Biology subjects

Zhu, B.

Publications and source records attributed to Zhu, B..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

Enhancing Proteoform Sequence Coverage Using Top-Down Mass Spectrometry with In-Source Fragmentation and Middle-Down Mass Spectrometry

The study of complex proteoforms with mutations and post-translational modifications has gained increasing attention with the advancement of mass spectrometry (MS)-based techniques. Achieving high proteoform sequence coverage by MS is essential for accurately characterizing these complex proteoforms. Extensive efforts have been made to increase proteoform sequence coverage using deep bottom-up and top-down MS strategies. In this study, we evaluated top-down and middle-down MS approaches for enhancing proteoform sequence coverage using three proteins: ubiquitin, myoglobin, and carbonic anhydrase II. In the top-down MS approach, we applied in-source fragmentation (ISF) to generate pseudo-MS3 spectra, thereby improving sequence coverage. For the middle-down MS strategy, we performed short-duration enzymatic digestions to produce longer peptides that preserve more proteoform sequence information. Our experimental results demonstrated that ISF and partial digestion significantly increased the sequence coverage of the proteins, achieving coverage greater than 90%.

bioinformatics↗

bioRxiv2025

Thymine DNA Glycosylase Binds to R-Loops and Excises 5-Formyl and 5-Carboxyl Cytosine from DNA/RNA Hybrids

R-loops are three-stranded nucleic acid structures consisting of a DNA/RNA hybrid and a displaced single-stranded DNA. Once considered rare byproducts of transcription, R-loops are now recognized as important regulators of various nuclear processes. In particular, evidence indicates a role for R-loops in regulating DNA methylation dynamics. R-loops have been shown to promote active DNA demethylation--the enzymatic reversal of 5-methylcytosine (5mC) back into cytosine--by recruiting associated proteins, providing an attractive targeting mechanism. Nevertheless, many important aspects of this process, including whether the associated proteins bind to and function on R-loops, remain to be substantiated. In this study, we demonstrate for the first time that thymine DNA glycosylase (TDG), a key enzyme in the active DNA demethylation pathway, binds tightly to R-loops in vitro and can excise DNA demethylation intermediates 5-formylcytosine (5fC) and 5-carboxycytosine (5caC) from DNA in DNA/RNA hybrid duplexes. We also show that R-loops guide the strand-specific activity of TDG at CpG sites, potentially explaining the asymmetric distribution of 5fC/5caC at gene promoters. Furthermore, we provide important mechanistic insights into base excision on DNA/RNA hybrid duplexes using 19F NMR. Finally, our findings suggest that TDG-R-loop interactions may be widespread in human cells. Collectively, our results provide strong evidence that R-loops play a critical role in DNA demethylation and support a mechanism in which 5fC/5caC are directly removed from DNA/RNA hybrids in cells. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/668694v1_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@1ac2095org.highwire.dtl.DTLVardef@18f4952org.highwire.dtl.DTLVardef@16c403org.highwire.dtl.DTLVardef@9ed96c_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

The neurotrophin DNT-2 regulates cell survival and connectivity via the Toll-2 receptor during visual system development of Drosophila

During development, neurons are produced in excess and those that receive trophic support are maintained, whereas excess neurons are eliminated, enabling the establishment of appropriate neural circuits. In vertebrates, neurotrophin ligands promote cell survival during periods of naturally occurring cell death, by signalling through p75 and Trk receptors. In the Drosophila optic lobe, a wave of apoptosis occurs during neural circuit development; however, whether this also involves neurotrophism remains unresolved. Drosophila neurotrophins (DNTs) are encoded by spatzle (spz) paralogue genes and bind Toll receptors instead. Here, we focused on DNT-3 (previously known as spz-3) and DNT-2 (also known as spz-5) to ask whether they underlie neurotrophism in visual system development. We show that DNT-3 (spz-3) and DNT-2 (spz-5) are both expressed in the retina and in medulla neurons, and multiple Tolls are expressed across lamina and medulla neurons. Over-expression of DNT-3 (spz-3) and DNT-2 (spz-5) could rescue natural occurring cell death, whereas their loss of function caused cell death, showing that DNT-3 and DNT-2 can, and are required to, promote cell survival during optic lobe development. Importantly, DNT-2 is expressed in Mi1 neurons and Toll-2 in connecting L1 neurons. We show that DNT-2 functions in concert with Toll-2, as Toll-2 RNAi knock-down prevented the rescue of apoptosis by DNT-2 over-expression and all Toll-2+ neurons were lost in DNT-2 mutants. Furthermore, alterations in DNT-2 or Toll-2 expression levels impaired connectivity of L1 neurons at the M1 medulla layer and altered dendritic morphology of L1 neurons. These data suggest that L1 neurons could take up DNT-2 secreted from medulla neurons during the establishment of connectivity patterns. As DNT-3 (spz-3) and DNT-2 (spz-5) are expressed in the medulla and they could influence both lamina and medulla neurons, this suggests that their function maintaining cell survival could enable the stabilisation or alignment of connected neurons across medulla columns.

developmental biology↗

bioRxiv2025

A Thiopurine-like Mutagenic Process Defines TGCT Subtypes

Testicular germ cell tumors (TGCTs) are the most common malignancy in young men, exhibit a unique developmental origin and exceptional chemosensitivity. However, the molecular distinctions between TGCT subtypes remain poorly understood. Here we present a comprehensive genomic analysis of 252 treatment-naive primary TGCTs, integrating deep whole-genome sequencing with matched transcriptomic and epigenomic data. We identify new driver genes and uncover defining features of TGCTs, including pervasive chromosome X amplification with subtype-specific X chromosome inactivation, and a germ cell-like transcriptional program. Although previously reported, whole genome doubling (WGD) in TGCTs is further characterized here as ubiquitous, developmentally early, and associated with age at onset. Seminomas are enriched for early driver mutations, secondary WGD events, sustained XIST expression and replication stress-associated indel mutational signatures, while non-seminomas show greater structural complexity, subclonal diversity, relatively earlier-onset WGD, extended tumor latency, and telomere elongation. Moreover, we identify a mutational signature, SBS87, that is exceptionally rare across cancers with exception of thiopurine-treated leukemia, but strikingly prevalent in TGCT, especially non-seminomas. SBS87 is linked to extended tumor latency and telomere elongation, implicating possible environmental or endogenous processes that mimic thiopurine-induced DNA damage in TGCT pathogenesis. Collectively, our findings define TGCTs as molecularly distinct tumors shaped by early genomic instability and highlight SBS87 as a novel mutational footprint with potential etiologic and clinical relevance.

genomics↗

bioRxiv2025

RNA structure directs RNA partitioning and is actively disrupted inside stress granules to enable cellular recovery

RNA structures play important roles in liquid-liquid phase separation. However, how it is regulated during stress response and stress granule formation is still under studied. Here, we performed in vivo RNA structure probing before and after sodium arsenite treatment, and in stress granules. While RNAs generally become more double-stranded upon stress, they maintain their single-strandedness inside stress granules. We showed that RNA single-strandedness enables increased inclusion inside stress granules and that stress granule-enriched RNAs form fewer intra- and intermolecular RNA-RNA interactions. Additionally, several RNA binding proteins including SRSF1 are enriched in differential structure regions. eCLIP analysis revealed that SRSF1 binds to single-stranded regions along RNAs, and increased SRSF1 binding enabled better inclusion of RNAs in stress granules, whereas depletion of SRSF1 decreased stress granule formation under mild oxidative stress. We also observed the active unwinding of RNAs inside stress granules regulated by helicases, including DDX3X, and showed that inhibition of DDX3X results in slower dissolution of stress granules during recovery. Our study reveals the existence of multiple mechanisms to maintain RNA single-strandedness inside stress granules and to allow reversibility of stress granule formation, highlighting the importance of regulating RNA structure to enable cellular plasticity and stress response.

molecular biology↗

bioRxiv2025

The hemagglutinin proteins of clades 1 and 2.3.4.4b H5N1 highly pathogenic avian influenza viruses exhibit comparable attachment patterns to avian and mammalian tissues

The global spread of the A/goose/Guangdong/1/96-lineage H5N1 highly pathogenic avian influenza (HPAI) viruses is accompanied by an expanded host range and the establishment of sustained viral transmission among dairy cattle. To evaluate if the evolving H5N1 viruses have changed tissue tropism over time, we compared the binding patterns of recombinant hemagglutinin (HA) proteins derived from clade 1 (A/Vietnam/1203/04, H5VN) and circulating clade 2.3.4.4b viruses detected from a wild bird (A/Eurasian Teal/Hong Kong/AFCD-HKU-23- 14009-01020/2023, H5HK) and dairy cattle (A/bovine/Ohio/B24OSU-439/2024, H5OH). The HA protein of A(H1N1)pdm09 virus was included for comparison. Using bio-layer interferometry, H1 protein preferentially bound to the 2,6-linked sialoside 6SLNLN while H5 proteins preferentially bound to the 2,3-linked sialoside 3SLN. H5OH showed higher binding affinity to 3SLN than H5HK and H5VN. The attachment pattern of H1 and H5 proteins to the respiratory tissues of different species and dairy cattle mammary glands were evaluated. Compared to the H1 protein, H5 proteins showed stronger binding to the lung epithelial cells of cat, cattle, chicken, ferret, human, and pig, and the clade 2.3.4.4b H5 proteins exhibited increased binding to pig and cattle bronchial epithelial cells. All H5 proteins attached to the alveolar and cistern epithelial cells in mammary glands where 2,3-linked and 2,6-linked sialyl glycans were detected by Maackia amurensis lectin II and Sambucus Nigra Lectin, respectively. Taken together, the HA proteins of clade 1 and 2.3.4.4b H5N1 viruses generally share comparable attachment patterns to avian and mammalian tissues, despite of evolving into antigenically distinct clades over the past 3 decades. IMPORTANCEThe outbreaks of H5N1 HPAI among US dairy cattle since 2024 have raised concerns of the potential changes in HA receptor binding specificity and tissue tropism. Using insect-cell expressed recombinant HA proteins derived from clade 1 and circulating clade 2.3.4.4b H5N1 viruses, we showed that the dairy cattle H5 protein retained binding specificity for the avian-like 2,3-linked sialoside 3SLN over the human-like 2,6-linked sialoside 6SLNLN, with higher binding affinity to 3SLN than the other H5 proteins. Clade 1 and 2.3.4.4b H5 proteins showed comparable attachment patterns to the mammary tissues of lactating dairy cattle, which showed high expression of 2,3-linked and 2,6-linked sialyl glycans. All H5 proteins also showed comparable attachment patterns to the lungs of cat, cattle, chicken, ferret, human, and pig. Our results suggest that the recent H5N1 outbreaks in dairy cattle may be related to ecological factors rather than changes in HA receptor binding specificity.

microbiology↗

bioRxiv2025

Inflamed Microglia like Macrophages in the Central Nervous System of Prodromal Parkinson's Disease

We hypothesized that prodromal Parkinsons Disease (PD), characterized by REM Sleep Behavior Disorder and hyposmia, is an inflammatory disorder initiated in the gut that evolves into a neurodegenerative process manifest as classical PD. To test this hypothesis, we performed single-cell RNAseq analysis of cerebrospinal fluid (CSF) and blood from 118 individuals, comparing healthy subjects with prodromal PD and manifest PD to patients with the central nervous system (CNS) autoimmune disease, multiple sclerosis (MS). Surprisingly, we identified increased numbers of immune cells in the CSF of patients with prodromal PD. Single-cell RNA sequencing revealed increases in CSF-specific microglia-like macrophages with enhanced JAK-STAT and TNF signaling signatures in prodromal PD. CSF macrophages exhibited similar transcriptional profiles to dural macrophages and gut muscularis macrophages from -synuclein-expressing PD model mice. Finally, shared memory T cell clones were found in humans between gut and CSF T cells. These findings uncover a myeloid-mediated TNF inflammatory process in the CNS of patients with prodromal PD, with an immunological linkage between the gut and the CNS.

immunology↗

bioRxiv2025

Robust Bayesian Integrative Modeling of Single Cell and Spatially Resolved Transcriptomics Data

A recent technology breakthrough in spatially resolved transcriptomics (SRT) has enabled comprehensive molecular characterization at the cellular level while preserving spatial information. However, many SRT technologies (e.g., spatial transcriptomics) cannot achieve single-cell resolution. Instead, they measure the average gene expression of a mixture of cells within a spot arranged on a lattice. Here, we introduce iMOSCATO, a fully Bayesian model that integrates single-cell RNA sequencing (scRNA-seq) data and SRT data to simultaneously decompose cell-type compositions of spots and identify the underlying spatial domains. We incorporate the lattice structure by employing a Markov random field prior, improving the accuracy of both cell-type deconvolution and spatial domain detection. Moreover, we use a zero-inflated Dirichlet distribution to capture cell-type sparsity. Finally, iMOSCATO shows competitive performance in accuracy compared to existing methods in both simulation studies and two real data applications.

bioinformatics↗

bioRxiv2025

Beige fat appearance during lipoatrophy reveals trade-offs between metabolic homeostasis and female fertility

White adipose tissue (WAT) meets the energetic demands required for estrous cycle regularity, ovulation, and reproductive success in women. Here, we report WAT loss and subsequent female-specific compensatory beige fat recruitment impact metabolic and reproductive outcomes in mice. Female mice that undergo progressive lipoatrophy ( fat-less) displayed disrupted estrous cycles, reduced ovarian reserve, and subfertility. These effects were attributable to an accumulation of thermogenic beige fat cells in residual subcutaneous WAT depots and greater energy expenditure. Conversely, high-fat diet lowered energy expenditure and rescued estrous cycle regularity among fat-less mice compared to littermate controls, despite profound insulin resistance and metabolic dysfunction. Together, these findings provide evidence of trade-offs between the maintenance of metabolic homeostasis and reproduction, where increased energy expenditure and beige fat cell emergence compensate for fat loss but occur at the expense of fecundity in female mice.

cell biology↗

bioRxiv2025

EssentCell: Discovering Essential Evolutionary Relations in Noisy Single-Cell Data

Single-cell sequencing (SCS) enables investigating tumor evolution at a single cell resolution. A common type of analysis to investigate evolutionary structure from an SCS experiment is to determine a phylogenetic tree structure from the data. This problem has been well-studied under the assumption that mutations only accumulate in the evolution of cancer and there is a simple characterization of when the data is compatible with a perfect phylogeny based on the absence of a special "conflict" submatrix. SCS data can be represented as a binary matrix, where the ij-th entry indicates whether cell i has mutation j. In practice, SCS data is noisy, so a natural question is what is the minimum number of entries to flip in the data matrix, in order that the matrix becomes "conflict-free" and thus compatible with a perfect phylogeny. Furthermore, the false positive rate is orders of magnitude smaller than the false negative rate, so that at most a few false positives occur with high probability. We consider a variation of the minimum-flip problem in which the number of false positives in the solution is a parameter. Often, there can be multiple optimal solutions, so a natural question is what relations are true among all optimal solutions for a small range of possible false positives values; we call such relations essential. In this work, we propose an efficient algorithm based on integer linear programming to determine all essential relations in the data. We test our software tool, EssentCell, on several data sets and discuss the results found.

cancer biology↗

bioRxiv2025

Highly sensitive chemiluminescence imaging of misfolded proteins in neurodegenerative models

Visualizing misfolded proteins would greatly facilitate early diagnosis, etiology elucidation, and therapy monitoring of neurodegeneration. Although several probes have been reported, simple and versatile detection in vivo is still challenging. We demonstrated that both generic and precise detection of misfolded proteins could be achieved with a chemiluminescence probe, ADLumin-1. For the generic aspect, ADLumin-1 was highly sensitive to various misfolded proteins, showing up to 127.73-fold higher signal-to-noise ratio than Thioflavin T. ADLumin-1 could also non-invasively visualize misfolded proteins in mouse models of Parkinsons disease, Alzheimers disease and amyotrophic lateral sclerosis. Furthermore, ADLumin-1 displayed precise detection value for -synuclein by combining with PMCA in vitro and bioorthogonal ChRET imaging technology in vivo. ADLumin-1 can selectively detect -synuclein in CSF at the femtomolar level and enables in situ monitoring of misfolded -synuclein in vivo. Combining generality and precision, our findings could be widely applied in preclinical and clinical studies of neurodegenerative diseases. TeaserA highly sensitive chemiluminescence probe enables both generic and precise detection of various misfolded proteins

biochemistry↗

bioRxiv2025

Divalent cations govern the activity of coronavirus nsp15

Coronavirus nsp15 is an endoribonuclease that limits the accumulation of viral dsRNA in cytosol and plays a crucial role in the evasion of host immunity. Here, we show that Co2+ or Ni2+ potently activates nsp15 of human and animal coronaviruses, including SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, MHV-A59, and PEDV. Whereas Zn2+ strongly inhibits nsp15 of these coronaviruses. Cryo-EM structures of the Co2+-bound SARS-CoV-2 nsp15/dsRNA complexes reveal that Co2+ binds to the nsp15 active site and is coordinated by three catalytic residues H234, H249, K289, and the 2 hydroxyl of the flipped base from dsRNA, suggesting that Co2+ stabilizes the catalytic core and increases substrate binding. Although our data indicated that Ni2+ and Zn2+ bind to the same site as Co2+, Zn2+ lacks the catalytic capability and inhibits nsp15. These findings reveal that divalent cations govern coronavirus nsp15s activity and provide basis for new therapeutic strategies.

molecular biology↗

bioRxiv2025

SMART platform for enzyme engineering: A novel single-molecule display approach for evolution and functional studies of D-amino acid oxidase

This study introduces SMART (Single Molecule Assay on Ribonucleic Acid by Translated Product), an innovative in vitro platform integrating mRNA display, next-generation sequencing, and bioinformatics to address several key challenges in enzyme engineering. The characteristic feature of SMART is its auxiliary unit, attached to the mRNA-displayed enzyme library to mediate the biotinylation of reactive enzyme variants and enable their enrichment by streptavidin beads pull-down. The unit comprises a hairpin single-stranded DNA that hybridizes with the mRNA and anchors a DNA-binding protein, single-chain Cro, which carries functional auxiliary molecules specific to the enzyme of interest. Here, we report on the establishment of SMART for the engineering of oxidases, specifically D-amino acid oxidase (DAAO) from Schizosaccharomyces pombe, with engineered ascorbate peroxidase 2 as its auxiliary enzyme. In a single selection round of a DAAO library with randomized catalytic residue Y232 and D-Alanine as the substrate, we identified several catalytically active enzyme variants with altered substrate specificity in a matter of days. This surpasses traditional enzyme engineering methods in speed, cost, library capacity, and precision. This research underscores the potential of SMART for the engineering of various oxidases, and other enzymes after the corresponding adjustment of the auxiliary unit.

bioengineering↗

bioRxiv2025

Deciphering lung adenocarcinoma evolution and the role of LINE-1 retrotransposition

Understanding lung cancer evolution can identify tools for intercepting its growth. In a landscape analysis of 1024 lung adenocarcinomas (LUAD) with deep whole-genome sequencing integrated with multiomic data, we identified 542 LUAD that displayed diverse clonal architecture. In this group, we observed an interplay between mobile elements, endogenous and exogenous mutational processes, distinct driver genes, and epidemiological features. Our results revealed divergent evolutionary trajectories based on tobacco smoking exposure, ancestry, and sex. LUAD from smokers showed an abundance of tobacco-related C:G>A:T driver mutations in KRAS plus short subclonal diversification. LUAD in never smokers showed early occurrence of copy number alterations and EGFR mutations associated with SBS5 and SBS40a mutational signatures. Tumors harboring EGFR mutations exhibited long latency, particularly in females of European-ancestry (EU_N). In EU_N, EGFR mutations preceded the occurrence of other driver genes, including TP53 and RBM10. Tumors from Asian never smokers showed a short clonal evolution and presented with heterogeneous repetitive patterns for the inferred mutational order. Importantly, we found that the mutational signature ID2 is a marker of a previously unrecognized mechanism for LUAD evolution. Tumors with ID2 showed short latency and high L1 retrotransposon activity linked to L1 promoter demethylation. These tumors exhibited an aggressive phenotype, characterized by increased genomic instability, elevated hypoxia scores, low burden of neoantigens, propensity to develop metastasis, and poor overall survival. Re-activated L1 retrotransposition-induced mutagenesis can contribute to the origin of the mutational signature ID2, including through the regulation of the transcriptional factor ZNF695, a member of the KZFP family. The complex nature of LUAD evolution creates both challenges and opportunities for screening and treatment plans.

genomics↗

bioRxiv2025

The conserved nature of nitrogen recycling within the bacterial pouch of Tetraponera ants

Microbial symbioses are fundamental to the nutrition of many animal groups, yet the mechanisms of nutrient provisioning remain poorly understood -- even in social insects, a diverse clade whose global ecological dominance and sophisticated caste systems make them disproportionately well-studied models for symbiosis research. Here, we investigate the functional significance of a specialized and unusual symbiotic organ-- the bacterial pouch--in four ant species within the Tetraponera nigra-group, focusing on the symbionts roles in nitrogen recycling and colony fitness. Bacterial pouch houses a microbial community consistently dominated by the core bacterial symbiont Tokpelaia. Metagenomic sequencing and targeted {superscript 1}N-labeled urea feeding experiments demonstrate that these symbionts assimilate nitrogen from urea into amino acids, transferring them to adult workers and developing larvae that incorporate recycled nitrogen into their tissues. Disruption of this nitrogen-recycling symbiosis severely impairs larval growth and overall colony fitness. Our study highlights the critical role of the bacterial pouch in sustaining colony fitness in nitrogen-limited environments, providing new insights into the pivotal role of microbial symbioses in the ecological success of social insects.

ecology↗

bioRxiv2025

An exonuclease safeguards cGAS immune signaling by degrading its 2'-5' phosphodiester intermediate

Excessive activation of cyclic GMP-AMP synthase (cGAS) drives inflammatory and autoimmune diseases, but complete inhibition of cGAS signaling can compromise host defence. Here we show that cGAS signaling can be tuned through a previously unrecognized regulatory checkpoint. Bacterial cGAS generates cGAMP through a two-step mechanism involving release and rebinding of a 2'-5'-linked linear dinucleotide intermediate. We identify a CBASS-associated exonuclease (Exo) that selectively degrades this catalytic intermediate, establishing a threshold for cGAS activation while preserving immune function. This thresholding mechanism is conserved across domains of life: Exo suppresses human cGAS activity in vitro and in mammalian cells and attenuates cGAS-driven pathology in vivo. Our findings identify the catalytic intermediate surveillance as a conserved strategy for safeguarding cGAS signaling.

biochemistry↗

bioRxiv2025

Lancet2: Improved and accelerated somatic variant calling with joint multi-sample local assembly graph

Here, we present Lancet2, an open-source somatic variant caller designed to improve detection of small variants in short-read sequencing data. Lancet2 introduces significant enhancements, including: 1) Improved variant discovery and genotyping through partial order multiple sequence alignment of assembled haplotype contigs and re-alignment of sample reads to the best supporting allele. 2) Optimized somatic variant scoring with Explainable Machine Learning models leading to better somatic filtering throughout the sensitivity scale. 3) Integration with Sequence Tube Map for enhanced visualization of variants with aligned sample reads in graph space. When benchmarked against enhanced two-tech truth sets generated using high-coverage short-read (Illumina) and long-read (Oxford Nanopore) data from four well characterized matched tumor/normal cell lines, Lancet2 outperformed other industry-leading tools in variant calling performance, especially for InDels. In addition, significant runtime performance improvements compared to Lancet1 ([~]10x speedup and 50% less peak memory usage) and most other state of the art somatic variant callers (at least 2x speedup with 8 cores or more) make Lancet2 an ideal tool for accurate and efficient somatic variant calling.

bioinformatics↗

bioRxiv2025

Spatial Total RNA Sequencing of Formalin-Fixed Paraffin Embedded Tissues by spRandom-seq

Current oligo(dT) primers-based spatial transcriptomic methods are limited to fresh or fresh-frozen samples due to the low efficiency of oligo(dT) primers in capturing RNAs in degraded or microbial samples. Here, we have developed a random primer-based spatial total RNA sequencing (spRandom-seq) technology for simultaneously capturing whole host and microbial RNAs in formalin-fixed paraffin-embedded (FFPE) tissues. spRandom-seq eliminated 3 or 5 gene-body biases and outperformed oligo(dT)-based 10X Visium with an 8-fold higher capturing rate for lncRNA and other non-polyadenylated RNA biotypes, including miRNA, snRNA and miscRNA. In the clinical FFPE sections of breast cancer, we revealed the inherent heterogeneity within the tumor region. We also simultaneously captured host and microbial RNAs in Klebsiella pneumoniae-infected sections. Totally, spRandom-seq provided a versatile tool for both clinical pathology and infection biology with established spatial platforms, ensuring ease of operation and large-scale applications.

genomics↗