Recent Publications

Protein-based biological nanopores have emerged as versatile tools for sequencing biopolymers, identifying molecules, and probing reactions at the single-molecule level. Many biological nanopores have been characterized, revealing the significance of nanopore size, shape, and chemical composition in molecular detection and discrimination. Advancements in understanding the nanofluidic properties of nanopores have complemented engineering approaches expanding the possible applications of nanopore......Read more

As a liquid approaches its glass transition, its dynamics turns heterogeneous: mobile and immobile regions coexist, and the four-point susceptibility χ (4) that quantifies this heterogeneity grows sharply. However, collective signals such as tracer trajectories, overlap functions, or mean fields are generated by the very particles they monitor. Here, we compute exactly what conditioning on such a shared record does to the population that produced it; the guiding example is a tagged particle and......Read more

Advances in single-cell sequencing and spatial omics technologies have enhanced our understanding of diverse cellular identities, compositions, architectures and functions. However, the single-cell three-dimensional (3D) organization of the epigenome is still not well understood, due to an absence of spatial single-cell methods that allow high-resolution, locus-specific detection of combinations of epigenetic marks while maintaining 3D genome organization. Here we develop epigenetic proximity......Read more

Transcription factors (TFs) that can recognize specific DNA sequences within nucleosomes play key roles in regulating cell fate transitions, but what regulates their condition-dependent binding to nucleosomes remains poorly understood. Here, we determined the direct effects of H3K27ac, H3K27me3 and H3K9me3 on binding by 7 human TFs to a highly diverse library of nucleosomes. We found that these marks influence the preference of FOXA1 and PAX7 to bind as monomers versus dimers to nucleosomes.......Read more

TFIID is instrumental in recognizing promoter sequences and initiating transcription, yet a cohesive understanding of how this complex engages 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 agree with cryo-EM structures and point to new structural details. At different promoter types, the......Read more

Noncoding sequences comprise most of the human genome; however, their functional significance remains largely elusive. Using CRISPR tiling mutagenesis with near-PAMless, high-fidelity SpRY-Cas9, we mapped the fitness landscape of an entire human gene, the 10-kb MYC locus, at base-pair resolution. This unbiased interrogation revealed that 67% of functionally essential base pairs are noncoding. Essential noncoding base pairs are under strong constraint in the human population but show limited......Read more

Genetic variation can alter organ structure and function. Comparative statistical analysis of organs across genetic backgrounds requires spatial, single-cell, atlas-scale data in replicates. Here we introduce atlas-scale transcriptome localization using aggregate signatures (ATLAS), a scalable tissue mapping method that learns transcriptional signatures from single-cell RNA sequencing data, encodes them in situ with tens of thousands of oligonucleotide probes and decodes them to infer cell types......Read more

RNA viruses encode multiple layers of information within a single molecule, which functions simultaneously as a template for protein synthesis and as a structured regulatory RNA. These structures control key aspects of viral gene expression, govern host interactions, and mediate immune evasion. Although the prevalence of RNA structure in viral genomes is now well established, only recently have functional and mechanistic studies begun to uncover how these elements act as regulatory modules that......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

Mutations in GRN and C9orf72 are common causes of frontotemporal dementia (FTD). While it is well-recognized that GRN or C9orf72 mutations also increase the risk of autoimmune diseases, the intersection of innate and adaptive immunity, neuroinflammation, and neurodegeneration in FTD remains unclear. Here we show that concurrent loss of Grn and C9orf72 breaks immune tolerance by activating the pattern recognition receptor, TLR7, leading to clonal expansion of age-associated B cells and activated......Read more

Noncoding regulatory elements in the human genome can control gene expression through distinct mechanisms, including acting as enhancers, silencers, or topological elements. However, we lack tools to identify all such classes systematically and quantify their effects on gene expression in an endogenous genomic context. Here we develop Swap-seq, a method that combines pooled twin prime editing with flow-sorting to delete hundreds of endogenous regulatory sequences and measure their quantitative......Read more

Centromeres play essential roles in chromosome segregation and genome stability, yet they remain among the least characterized regions of the human genome. Despite advances in long-read sequencing and complete genome assembly, the extreme repetitiveness and structural complexity of these regions still challenge population-scale analysis, obscuring their mutational dynamics. The Human Pangenome Reference Consortium has now accurately assembled over 6,000 centromeres, providing an opportunity to......Read more

All-atom molecular dynamics has become an indispensable tool in the 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. Alone, such experiments 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......Read more

Pseudouridine is the most abundant RNA base modification due to its prevalence in tRNA and rRNA, where it serves as a key modulator of structure and function. Yet, in the widely used model organism, the budding yeast Saccharomyces cerevisiae, tRNA pseudouridine sites have not been comprehensively annotated. Here, we performed two high-throughput methods established for detecting tRNA pseudouridylation positions: Nanopore direct RNA sequencing (DRS) and 2-bromoacrylamide-assisted cyclization......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 (adenosine deaminases acting on RNA [ADAR]-modulatable aptamer), a compact (<120 base pair) RNA switch that couples FDA-approved small-molecule-responsive aptamers with endogenous ADAR-mediated RNA editing to......Read more

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

The human microbiome exerts broad influence in health and disease with associative studies implicating the microbiome in influencing immunity, cancer outcomes, and neurodegeneration. However, the molecular mediators of microbe-host communication remain poorly defined. Bacterial microproteins from the microbiome represent a largely uncharacterized class of potential regulators of host immunity. Here, we utilize functional genomics to interrogate 3,552 microproteins in order to identify novel......Read more

Protein-coding genes mapping to high-identity segmental duplications (SDs) have been difficult to annotate and characterize and are the source of most previously unknown protein-coding genes being discovered as part of the human pangenome. Here, we combine long-read assembled human genomes (298) and long-read transcriptome data (5.6 billion full-length cDNA from 83 tissues) to phylogenetically interrogate 493 gene families discovering 2713 potentially copy number polymorphic genes not present in......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

Low-affinity transcription-factor (TF) motifs are an important element of the cis-regulatory code, yet they are notoriously difficult to map and mechanistically incompletely understood, limiting our ability to interpret non-coding variation in development, evolution, and disease. Here, we investigate their role in pioneering and leverage sequence-to-profile models of chromatin accessibility in mouse embryonic stem cells to reliably map and interpret low-affinity motifs across the genome. We find......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

With this status report, we aim to provide a timely snapshot of the protein engineering field as a broad and rapidly advancing discipline that integrates computational, molecular biology, structure-guided, evolutionary, and synthetic approaches to create new and improved proteins with tailored structures and useful functions. The report is organized into eight thematic areas spanning core methodologies and major application domains, including enzymes, therapeutics, detection, synthetic biology,......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