areas. Conflict of interest statementThe authors declare no competing financial interests.

neuroscience↗

bioRxiv2024

Metabolomics profiling reveals association of fermented herbal feed additive, growth performance and gut microbiota in piglets

Fermented Chinese medicine (FCM), as a functional feed additive, has been widely recognized to play a significant role in protecting the intestinal health of piglets and enhancing productive performance. However, the relationship between the active components of FCM, gut microbiota, and their beneficial effects on animal performance remains unclear. In this study, metabolomics analysis revealed a significant increase in the main contents of lactic acid and propionic acid in FCM, while most glycosides and their derivatives decreased after three days of microbial fermentation. Subsequently, piglets were fed a basic diet supplemented with 1% FCM, while the control group received only the basic diet. The results indicated a significant increase in feed intake and average daily gain within 14 days (P<0.05) due to FCM supplementation. Additionally, FCM significantly improved feed efficiency from 1.76 to 1.50 (P<0.05). Meanwhile, piglets in the FCM group exhibited significantly lower frequencies of diarrhea and coughing, indicating better health. Moreover, high-throughput sequencing analysis revealed higher abundances of Lactobacillus agilis, Megasphaera elsdenii, Ligilactobacillus, and Veillonellaceae in piglets fed with FCM. In summary, FCM increased the content of active ingredients through microbial fermentation and regulated the intestinal microbiota to improve the health of piglets. FCM offers a promising potential for enhancing production performance and economic efficiency in the livestock industry.

microbiology↗

bioRxiv2024

Deep-Tissue Spatial Omics: Imaging Whole-Embryo Transcriptomics and Subcellular Structures at High Spatial Resolution

The inherent limitations of fluorescence microscopy, notably the restricted number of color channels, have long constrained comprehensive spatial analysis in biological specimens. Here, we introduce cycleHCR technology that leverages multicycle DNA barcoding and Hybridization Chain Reaction (HCR) to surpass the conventional color barrier. cycleHCR facilitates high-specificity, single-shot imaging per target for RNA and protein species within thick specimens, mitigating the molecular crowding issues encountered with other imaging-based spatial omics techniques. We demonstrate whole-mount transcriptomics imaging of 254 genes within an E6.5[~]7.0 mouse embryo, achieving precise three-dimensional gene expression and cell fate mapping across a specimen depth of [~] 310 {micro}m. Utilizing expansion microscopy alongside protein cycleHCR, we unveil the complex network of 10 subcellular structures in primary mouse embryonic fibroblasts. Furthermore, in mouse hippocampal slice, we image 8 protein targets and profile the transcriptome of 120 genes, uncovering complex gene expression gradients and cell-type specific nuclear structural variances. cycleHCR provides a unifying framework for multiplex RNA and protein imaging, offering a quantitative solution for elucidating spatial regulations in deep tissue contexts for research and potentially diagnostic applications.

developmental biology↗

bioRxiv2024

Recognition of RNA secondary structures with a programmable peptide nucleic acid-based platform

RNA secondary structures comprise double-stranded (ds) and single-stranded (ss) regions. Antisense peptide nucleic acids (asPNAs) enable the targeting of ssRNAs and weakly formed dsRNAs. Nucleobase-modified dsRNA-binding PNAs (dbPNAs) allow for targeting of relatively stable dsRNAs. A programmable RNA structure-specific targeting strategy is needed for simultaneous recognition of dsRNAs and ssRNAs. Here, we report on combining dbPNAs and asPNAs (designated as daPNAs) for the targeting of dsRNA-ssRNA junctions. Our binding and modeling data suggest that combining traditional asPNA (with a 4-letter code: T, C, A, and G) and dbPNA (with a 4-letter code: T or s2U, L, Q, and E) scaffolds facilitates RNA structure-specific tight binding (nM to M) under physiologically-relevant conditions. We further applied our daPNAs in substrate specific inhibition of Dicer acting on pre-miR-198 in a cell-free assay and regulating ribosomal frameshifting induced by model hairpins in both cell-free and cell culture assays. daPNAs would be a useful platform for developing chemical probes and therapeutic ligands targeting RNA. HighlightO_LIWe demonstrated that sequence- and structure-specific targeting of RNA can be facilitated by nucleobase-modified dsRNA-binding PNAs (dbPNAs) platform in combination with antisense PNAs (asPNAs). We name the novel PNAs as daPNAs. C_LIO_LIdaPNAs can be used in a programmable way for targeting RNAs by formation of a short triplex next to a short duplex at a dsRNA-ssRNA junction. C_LIO_LIWe applied our daPNAs in substrate specific inhibition of Dicer acting on pre-miR-198 in a cell-free assay and regulating ribosomal frameshifting induced by model hairpins in both cell-free and cell culture assays. C_LIO_LIThe daPNAs platform would serve as useful junction-specific molecular glues for the targeting of many biologically important RNA structures in transcriptomes. C_LI

biochemistry↗

bioRxiv2024

Enhanced Recognition of a Herbal Compound Epiberberine by a DNA Quadruplex-Duplex Structure

The small molecule epiberberine (EPI) is a natural alkaloid with versatile bioactivities against several diseases, including cancer and bacterial infection. EPI can induce the formation of a unique binding pocket at the 5' side of a human telomeric G-quadruplex (HTG) sequence Q4, resulting in a nanomolar binding affinity (KD approximately 26 nM) with significant fluorescence enhancement upon binding. It is important to understand (1) how EPI binding affects HTG structural stability and (2) how enhanced EPI binding may be achieved through the engineering of the DNA binding pocket. In this work, the EPI binding-induced HTG structure stabilization effect was probed by a peptide nucleic acid (PNA) invasion assay in combination with a series of biophysical techniques. We show that the PNA invasion-based method may be useful for the characterization of compounds binding to DNA (and RNA) structures in physiological conditions without the need to vary the solution temperature or buffer components, which are typically needed for structural stability characterization. Importantly, the combination of theoretical modeling and experimental quantification allows us to successfully engineer the Q4 derivative Q4-ds-A by a simple extension of a duplex structure to Q4 at the 5' end. Q4-ds-A is a superb EPI binder with a KD of 8 nM, with the binding enhancement achieved through the preformation of a binding pocket and a reduced dissociation rate. The tight binding of Q4 and Q4-ds-A with EPI allows us to develop a novel magnetic bead-based affinity purification system to effectively extract EPI from Rhizoma coptidis (Huang Lian) extracts.

biochemistry↗

bioRxiv2023

Learning spatio-temporal V1 cells from diverse LGN inputs

Since Hubel and Wiesels discovery of simple cells and complex cells in cats primary visual cortex (V1), many experimental studies of V1 cells from animal recordings have shown the spatial and temporal structure of their response properties. Although numerous computational learning models can account for how spatial properties of V1 cells are learnt, how temporal properties emerge through learning is still not well understood. In this study, a learning model based on sparse coding is used to show that spatio-temporal V1 cells, such as biphasic and direction-selective cells, can emerge via synaptic plasticity when diverse spatio-temporal lateral geniculate nucleus (LGN) cells are used as upstream input to V1 cells. We demonstrate that V1 cells with spatial structures and temporal properties (such as a temporal biphasic response and direction selectivity) emerge from a learning process that promotes sparseness while encoding upstream LGN input with spatio-temporal properties. This model provides an explanation for the observed spatio-temporal properties of V1 cells from a learning perspective, enhancing our understanding of how neural circuits learn and process complex visual stimuli.

neuroscience↗