Recent Publications

Pseudouridine (psi) is one of the most abundant mRNA modifications, yet its impact on translation is unclear, in part because existing modification maps are inconsistent, curated comparisons across cell types are lacking, and paired analyses with translation are limited. Using direct RNA nanopore sequencing coupled with our Mod-p ID analytical framework, we mapped psi at single-nucleotide resolution across six immortalized human cell lines. Nanopore sequencing provided single-molecule......Read more

Pseudouridine (Ψ) is an abundant post-transcriptional modification found across all classes of RNA. It is widely speculated that Ψ inclusion in messenger RNAs (mRNAs) might provide an avenue for cells to control gene expression post-transcriptionally. Here we demonstrate that one of the principal mRNA pseudouridylating enzymes, pseudouridine synthase 7 (PUS7), exhibits a stress-induced accumulation in the cytoplasm of yeast and human epithelial lung cells. Stress-induced and cytoplasmic......Read more

The principle that form follows function has long guided thinking in biology and architecture alike. In the nervous system, however, form does more than reflect function: neuronal morphology actively constrains input patterns, synaptic integration, and circuit wiring. During postnatal neurodevelopment, dendritic architectures are assembled and remodeled through genetically encoded programs and activities, transforming molecular programs into circuit architecture. However, dendritic morphogenesis......Read more

SUMMARY: Nanopore sequencing measures ionic current as native DNA or RNA molecules move through a biological pore. The resulting ionic current changes are inferred into sequence by Oxford Nanopore Technologies' Dorado basecaller. These data permit direct analysis of nucleotide sequences and modifications. The Dorado basecaller also outputs a move-table that contains approximate mappings between ionic current signal and basecalled sequence. This ionic current information can be visualized at......Read more

Spatial transcriptomics (ST) enables gene expression profiling while preserving the spatial architecture of intact tissue. Analyzing ST data often proceeds by first extracting cell-level information, typically through cell segmentation or cell-type deconvolution. However, a critical oversight has been that a substantial portion of molecular expression is systematically lost or unannotated by these methods. This lost expression can arise from diverse and biologically important sources like......Read more

Biological structures and the epigenome are intertwined. For example, complex tissues are often the combined products of various groups of spatially patterned cell types with distinct epigenetic states. Furthermore, chromatin at various subnuclear locations within a cell often differ in their epigenetic properties. Thus, a systematic understanding of the relationship between the epigenome and its spatial distribution across biological scales would inform tissue and cellular functions as well as......Read more

Circulating cell-free nucleic acids (cfDNA and cfRNA) found in blood and other biofluids are promising biomarkers for cancer. However, current methods exploiting tumor-derived cfDNA (ctDNA) are not sensitive enough in detecting minimal residual disease and early stages of cancer when it is more treatable. Small RNAs and RNA fragments (sRNA) can potentially provide higher detection sensitivity and specificity than ctDNA. Sequencing analysis of the variety of sRNAs representing the entire RNA......Read more

Post-transcriptional regulation of gene expression is orchestrated by RNA-binding proteins (RBPs), which regulate key aspects of the RNA life cycle including splicing, localization, translation, and decay. Although RBPs have been initially considered as isolated regulators, it is becoming clear that RNA molecules are commonly bound by several RBPs whose coordination directs their fate. These combinatorial interactions produce complex, context-dependent post-transcriptional regulatory networks......Read more

Long-read RNA sequencing is a powerful technology to link transcript structures to genetic variants, but this type of analysis is not often performed owing to the lack of end-user tools. Here we introduce longcallR for joint single-nucleotide polymorphism calling, haplotype phasing and allele-specific analysis, which achieves high accuracy on benchmark datasets. Applied to 202 human samples, longcallR identified 88 significant allele-specific splicing events per sample on average, of which 46%......Read more

In vivo CRISPR screening with joint transcriptomic and chromatin readouts has been limited by inefficient recovery of gRNAs from nuclei. Here, we developed in vivo multiomic Perturb-seq, an effective platform combining nuclear transcript anchoring with gRNA-specific capture and amplification to enable high-fidelity, high-recovery gRNA assignment and scalable perturbation-resolved single-nucleus multiomics. Applying this platform to interrogate neurodevelopmental disorder risk genes in the......Read more

Transfer RNAs (tRNAs) are utilized by the ribosome to decode the nucleic acid alphabet. tRNA structure, stability, aminoacylation efficiency, and decoding efficacy are governed by their extensive post-transcriptional modifications. In most studies, individual tRNAs are generated using in vitro transcription, which produces tRNAs devoid of these critical site-specific modifications, negatively affecting translation yields and fidelity. To address this challenge, we have developed a purification......Read more

Protein macrocyclization is a pivotal process in the stabilization of protein structures, significantly enhancing their proteolytic stability and thermostability. While nature elegantly accomplishes this through a diverse family of ligases, laboratory methods typically rely on recombinant proteins engineered with unnatural amino acids and cysteine crosslinkers. Herein, we present a biological metabolite 4-hydroxynonenal (4-HNE) to selectively modify nucleophilic amino acids, cysteine (Cys),......Read more

The interplay of genetic and epigenetic information shapes cell identity, development, and disease. However, standard methods for profiling DNA modifications (e.g., bisulfite sequencing) rely on selective C-to-T conversions, hindering the simultaneous examination of both genetic and epigenetic information. Here, we introduce Integrated Sequencing, which provides high-fidelity mapping of DNA modifications while preserving the native four-base genetic code in single DNA molecules. Integrated-Seq......Read more

Motor enzymes that interact with DNA are essential for replicative biological processes. In nanopore sequencing, a motor enzyme controls the motion of a nucleic acid through a protein nanopore, and sequence-dependent blockages of an ion current flowing through the nanopore are used to decode the DNA sequence. The kinetics of these enzymes are sequence-dependent and can serve as an additional source of information during sequencing. Here, we use Mutual Information (MI) to quantify the......Read more

The brain displays the richest repertoire of post-transcriptional mechanisms regulating mRNA translation^(1-11). Among these, alternative splicing has been shown to drive cell-type specificity and, when disrupted, is strongly linked to neurological disorders^(12-17). However, genome-wide measurements of mRNA translation with isoform sensitivity at single-cell resolution have not been achieved. To address this, we deployed Surveying Ribosomal Targets by APOBEC-Mediated Profiling (Ribo-STAMP)......Read more

Recent studies have revealed many alternative exons differentially spliced across diverse neuron types in the mammalian brain, but their links to neuronal physiology remain unclear. Here we characterize a deeply conserved microexon E35a in Ank3 encoding ankyrin-G (AnkG), a multifaceted adaptor protein best known as a master organizer of the axon initial segment (AIS) and as a leading genetic risk factor for bipolar disorder. E35a is predominantly skipped in cortical glutamatergic neurons but......Read more

The CRISPR-Cas9 system has become a widely used gene-editing tool. Here, we present a new method for small-molecule control of CRISPR-Cas9 using bio-orthogonal chemistry between tetrazine (Tz) and trans-cyclooctene (TCO). We carried out molecular modeling studies and identified a unique position on single guide RNA (sgRNA) that can be site-specifically tagged with Tz without disrupting its activity. We also synthesized a series of TCO-modified CRISPR suppressors. When exogenously added, they......Read more

Molecular recording is an emerging paradigm for measuring biology over time. Enhancer-mediated genomic recording of activity in multiplex (ENGRAM) is a recently described synthetic biology circuit architecture that converts the transient activity of cis-regulatory elements (CREs) into stable genomic records that can be retrospectively recovered via DNA sequencing. Here we provide a step-by-step protocol for conducting ENGRAM experiments and analyzing the resulting data. We also describe key......Read more

Advanced nanopore measurements allow structural probing of molecules with high spatial and temporal resolution. We report high signal-to-noise, 1-10 MHz bandwidth, translocation measurements of the multistate folding of heme protein cytochrome c in KCl solution through optimally designed silicon nitride pores of 2.3 - 3.3 nm diameter and 3.6-3.8 nm effective thickness, and an optimal concentration of a denaturant (Gdm-Cl). The pore diameter is slightly smaller than the protein's size, forcing......Read more

CRISPR screens have become essential tools for systematically probing gene function from basic biology to drug discovery, yet important frontiers remain beyond genome scale. Probing regulatory elements, interpreting genetic variants, and mapping genetic interactions all challenge the sensitivity and scalability of existing approaches. Here we introduce two synergistic technologies to address these limitations. PORTAL (Perturbation Output via Reporter Transcriptional Activity in Lineages) shifts......Read more

The nucleosome acidic patch is a hub of coordinated engagement by proteins that regulate genomic function. Here we report that S. cerevisiae Dot5 contains an arginine-rich HMGN-like motif that mediates nucleosome acidic patch binding and is required for the cell growth, DNA repair and heterochromatin defects exhibited when the protein is overexpressed. The heterologous expression of camelid single chain antibodies to the nucleosome acidic patch confers a similar range of phenotypes, with the......Read more

TFIID is instrumental in recognizing promoter sequences and initiating transcription, yet a cohesive understanding of how this complex interacts with and functions at different promoter types in vivo is still lacking. Here, we employed ChIP-nexus to capture high-resolution binding footprints of all Drosophila TFIID subunits across the genome. These footprints reveal TFIID sub-modules whose DNA contacts suggest new structural details. At different promoter types, the footprints of the TAFs are......Read more

Nociceptive afferent neurons within the dorsal root ganglion (DRG) detect and relay painful peripheral stimuli, and the malfunctioning of this process leads to sustained pain states. Animal model studies have been invaluable for demonstrating the importance of the DRG nociceptor in pain sensation and the development of related analgesic targets. However, a human in vitro model of nociception is essential to confirming the relevance of preclinical findings for therapeutic drug development. We......Read more

As DNA sequencing technologies improve, it is becoming easier to sequence and assemble new genomes from non-model organisms. However, before a newly assembled genome sequence can be used as a reference, it must be annotated with genes and other features. This can be conducted by individual laboratories using publicly available software. Modern genome annotations integrate gene predictions from the assembled DNA sequence with gene homology information from other high-quality reference genomes and......Read more

The ability to design and engineer mammalian genomes across arbitrary length scales would transform biology and medicine. Such capabilities would enable the systematic dissection of mechanisms governing gene regulation and the influence of complex haplotypes on human traits and disease. They would also facilitate the engineering of disease models that more faithfully recapitulate human physiology and of next-generation cell therapies harboring sophisticated genetic circuits. Over the past......Read more

Human embryonic stem cells (hESCs) provide a powerful in vitro model to study lineage specification and the regulatory programs underlying early human development. Here, we present a high-resolution, temporal multi-omics dataset tracking mRNA, translation, and protein expression dynamics during hESC differentiation into definitive endoderm and subsequent polyhormonal (PH) cells, a key pancreatic lineage. RNA-seq, ribosome profiling, and quantitative mass spectrometry-based proteomics were......Read more

CONCLUSION: CIPHER formulates aggregate signature design as a joint optimization problem over decoding accuracy and experimental measurability. This enables systematic, scRNA-seq-aligned feature design for scalable spatial transcriptomics based on aggregate measurements....Read more

Ribosomal RNA (rRNA) methylation is conserved across biology, yet the effect of rRNA methylation on ribosome function is poorly understood. In this work, we identify a biological function for the rRNA 2'-O-methylcytidine methyltransferase TlyA, conserved between Bacillus subtilis and Mycobacterium tuberculosis (Mtb). The tlyA deletion in B. subtilis confers a cold sensitive phenotype and resistance to aminoglycoside and cyclic polypeptide antibiotics. We show that ∆tlyA cells have ribosome......Read more

Structure-switching aptamers transduce target-induced conformational changes into detectable signals, enabling the specific detection of small molecules with limited surface area and charge. Understanding these structural transitions is critical for the rational design of aptamers in downstream biosensing. However, current methods lack the scalability and high spatiotemporal resolution to characterize and resolve these structural dynamics within a single unified platform. Here, we report a......Read more

Precise, reversible control of transgene expression is essential for safe and durable gene therapy, yet current inducible systems remain difficult to translate in vivo due to large size, limited induction duration, and dependence on immunogenic regulators. Here we present the adaptamer (ADAR modulatable aptamer), a compact (<120 bp) RNA switch that couples an FDA-approved small-molecule-responsive aptamer with endogenous ADAR-mediated RNA editing to regulate expression. Ligand binding stabilizes......Read more

We describe application of Nanopore direct tRNA sequencing and ionic current signal for isodecoder-level analysis of bacterial and yeast tRNA. The computational advance is the application of deep learning for predicting tRNA isodecoders directly from raw nanopore ionic current signals. We then combined ionic current analysis with a pairwise sequence alignment strategy to improve tRNA sequence alignments. We applied this strategy to Nanopore data for E. coli and S. cerevisiae tRNA and aligned......Read more

TFIID is instrumental in recognizing promoter sequences and initiating transcription, yet a cohesive understanding of how this complex interacts with and functions at different promoter types in vivo is still lacking. Here, we employed ChIP-nexus to capture high-resolution binding footprints of all Drosophila TFIID subunits across the genome. These footprints reveal TFIID sub-modules whose DNA contacts suggest new structural details. At different promoter types, the footprints of the TAFs are......Read more

Screening soluble protein ligands is essential for understanding signaling interactions and enabling drug discovery. Currently, screens require arrayed formats because ligand diffusibility causes non-cell-autonomous effects. To enable multiplexed pooled assaying, we developed Obligate Autocrine Signaling In situ Screening (OASIS). Using lentiviral delivery of genetically barcoded ligands fused to a tethering domain, we anchor proteins to the expressing cell's outer membrane, exclusively......Read more

Deep mutational scanning couples a protein's activity to DNA sequencing for high-throughput assessment of the effects of all single amino acid substitutions, but it largely uses indirect assays, like cell proliferation, as proxy for protein activity. Here, we covalently link variant proteins in vivo to an RNA barcode by fusing them to Escherichia coli tRNA (m(5)U(54)) methyltransferase TrmA (E358Q). This methyltransferase mutant forms a covalent bond with a tRNA T-arm stem-loop sequence, which......Read more

Pedigree analysis remains the gold standard for rare disease diagnostics, yet whole genome sequencing studies typically omit critical regions like centromeres, telomeres, and acrocentric chromosome p-arms. Here, we present telomere-to-telomere (T2T) reference genomes for four self-identified African American individuals of admixed ancestry spanning three generations. Our parent-of-origin assigned, chromosome-level assemblies revealed precise meiotic recombination breakpoints in previously......Read more

Although there are many known risk alleles associated with adult-onset psychiatric disorders such as schizophrenia [1-4], bipolar disorder [5-7], and major depressive disorder [8-10], the mechanistic links between these risk alleles and disease pathology, especially on a circuit-level, remain unclear. In vivo pooled CRISPR screening with single‑cell readout (in vivo Perturb‑seq) has begun to fill this gap by mapping causal genes to defined cell states directly in animal tissues [11-14]. Here, we......Read more

This work aims to improve RNA synthesis and manufacturing, exemplified by T7 RNA polymerase-driven in vitro transcription. We developed a novel, plasmid-compatible co-tethering strategy that functionally couples RNA polymerase to its promoter DNA immobilized on a solid matrix. As demonstrated recently, co-tethering enhances promoter binding, increases RNA yield, and suppresses RNA re-binding, especially under high-salt conditions, thereby reducing double-stranded RNA by-products. The system......Read more

Frailty is an age-related geriatric syndrome with largely unknown mechanisms. We conducted a longitudinal study of aging C57BL/6JNIA mice (females; n = 40, male; n = 49), measured frailty index and derived DNA methylation data from PBMCs. We selected frailty-related differentially methylated CpGs and determined differentially methylated regions (DMRs), focusing on both age-independent and -dependent frailty, and using both mixed-sex and sex-stratified subgroups. We propose a joint set of 925......Read more

Advanced nanopore measurements allow structural probing of molecules with high spatial and temporal resolution. We report high signal-to-noise, 1-10 MHz bandwidth, translocation measurements of the multi-state folding of heme protein cytochrome c in KCl solution through optimally designed silicon nitride pores of 2.3-3.3 nm diameter and 3.6-3.8 nm effective thickness, and an optimal concentration of a denaturant (Gdm-Cl). The pore diameter is slightly smaller than the protein size, forcing the......Read more

Recent studies have revealed many alternative exons differentially spliced across diverse neuron types in the mammalian brain, but their links to neuronal physiology remain unclear. Here we characterize a deeply conserved microexon E35a in Ank3 encoding ankyrin-G (AnkG), a multifaceted adaptor protein best known as a master organizer of the axon initial segment (AIS) and as a leading genetic risk factor for bipolar disorder. E35a is predominantly skipped in cortical glutamatergic neurons but......Read more

Despite recent advances in technology to map RNA chemical modifications transcriptome-wide, the distribution of N¹-methyladenosine (m¹A) in mRNA remains contested, hindering a clear understanding of its function. Additionally, the enzyme(s) that installs the majority of reported mRNA m¹A sites has yet to be identified. In this study, we characterized TRMT61B, an m¹A methyltransferase known to methylate mitochondrial RNAs, but whose sequence preferences have been underexplored. By integrating......Read more

While most RNA-seq methods sequence amplified cDNA molecules, the advent of direct RNA sequencing (DRS) empowered the scientific community to read native RNA. This technology unlocked characterization of natural RNA modifications and long RNA isoforms without the inherent biases of PCR amplification. In the library preparation prior to Oxford Nanopore (ONT) sequencing, polyadenylated RNAs are copied by a reverse transcriptase (RT) to generate an RNA-cDNA hybrid. The step aims to eliminate the......Read more

Recent studies have uncovered a number of functional RNA structures in RNA viruses, yet their regulatory roles remain poorly understood. Here, using an unbiased proteomic approach alongside targeted biochemical assays, we investigate a previously uncharacterized functional pseudoknot (pk1) within the hepatitis C virus coding region and show that it stably interacts with host ribosomes, inhibiting translation and potentially acting as a regulator between viral translation and RNA genome......Read more

RNA-binding proteins (RBP) are important regulators of posttranscriptional gene expression. Understanding which and how RBPs promote cancer progression is crucial for cancers that lack effective targeted therapies, such as triple-negative breast cancer (TNBC). In this study, we employed both in vitro and in vivo pooled CRISPR/Cas9 screening to identify 50 RBP candidates essential for TNBC cell survival. Integrated enhanced cross-linking and immunoprecipitation and RNA sequencing analysis......Read more

Metastasis to the liver drives mortality in pancreatic ductal adenocarcinoma (PDAC), yet mechanisms of colonization remain unclear. Using genomic barcoding, we developed a clonal competition model under immune surveillance, isolating murine PDAC subclones with high or low liver-colonization potential. Combined transcriptome and chromatin-accessibility analyses revealed a distinct "metastatic-potential axis," separate from the normal-to-PDAC and classical-basal axes. We established "MetScore" as......Read more

Transfer RNA-derived small RNAs (tsRNAs) have emerged as crucial players in diverse biological processes. Yet, their involvement in lipid metabolism and cardiovascular disease remains elusive. Using an advanced PANDORA-seq method, we identify tsRNA-Glu-CTC as the most abundant tsRNA in mouse liver. Intriguingly, tsRNA-Glu-CTC is cholesterol responsive. Overexpression of tsRNA-Glu-CTC elicits hypercholesterolemia and hepatic steatosis, whereas its knockdown protects against diet-induced......Read more

Advances in spatially resolved transcriptomics provide unprecedented opportunities to characterise intercellular communication pathways. However, robust and computationally efficient incorporation of spatial information into intercellular communication inference remains challenging. Here, we present LARIS (Ligand And Receptor Interaction analysis in Spatial transcriptomics), an accurate and scalable method that identifies cell type-specific and spatially restricted ligand-receptor (LR)......Read more

Surface-enhanced Raman scattering (SERS) on lithographically fabricated structures has been extensively explored. The consensus is that non-resonant single-molecule sensitivity (SMSERS) is not achievable with isolated structures. Difficult to manufacture closely spaced pairs of structures, akin to the closely approaching (< 1 nm separation) spheres identified in colloidal clusters, are thought to be required. This scale is beyond the capability of current lithographic approaches. FDTD modeling......Read more

CONCLUSION: SVs are enriched and difficult to call in LCRs. Special care needs to be taken for calling and analyzing these variants....Read more

SYNGAP1 regulates synaptic plasticity through interactions with scaffold proteins and modulation of Ras and Rap GTPase signaling. Human SYNGAP1 mutations are linked to intellectual disability, epilepsy, and autism. In mice, Syngap1 haploinsufficiency causes impaired LTP, premature maturation of dendritic spines, learning disabilities, and seizures, reflecting the human phenotypes of SYNGAP1 syndrome. Recently, SYNGAP1 was shown to influence cortical neurogenesis and progenitor proliferation in......Read more