Recent Publications

The nucleosome acidic patch is a hub of coordinated engagement by proteins that regulate genomic function. Here, we report that Saccharomyces 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,......Read more

The rapid growth of RNA-based therapeutics demands accurate sequencing of all RNA species, including minor and modified variants. Conventional LC-MS/MS typically confirms only a predefined target sequence rather than determining RNA sequences de novo from the analyzed sample, thereby overlooking coexisting impurities and modifications. Here, we present 3D NGMS-Seq, a three-dimensional next-generation mass spectrometry-based sequencing platform for de novo direct sequencing of mixed RNA samples......Read more

Comprehensively mapping the relationship between genotype and phenotype offers essential insights into how a cell's state arises from its genetic components. Toward this goal, we generated an expressed genome-scale CRISPRi perturbation cell atlas in KOLF2.1J human induced pluripotent stem cells, mapping transcriptional phenotypes associated with 11,692 perturbed genes across >2.5 million single cells. Using correlations among perturbed phenotypes, we created a cell map of the pluripotent state,......Read more

SUMMARY: Association measurements, such as mutual information (MI), are fundamental in the analysis of cancer multi-omics data for identifying cancer-related genes, gene signatures, and gene regulatory networks, thereby shedding light on tumor development, progression, and treatment. Confounding factors, including tumor purity and mutation burden, can bias association measurements in MI, potentially leading to the misclassification of passenger events as drivers. Conditional mutual information......Read more

Endogenous gene tagging using CRISPR has changed the understanding of the role played by different proteins due to the ability to track and study proteins in their natural state. With CRISPR-based gene tagging, it is possible to insert fluorescent, luminescent, epitope, affinity, and proximity labels into the target protein at its endogenous genomic location without affecting its physiological expression and dynamics. Here, we discuss the DNA-repair mechanisms employed in endogenous gene......Read more

Lipid peroxidation products modify proteins during oxidative stress, but the residue-pair connectivity and structural consequences of these reactions remain difficult to define. Here, we redefine the lipid peroxidation product 4-oxo-2-nonenal (4-ONE) from a damaging electrophile into a chemoselective Cys-Lys covalent crosslinker. Through a chemoselective, two-step pathway, Michael addition to cysteine activates a latent aldehyde that cyclizes with lysine to form a stable pyrrole linkage under......Read more

N⁶-methyladenosine (m⁶A) on RNA plays diverse regulatory roles, yet its spatial distribution within tissues remains largely unexplored. Here we introduce m⁶A-ARTR-DBiT, a spatial m⁶A profiling assay that leverages reverse-transcription-based detection and deterministic barcoding in tissue to map transcriptome-wide m⁶A distribution while preserving native tissue context. Applying m⁶A-ARTR-DBiT to mouse embryonic tissues and adult brains generates spatially resolved m⁶A landscapes and reveals......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

Optical pooled screening (OPS) has emerged as a powerful technique for functional genomics, enabling researchers to link genetic perturbations with complex cellular morphological phenotypes at scale. However, OPS data analysis presents challenges due to massive datasets, complex multi-modal integration requirements, and the absence of standardized frameworks. Here, we present Brieflow, a computational pipeline for end-to-end analysis of fixed-cell optical pooled screening data. We demonstrate......Read more

Pangenome projects have enhanced our understanding of human genomic and genetic diversity, but repetitive regions are still challenging to assemble and yet medically important. Here we generate 20 near-complete haplotypes from 10 Japanese male individuals using three complementary long-read and long-range datasets and construct a pangenome graph from these haplotype-resolved assemblies. All haplotypes achieve an N50 value exceeding 100 Mbp for gapless contigs. We substantially improve the......Read more

Many anti-cancer clastogens are known to induce DNA replication stress and ultimately DNA double-strand breaks (DSBs), one of the most deleterious forms of DNA damage and threat to genome integrity. However, the mechanism(s) by which these chemicals cause DSB is unclear. Our previous work suggested that they induce DSB at transcriptionally up-regulated genes, particularly those oriented in a head-on configuration with respect to incoming replication. Here, we rigorously test this model by......Read more

CONCLUSIONS: The transition from RNA002 to RNA004 substantially improves sequencing accuracy and modification calling. However, Nanopore direct RNA sequencing-based RNA modification detection requires careful validation. We recommend combining Nanopore direct RNA sequencing with orthogonal methods and appropriate filtering strategies for increased confidence in modification calls....Read more

Aneuploidy is common in cancer and has been implicated in promoting tumor progression, yet the underlying mechanisms remain poorly understood. By generating models of aneuploidy, we found that aneuploidy confers resistance to reactive oxygen species (ROS)-mediated cell death, independent of the specific chromosomes gained or lost. Mechanistically, poly(ADP-ribose) polymerase 1 (PARP1) is suppressed in aneuploid cells, which inhibits PARP1-mediated cell death (parthanatos). We validated......Read more

Because all known living organisms are made from at least 20 canonical amino acids, the feasibility of life using a more simplified alphabet remains unclear. In this work, we leveraged computational design and synthetic biology to explore building a cell from a 19-amino acid alphabet. Initial analyses suggested that isoleucine (Ile) may be dispensable, which we confirmed by directly replacing Ile residues in essential proteins in Escherichia coli. Critically, protein language models and......Read more

Despite recent advances in technology to map RNA chemical modifications transcriptome-wide, the distribution of N1-methyladenosine (m1A) in messenger RNA (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......Read more

Adeno-associated viruses (AAVs) are preferred gene therapy vectors because of their versatility, durability and safety profile. Here, we demonstrate extensive chimerism, manifesting as pervasive barcode swapping, among complex recombinant AAV (rAAV) libraries that are packaged as a pool. The observed chimerism is length and homology dependent but capsid independent, in some cases affecting the majority of packaged rAAV genomes. These results have implications for the design and deployment of......Read more

RNA-binding proteins (RBPs) orchestrate post-transcriptional processes, including splicing, cleavage and polyadenylation, and translation. Our updated RBP resource integrates data from 92 additional RBPs (286 in total) profiled by enhanced CLIP (eCLIP), enabling comprehensive characterization of RNA elements within human K562 and HepG2 cells. To interrogate RBP-binding syntax, we trained deep-learning models on eCLIP profiles, allowing us to score genetic variants and quantify constraints on......Read more

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

Chemoproteomic strategies have revolutionized proteome annotation by targeting nucleophilic and redox-active side chains. However, the primary amides of asparagine (Asn) and glutamine (Gln) have long lacked robust chemical tools for proteome-wide interrogation. We report a chemoselective palladium-mediated dehydration that converts Asn/Gln amides to nitriles under mild aqueous conditions. This transformation enables the first proteome-wide mapping of chemically addressable Asn/Gln sites in......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

Small RNAs and RNA fragments (sRNAs) found in blood and other biofluids have emerged as promising biomarkers for cancer and other pathologies. Sequencing analysis of sRNAs representing the entire RNA fragmentome could improve understanding of their roles in cancer development and be used for discovery of new biomarkers, cancer detection, and personalized treatment management. Conventional methods of sRNA-seq library preparation are limited to detection of sRNAs with 5'-P and 3'-OH ends (sRNA......Read more

Reactive aldehyde metabolites are commonly viewed as drivers of nonspecific protein damage and stochastic cross-linking. Here, we show that cooperative aldehyde chemistry can generate multicomponent, mass-consistent electrophilic intermediates in water with strong lysine bias and site selectivity. Specifically, malondialdehyde (MDA) couples with monoaldehydes (e.g., acetaldehyde and benzaldehyde) to form a cooperative intermediate that channels reactivity toward lysine, yielding chemically......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

Transfer RNA has long served as an exemplar of a thermodynamically stable, structured RNA. Yet, it undergoes significant structural changes upon binding and catalysis by diverse modification enzymes. We leveraged optical binding assays and single-molecule FRET to observe the structural dynamics of two yeast tRNAs, in isolation, and upon interaction with the conserved pseudouridine synthase Pus4/TruB. We show that unmodified and pseudouridylated tRNA^(eMet(CAU)) and tRNA^(Thr(AGU)) all sample......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

Amides are ubiquitous in pharmaceuticals, natural products, and biomolecules, owing to their exceptional stability and hydrogen-bonding capacity. Among the amino acids, asparagine (Asn) and glutamine (Gln) contain neutral primary amide side chains and constitute over 8% of the human proteome. Despite their abundance, these residues have remained largely inaccessible to selective chemical modification due to their low intrinsic reactivity and the propensity of proteinogenic side chains to poison......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

LINE-1 (L1) retrotransposons are increasingly recognized as key players in cancer biology. While traditionally viewed as mutators through their endonuclease (EN) activity, recent findings show that L1 elements can also activate innate immune pathways independently of EN activity, particularly type I interferon signaling via "viral mimicry." These dual functions position L1 at the intersection of genome regulation and innate immune response. From this perspective, this mini-review discusses......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