Recent Publications

Nanopore sensors have demonstrated single-molecule resolution for nucleic acids and small molecules, yet the discrimination and detection of folded proteins in complex biological matrices remains a challenge. Insulin is a demanding test case: it circulates in blood at picomolar concentrations, differs from clinically used pharmaceutical analogues by as little as a single amino acid, and its continuous monitoring would close the remaining open loop in artificial pancreas systems. Here, we show......Read more

In most cells, Malat1 long noncoding RNA localizes to the nucleus where it affects splicing and chromatin function. In mouse neurons, Malat1 is exported to the cytoplasm where it is translated to generate the M1 micropeptide. Here, we characterize an internal ribosome entry site (IRES) required for M1 translation that is sufficient to induce cap-independent expression of a downstream open reading frame. In vivo chemical probing and structural modeling defined a three-stem-loop RNA secondary......Read more

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Stem cells self-organize into models of early development, but even under carefully controlled conditions, the resulting organoids exhibit substantial "inter-individual" heterogeneity. To explore its origins, we generated monoclonal gastruloids, each derived from a single mouse embryonic stem cell (mESC). Molecular profiling revealed extensive heterogeneity: some gastruloids failed to develop, others were biased toward mesodermal or neural fates, and yet others contained cell types atypical of......Read more

N ⁶-methyladenosine (m⁶A) regulates nearly every aspect of messenger RNA (mRNA) processing and function, impacting downstream gene expression programs. Changes in m⁶A have been implicated in many different types of cancer, and inhibition of m⁶A installation is emerging as a cancer therapeutic strategy. However, chemoresistance remains a significant clinical challenge in the treatment of glioblastoma (GBM). We established GBM cell culture models of acquired temozolomide (TMZ) resistance, analyzed......Read more

Machine learning models in single-cell biology increasingly forecast differentiation, reprogramming and therapeutic response from early transcriptomic profiles. Testing whether a model has learned real biology requires held-out cells. Single-cell data, however, are grouped: cells from the same clone, patient or batch share the same label. A random split therefore places relatives of each test cell, carrying its label, in the training set, and a model can score well by memorizing a relative......Read more

RNA-binding proteins (RBPs) are key regulators of gene expression that shape cellular function in health and disease. However, the roles of RBPs in immune cells within the central nervous system (CNS) remain poorly understood. Here, we identify ARID5A as an RBP highly expressed in microglia and uncover its RNA-mediated regulatory functions using integrated multi-omics analyses of its RNA, DNA, and protein interactions. ARID5A regulates the splicing and translation of its RNA targets, many of......Read more

Post-transcriptional RNA modifications modulate diverse aspects of RNA metabolism. N6-methyladenosine (m6A), one of the most abundant internal RNA modifications, is deposited by the core methyltransferase complex, METTL3 and METTL14. Oxford Nanopore Technologies (ONT) platform permits direct, single RNA molecule sequencing while preserving native modifications. However, without rigorous benchmarking, the accuracy and reproducibility of modification detection remain uncertain. Here, we leveraged......Read more

We report in situ synthesis of iron oxide particles inside silicon nitride nanopores via a chemical reaction, monitored by current readout. Nanopores were formed by electroporation on glass chips (diameters from 1.7 to 11.3 nm), transmission electron microscopy (TEM) drilling (diameters from 6.5 to 64.6 nm), or hydrofluoric acid etching (diameters from 12.6 to 36.2 nm) in 5-20 nm thick membranes. Nanopores seal on time scales from ∼1 ms to ∼3.6 s, across a range of sizes and concentrations. We......Read more

Genomic recording enables transient biological signals to be indelibly captured through DNA alterations, creating a permanent record of cellular history retrievable by sequencing. However, current methods are limited by scarce writing space, typically targeting only one or a few amenable genomic sites and requiring large cell populations for signal reconstruction. Here, we establish Repeats for Genomic Recording (RGRs): sequences with up to 400 copies targetable by a single CRISPR guide RNA,......Read more

Appendage regenerative capacity varies dramatically across species, developmental stages, and anatomical sites, yet comparing functional transitions across organisms remains difficult because gene vocabularies diverge. Cross-species single-cell analysis conventionally collapses divergent genomes to one-to-one orthologs-a reduction that is not neutral. Here, we construct a species-native input representation for allotetraploid Xenopus laevis that preserves duplicated L and S homeologs (96.39%......Read more

Viral genomes encode regulatory RNA structures that orchestrate key steps of viral replication and gene expression. Although these structures are increasingly recognized as critical regulators of viral function, their systematic characterization in an infection context and roles in regulating viral fitness and immune recognition in vivo remain limited. Here, we systematically map and functionally interrogate structured RNA elements across the murine norovirus genome using orthogonal in-cell......Read more

Centromeres ensure chromosome segregation, but their chromatin organization within repetitive alpha-satellite DNA has been difficult to resolve. To address this, we generated haplotype-resolved satellite DNA annotations for the complete diploid T2T-HG002 human genome assembly, then we mapped centromere protein A (CENP-A), H3K9me3, and CpG methylation on ultra-long, adaptively sampled nanopore reads using directed methylation with long-read sequencing (DiMeLo-seq). We find that CENP-A occupies......Read more

Human genome sequencing typically relies on mapping reads to a reference genome to call variants, but this approach introduces technical biases, excluding duplicated and structurally polymorphic regions of the genome. To overcome this, we present a telomere-to-telomere genome benchmark with near-perfect accuracy across 99.4% of the diploid HG002 genome. This benchmark adds 701.4 Mb of autosomal sequence and both sex chromosomes (216.8 Mb), which were absent from prior benchmarks. We annotated......Read more

Centromere identity is specified by CENP-A, a histone H3 variant that epigenetically defines centromere position. How CENP-A is maintained at one location in rapidly evolving centromeric DNA is unknown. Using single-cell-derived clones of human cell lines, we demonstrate heterogeneity in CENP-A position within cell populations at neocentromeres and a native centromere. CENP-A heterogeneity is accompanied by heterogeneous DNA methylation patterns, with DNA methylation shifting according to CENP-A......Read more

Nanopore technology has revolutionized single-molecule science by enabling ultrasensitive characterization of biomolecular structure and interactions via accessible, physics-based tabletop measurements. In a typical experiment, the chemical structure of a biomolecule is determined by measuring a transient blockade of the nanopore ionic current produced by the nanopore passage of the molecule. Nanopore sensing can now deliver a telomere-to-telomere sequence of a human chromosome and is poised to......Read more

Interactions among c is-regulatory elements (CREs) are central to mammalian gene regulation. Long @$$ massively parallel reporter assays (LAMPRAs) integrate combinatorial cloning, molecular barcoding and long- and short-read sequencing, to scalably measure how CRE identities, numbers, spacings, orders, orientations, and interactions shape regulatory output at multi-kilobase length scales. As a proof-of-concept, we assay 36,000 × 5-kb synthetic c is-regulatory loci (sCRLs), each a 5 × 1-kb random......Read more

A pangenome reference overcomes the inherent limitation of any individual reference genome by integrating the variation present in a population. We present the Human Pangenome Reference Consortium's (HPRC) Release 2 (HPRC2), an openly available, second phase pangenome that is an approximately fivefold expansion in genome number over HPRC Release 1 (HPRC1) and measurable improvement in genome completeness, contiguity, and accuracy. Selecting samples with a principled algorithm prioritising common......Read more

RNA modification analysis by LC-MS/MS is central to epitranscriptomics, yet quantitative comparison across laboratories and instrument platforms remains poorly standardized. Here, we performed a community-driven benchmarking study during the first Human RNome Project workshop to systematically evaluate cross-platform reproducibility of ribonucleoside mass spectrometry workflows. Using the same analytical column and gradient, standardized RNA samples, and shared reagents, we compared nucleoside......Read more

Centromeres are essential chromosomal regions that ensure accurate chromosome segregation during cell division, yet their highly repetitive sequence has historically hindered their complete assembly and characterization¹. Consequently, the full spectrum of centromere diversity across individuals, populations and evolutionary contexts remains largely unexplored. Here we address this gap in knowledge by assembling and characterizing 2,110 centromeres from diverse individuals representing 5......Read more

Photochemical control of CRISPR/Cas9 gene editing technology is an important tool for a variety biochemical applications. In the past, either photo-activation or photo-inactivation has been achieved using UV-light responsive chromophores, which are not ideal for in vivo applications. In this work, we describe a coumarin containing phosphoramidite that can be site-specifically incorporated into guide RNA using solid phase synthesis. It will facilitate regulation of Cas9-enabled nuclease activity......Read more

5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) regulate gene expression and exhibit dynamic levels during development and disease. While high-depth, base-resolution studies offer the most detailed view of epigenetic landscapes, many open questions are answered by surveying changes in 5mC/5hmC levels across larger cohorts. Nonetheless, current global quantification methods, including mass spectrometry, are typically limited in accessibility, accuracy, or throughput. Here, to evaluate......Read more

Saccharomyces cerevisiae is an invaluable model in the study of mitochondrial tRNA biology. Yet the positions of modified bases in all yeast mitochondrially encoded tRNAs (mt-tRNAs) are still not fully mapped. We performed Nanopore direct RNA sequencing (DRS) on tRNAs from the crude mitochondrial fraction of yeast to map base modifications across all 24 mt-tRNA isoacceptors. Additionally, we adapted the "D-seq" method to detect dihydrouridine sites in tRNAs, where chemical reduction of......Read more

B cell maturation within the germinal center tissue microenvironment involves immunoglobulin gene diversification by somatic hypermutation (SHM). How three-dimensional (3D) genome architecture influences SHM is not fully understood. We leveraged sequencing-based and image-based 3D genomics and transcriptomics to map single-cell 3D genome organization and gene expression across cell types and states in human tonsils and in B cell lymphoma cell lines. These analyses revealed trajectories of......Read more

Higher-order chromatin structure and DNA methylation are critical for gene regulation, but how these vary across the human body remains unclear. We performed multiomic profiling of three-dimensional (3D) genome structure and DNA methylation for 86,689 single nuclei across 16 tissues, identifying 35 major and 206 cell subtypes. We revealed extensive changes in CG and non-CG methylation across cell types and characterized 3D chromatin structure at an unprecedented cellular resolution. Extensive......Read more

Cell-cycle dynamics of gene expression are fundamental to life, yet their mechanistic basis remains unclear. A prevailing model, derived from cancer cells, posits that RNAs transcribed in one cell-cycle phase peak in abundance in subsequent phases, suggesting temporal segregation of transcriptional regulation and RNA maturation during the cell cycle. However, whether such extensive transcription-to-maturation lags occur in normal cells remains unclear. Here, we report a lack of evidence for......Read more

Multimodal mapping of subcellular protein organization through imaging and interaction proteomics has been mostly confined to large consortia, owing to high per-target costs, reliance on target-specific antibodies or libraries of epitope-tagged cDNAs, and the lack of unified pipelines for coordinated data acquisition. Here, we present HIT-MAP (high-throughput integrated tagging for cell mapping), an end-to-end framework that couples endogenous epitope tagging with optimized wide-field......Read more

MOTIVATION: Existing pangenome file formats are designed for batch processing. Graphs must be loaded into memory, and alignment files must be read sequentially. Indexed file formats that can be used directly from disk would be more appropriate for interactive applications....Read more

Existing notions of pangenome coordinates rely on hard-to-compute multiple sequence alignments. On the other hand, pangenome-wide exact unique matches (multi-MUMs) can be computed efficiently, and represent conserved stretches of columns in the underlying MSA. We introduce Shredtools, which uses multi-MUMs as pangenome waypoints and allows for sophisticated queries in pangenome coordinates. Its primary query is extract, which takes an interval of one sequence and extracts the smallest window......Read more

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

Ion transport in artificial channels that mimic biological ion channel behavior can enable advances in neuromorphic computing and biomolecular sensing. We fabricate atom-scale artificial ion channels in multilayer hexagonal boron nitride (hBN), with dimensions comparable to biological ion channels, and demonstrate a distinct regime of ion transport. Channels ranging from 0.5 to 1.5 nm in diameter reveal unconventional ion transport under conditions of extreme confinement: the conductance G......Read more

G-quadruplexes (G4s), non-canonical DNA structures whose sequence motifs occupy approximately 1% of the human genome, are important for myriad cellular functions, including regulating transcription and replication. Yet they also contribute to genomic instability by increasing mutations and structural variation. Despite their significance, G4 motifs have not been studied in detail across multiple human genomes. Here, we conducted a comprehensive analysis of presence/absence and sequence......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

We introduce SCRIVENER (sequential conjugation and recombination for in vivo elongation of nucleotides with low errors), an in vivo DNA assembly platform that streamlines and scales DNA engineering. SCRIVENER combines bacterial conjugation, in vivo DNA cutting, and homologous recombination to stitch DNA blocks together by mating E. coli in large arrays or pools. This approach is simpler, cheaper, and higher throughput than methods requiring DNA to be moved in and out of cells. We performed over......Read more

Understanding organ formation requires capturing molecular information simultaneously in three-dimensional (3D) space and across developmental time. To this end, we developed 3D DNase-Enhanced Expression Profiling (3DEEP), a tissue-clearing approach that removes genomic DNA to extend spatial transcriptomic profiling hundreds of microns into intact tissues. We applied 3DEEP to neonatal mouse skin, capturing hundreds of developing hair follicles across their organogenesis trajectory. Ordering......Read more

High-throughput screening of protein domains enables the systematic discovery of protein sequences that encode specific cellular functions. Fluorescence-activated cell sorting-based assays have long been the standard readout for such screens but remain time- and resource-intensive, imposing practical limits on library size and coverage. Here we describe a scalable magnetic separation-based workflow that provides an alternative to fluorescence-activated cell sorting for screening large protein......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

Nanopores are compelling tools for ultrafast single-molecule protein analysis. Recent investigations into both enzyme-free and enzyme-regulated transport mechanisms suggest that the faster kinetics of enzyme-free transport enable higher throughput, albeit requiring speed control for accurate protein characterization. Here we investigate the signals obtained from chemically unfolded proteins when transported through wild-type cytotoxin K (CytK) in an enzyme-free manner. We designed, expressed,......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

Biological nanopores can be used to monitor the conformational dynamics of enzymatic reactions in real time and at the single-molecule level. However, the molecular basis of this process and the requirements for efficiently engineering a nanopore for measuring conformational transitions in proteins remain poorly understood. Here, we combine all-atom molecular dynamics simulations and electrophysiology experiments to determine the molecular mechanism and improve the resolution for the recognition......Read more

All-atom molecular dynamics has become an indispensable tool in development of nanopore sensors of biological information. In a typical nanopore experiment, measurements of ionic current flowing through a nanopore report on the chemical structure of biomolecules that pass through the nanopore. Such experiments alone are often insufficient to relate the structure of the biomolecules to the ionic current modulations. The molecular dynamics method can establish such a relationship directly through......Read more

Centromeres safeguard genome integrity, yet their roles in human disease remain understudied because of their repetitive sequence. We present HOROSCOPE, a k-mer-based framework that infers centromere architecture and generates centromere length estimates directly from short-read data. Using a reference atlas of 11,836 centromeres from telomere-to-telomere haplotypes, we derive diagnostic k-mer signatures that classify chromosome-specific architectures with 98.2% precision and 99.1% recall.......Read more

Genomic DNA used as a template for in vitro transcription of RNA can serve as a true negative control for benchmarking RNA modification detection by Nanopore direct RNA sequencing (DRS) models. We generated DRS data for in vitro transcribed (IVT) RNA composed of canonical nucleotides using genomic DNA from a human cell line. We applied Dorado modification calling models to these data, and calculated 9-mer specific false-positive rates for eight RNA modifications as a comparison point for future......Read more

What fraction of the human genome is essential for cellular viability? To date, essentiality in human cells has been mapped almost exclusively at the level of individual open reading frames (ORFs). Whether non-coding regions and broader architectural features of the genome are required for human cells to remain viable, and where the boundaries of any such regions lie, remains largely unexplored. Here we introduce Shred-seq, which couples Type I-C CRISPR-Cas3-mediated deletions with phage......Read more

RNA modifications contribute to the regulatory and structural complexity of RNA molecules, influencing key biological processes. Nanopore direct RNA sequencing (DRS) enables the detection of these modifications at single-molecule resolution without chemical conversion. Oxford Nanopore Technologies recently introduced RNA004 sequencing chemistry and the Dorado basecaller, which improves accuracy and enables the identification of eight RNA modifications. However, the reliability of these......Read more

Epigenetic state modulates gene regulation in a manner not always predictable from DNA sequence alone, yet current genomic deep learning models do not leverage epigenetic state as input. We present MethylSeqNet, a model that conditions pretrained sequence embeddings on CpG methylation, a stable epigenetic mark increasingly available from long-read sequencing data. Using a novel conditioning mechanism enabling scalability and interpretability, MethylSeqNet improves predictions in cases where......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

Understanding how cells change state, interact with their neighbors, and organize into tissues requires recording of cellular lineage history in native spatial context. Here, we present SPACE-seq (spatial tracing enabled by CRISPR-based barcodes and slide-seq), a versatile platform that integrates CRISPR-based lineage recording with spatial transcriptomics to jointly resolve lineage, cell state, and tissue architecture at near-cellular resolution in situ. Using SPACE-seq, we uncovered intratumor......Read more