Recent Publications

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

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

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

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

CRISPR-Cas12a is a versatile biosensing platform that detects sequence-specific DNA or RNA targets via a CRISPR RNA (crRNA) guide. While Cas12a's specificity is dictated by its crRNA, chemical modifications within the crRNA can influence nuclease performance. Here, we examined the effects of two well-known RNA modifications, N⁶-methyladenosine (m⁶A) and 5-methylcytosine (m⁵C), introduced into the different positions of the guide region of a crRNA. Melting temperature (T(m)) analysis showed that......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

CONCLUSION: GLP-1R shows tissue-specific expression and prognostic relevance across cancer types. The low expression of GLP-1R across cancer types and the low frequency of genetic alterations suggest a limited direct role of GLP-1R mutations in cancer progression, but its variable expression may influence tumor behavior. Further research is needed to clarify both the functional role of GLP-1R in cancer biology and the safety and therapeutic potential of GLP-1R agonists in oncology....Read more

Silencing remains a significant challenge for exogenous gene expression, limiting both the penetrance and expressivity of transgenes. In particular, silencing of Cas9 expression is a major technical limitation for many gene editing and CRISPR screening applications. Here, we demonstrate that including introns in Cas9 expression cassettes significantly reduces silencing across multiple cell lines. Notably, the incorporation of an intron into a CRISPRa construct results in reduced silencing,......Read more

MOTIVATION: Allele typing for Human Leukocyte Antigen (HLA) genes has many important clinical applications. Popular short-read typing can only accurately distinguish alleles at the peptide level, which potentially limit our understanding of the effect of variants in non-coding region. Recently, a few methods were declared to distinguish full-field HLA alleles from de-novo assemblies, which motivates us to develop an accurate HLA typing method directly from long reads....Read more

Brain aging is a complex process with profound health and societal consequences. However, the molecular and cellular pathways that govern its temporal progression-along with any cell type-, region-, and sex-specific heterogeneity in such progression-remain poorly defined. Here, we present a transcriptomic atlas of 5.3 million cells from 582 samples spanning 11 brain regions of 55 rhesus macaques (29 female, 26 male), aged 5 months (early life) to 21 years (late adulthood). We annotate 12 major......Read more

Programmable control of gene expression in specific cell types is essential for both basic discovery and therapeutic intervention, yet current strategies lack scalability across diverse cellular contexts. Here, we introduce SPICE (Splicing Proportions In Cell types), an integrated experimental and computational framework that harnesses alternative RNA splicing as a programmable modality for cell type-specific gene regulation. To power SPICE, we constructed a massively parallel reporter assay......Read more

Although we now have a rich toolset for genome editing, an equivalent framework for manipulating the proteome with a comparable flexibility and specificity remains elusive. A promising strategy for "proteome editing" is to use bifunctional molecules (e.g. PROteolysis-Targeting Chimeras or PROTACs¹) that bring a target protein into proximity with a degradation or stabilization effector, but their broader application is constrained by a limited repertoire of well-characterized target or effector......Read more

RNA polymerase I inhibition affects rRNA synthesis from rDNA clusters residing in nucleolar organizer regions (NORs). Here we have demonstrated RNA Pol I inhibition disrupts nucleolar architecture, NPM1 localization, and alters the chromatin landscape using coordinated eraser and writer enzymes. siRNA mediated depletion or dissociation of NPM1 allows HDAC1 loading on the chromatin. HDAC1 mediated deacetylation of H3K9ac creates H3K9 that undegoes SUV39H1-mediated methylation. The stripping of......Read more

The ability to map messenger RNA (mRNA) molecules from individual cells using next-generation sequencing technologies, known as single-cell RNA-seq (scRNA-seq), is transforming biology by redefining cellular identities with unmatched detail. However, all current protocols depend on copying RNA into complementary DNA with a single reverse transcriptase (RT) derived from murine leukemia virus, which is an RT enzyme known for low processivity and limited ability to unfold complex RNA structures.......Read more

L1 comprises 17% of the human genome, with 50-150 full-length sequences capable of retrotransposition. Although largely inactive in normal somatic tissues, tumorigenesis leads to L1 derepression and overexpression of its RNA binding chaperone protein, ORF1p. A potential cancer biomarker, ORF1p expression is a hallmark of multiple cancers and an early event in precursor lesions. Our study provides a comprehensive pan-cancer analysis of ORF1p using CPTAC proteogenomic data, revealing a dichotomous......Read more

Metagenomic sequencing and metabolomics of fecal matter have revealed the impact of the gut microbiome on health and disease. In addition to microbiota, feces also contain shed or exfoliated host epithelial, secretory and immune cells, but RNA profiling of these cells is challenging owing to degradation and cross-contamination. Here we introduce exfoliome sequencing (Foli-seq) to profile fecal exfoliated eukaryotic messenger RNAs (feRNAs) originating from the upper and lower gastrointestinal......Read more

A standardized 'parts list' of sequences for genetic engineering of microbes has been indispensable to progress in synthetic biology, but few analogous parts exist for mammalian systems. Here we design libraries of extant, ancestral, mutagenized or miniaturized variants of polymerase III promoters and guide RNA (gRNA) scaffolds and quantify their abilities to mediate precise edits to the mammalian genome through multiplex prime editing. We identify thousands of parts for reproducible editing in......Read more

We describe the synthesis and application of a coumarin-based photocleavable anchor for solid-phase synthesis and nonchromatographic purification of RNA oligonucleotides. In contrast to standard nitroaryl anchors, which are cleaved with ultraviolet (UV) light, the described construct requires visible light (λ = 456 nm) for photocleavage. This is especially important for RNAs that contain a phosphorothioate backbone, which is not compatible with UV light. We optimized the synthesis and......Read more

Chromatin is more than a simple genome packaging system but rather locally distinguished by histone post-translational modifications (PTMs) that can directly change nucleosome structure and/or be "read" by chromatin-associated proteins to mediate downstream events. An accurate understanding of histone PTM binding preference is vital to explain normal function and pathogenesis and has revealed multiple therapeutic opportunities. Such studies most often use histone peptides, though these cannot......Read more

An exciting feature of nanopore sequencing is its ability to record multiomic information on the same sequenced DNA molecule. Well-trained models allow the detection of nucleotide-specific molecular signatures through changes in ionic current as DNA molecules translocate through the nanopore. Thus, naturally occurring DNA modifications, such as DNA methylation and hydroxymethylation, may be recorded simultaneously with the genetic sequence. Additional genomic information, such as chromatin state......Read more

We describe an acid-mediated chemoselective method for the targeted modification of arginine residues in peptides. Malonaldehyde efficiently converts guanidinium side chains into amino pyrimidine moieties with near-quantitative conversion across diverse substrates. Side products are reversible with butylamine, underscoring the method's robustness. The resulting amino pyrimidine peptides exhibit enhanced cellular permeability and allow late-stage diversification into imidazo[1,2-a]pyrimidinium......Read more

The human RNome, the complete set of RNA molecules in human cells, arises through complex processing and includes diverse molecular species. While research traditionally focuses on four canonical nucleotide residues, the RNome, encompassing over 180 distinct modifications across organisms, with at least 50 in humans, is increasingly recognized. These modifications play critical roles in regulating RNA structure, stability, and function, yet the rules linking their precise locations to biological......Read more

In mammalian cells, gene copy number is controlled to maintain gene expression and genome stability. However, a common molecular feature across cancer types is oncogene amplification, increasing the copy number and expression of tumor-promoting genes and thus promoting cancer progression. For example, in tyrosine kinase inhibitor (TKI)-resistant lung adenocarcinoma (LUAD), oncogene amplification is frequent. Despite the prevalence of oncogene amplification in TKI-resistant tumors, the underlying......Read more

Transcription factor (TF) paralogs provide unique insights into how DNA-binding specificity evolves and diversifies. While paralogous TFs share conserved, highly similar DNA-binding domains, they achieve distinct regulatory functions through mechanisms that are now being elucidated. This review examines how sequence variations between paralogs translate into functional diversity, including how mutations distant from the DNA interface can allosterically modulate binding specificity. We focus on......Read more

Recognition of foreign RNA is critical for the innate immune response to viruses. Interferon (IFN)-induced proteins with tetratricopeptide repeats (IFIT) 2 and 3 are highly upregulated following viral infection, but mechanistic insight into their antiviral role is lacking. Here we demonstrate that short 5' untranslated regions (UTRs), a characteristic of many viral mRNAs, can serve as a molecular pattern for innate immune recognition via IFIT2 and IFIT3. Structure determination of the......Read more

CONCLUSIONS: Our work reveals how tissue, sex, and age jointly govern the dynamics of the two cytosine modifications, augments the scope of DNA modification biomarker discovery, and provides a reference atlas to explore epigenetic dynamics in development and disease....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 growth, as proxy for protein activity. Here, we covalently link variant proteins in vivo to an RNA barcode by fusing them to E. coli tRNA (m(5)U(54)) methyltransferase TrmA (E358Q), which forms a covalent bond with a tRNA stem-loop. Following cell lysis, variant proteins are separated in vitro......Read more

Pseudouridine (Ψ) is an abundant post-transcriptional modification found across all classes of RNA. It has been widely speculated that Ψ inclusion in 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 localization of......Read more

Human gastruloids are a powerful class of stem cell-derived models that recapitulate key features of early embryonic development, including symmetry breaking and the emergence of three germ layers^(1-3). However, they lack anterior embryonic structures and coordinated axial organization^(4-6). To address this limitation, we pre-patterned human pluripotent stem cells (hPSCs) by exposing them to either anterior (FGF2) or posterior (CHIR99021 [CHIR] & retinoic acid [RA]) cues. Upon mixing, these......Read more

Recent breakthroughs in spatial transcriptomics technologies have enhanced our understanding of diverse cellular identities, spatial organizations, and functions. Yet existing spatial transcriptomics tools are still limited in either transcriptomic coverage or spatial resolution, hindering unbiased, hypothesis-free transcriptomic analyses at high spatial resolution. Here, we develop reverse-padlock amplicon-encoding fluorescence in situ hybridization (RAEFISH), an image-based spatial......Read more

In the expanding field of spatial genomics, numerous methods have emerged to decode biomolecules in intact tissue sections. Advanced techniques based on combinatorial decoding can resolve thousands of features in a reasonable time but are often constrained by either the prohibitive costs associated with commercial platforms or the complexity of developing custom instruments. Alternatively, sequential detection methods, like single-molecule FISH, are easier to implement but offer limited......Read more

Tardigrades are remarkable in their ability to survive extreme environments. The damage suppressor (Dsup) protein is thought to contribute to their extreme resistance to reactive oxygen species (ROS) generated by irradiation. Here we show that expression of Ramazzottius varieornatus Dsup in Saccharomyces cerevisiae reduces oxidative DNA damage and extends lifespan in response to chronic oxidative genotoxicity. Dsup uses multiple modes of engagement with the nucleosomal H2A/H2B acidic patch,......Read more

In single-cell data analysis, addressing sparsity often involves aggregating the profiles of homogeneous single cells into metacells. However, existing metacell partitioning methods lack checks on the homogeneity assumption and may aggregate heterogeneous single cells, potentially biasing downstream analysis and leading to spurious discoveries. To fill this gap, we introduce mcRigor, a statistical method to detect dubious metacells, which are composed of heterogeneous single cells, and optimize......Read more

CONCLUSIONS: We here provide CREPE, a software platform that allows for parallelized primer design for PCR applications and that is optimized for targeted amplicon sequencing....Read more

Human genome resequencing typically involves mapping reads to a reference genome to call variants; however, this approach suffers from both technical and reference biases, leaving many duplicated and structurally polymorphic regions of the genome unmapped. Consequently, existing variant benchmarks, generated by the same methods, fail to assess these complex regions. To address this limitation, we present a telomere-to-telomere genome benchmark that achieves near-perfect accuracy (i.e. no......Read more

N ⁶-methyladenosine (m⁶A) is a critical regulator of mRNA processing and function, impacting nearly every step of the mRNA lifecycle. Changes in m⁶A status have been implicated in many different types of cancer, including glioblastoma where acquired resistance to temozolomide remains a major clinical challenge. We established glioblastoma cell culture models of acquired temozolomide resistance and analyzed the role of m⁶A in controlling resistance-associated pathways. We show that m⁶A stabilizes......Read more

RNA is at the forefront of therapeutics and gene editing technologies. Yet, RNA synthesis remains expensive and low-yield. Consequently, most oligo manufacturers abstain from synthesizing RNA oligos longer than 60-mers. Solid-phase synthesis is the current standard production method but is often fraught with low coupling yields for canonical nucleotides and even poorer coupling for modifications. This results in high levels of byproducts such as truncations and RNA infidelity. Existing......Read more

Cell-cycle dynamics of gene expression are fundamental to life, yet their origin remains unclear. A prevailing model, derived from cancer cells, posits that transcription occurs in one cell cycle phase, while mature RNA accumulates in subsequent phases, suggesting temporal segregation of transcriptional regulation and RNA abundance. Whether this paradigm applies to normal human cells is unclear. Here, we co-profiled nascent transcription and mature RNAs in cycling human fibroblasts. The two......Read more

Recent advances in single-molecule technologies are transforming the field of protein analysis. Solid-state nanopores provide an effective method to linearize and thread full-length proteins in a single file. However, slowing their rapid translocation remains a challenge for accurate, time-resolved ion-current-based fingerprinting. In this work, we present a click-chemistry-based strategy for covalently attaching short oligonucleotides to cysteine residues on denatured proteins across a broad......Read more