Biology subjects

Tzadikario, T.

Publications and source records attributed to Tzadikario, T..

6 recordsLinked to original sources

bioRxiv2026

Intact and single-molecule analysis of heparan sulfate

Establishing tools to couple biological processes to a DNA sequence has transformed our ability to monitor life at the molecular scale due to the scalability, flexibility, and low cost of DNA sequencing. Key examples include DNA-protein (ChIP-seq1), RNA-protein (CLIP-seq2), protein-protein (proximity ligation assay3), and Cas-based recording of cellular events4. In contrast, this paradigm has not yet significantly enhanced studies of glycans, which are mostly limited to non-DNA based chemical and biochemical assays. While classical asparagine-linked and serine/threonine-linked glycans can be directly sequenced using mass spectrometry, glycosaminoglycans - notable players in the extracellular matrix - cannot be easily analyzed in their full-length form. Here we introduce HS-nano-seq, a generalized framework to selectively label, process, and detect features of heparan sulfate on a nanopore sequencing platform. Recognizing that heparan sulfate is biochemically analogous to a nucleic acid, we report purification techniques using rapid nucleic acid strategies and conjugation methods to couple DNA adapters, generating HS-DNA chimeras resolved as discrete species by capillary electrophoresis (CE). The CE assay can distinguish features of chain length and sulfation patterns. At the single-molecule level enabled by nanopore sensing, we classify a library of synthetic heparan sulfate standards and demonstrate that nanopore ionic current fingerprints encode sulfation-dependent structural features of individual HS chains. Analysis of intact, cell-derived HS could discriminate features of individual chains with different sulfation patterns, defining the heterogeneity of binding motifs across cell types and how cells organize and program the tethered extracellular matrix. More broadly, HS-nano-seq establishes a framework for achieving full-length readouts of ECM glycopolymers that are amenable to the same biological interrogation as nucleic acids.

biochemistry↗

bioRxiv2025

tRNA isodecoder analysis using Nanopore ionic current signals and deep learning

tRNA are short non-coding RNA characterized by their distinct tertiary structure and abundant chemical modifications. Conventional analysis strategies do not fully characterize tRNA isodecoders. We demonstrate that this limitation can be resolved for tRNA using nanopore ionic current data. We developed tRNAZAP, a deep learning strategy that uses nanopore ionic current signal information to classify native tRNA strands at isodecoder-level resolution without relying on sequence information. Additionally, the ionic current level classification allows for pairwise alignment of read sequences to reference sequences, producing optimal tRNA alignments. We applied tRNAZAP to direct tRNA sequencing data from Escherichia coli and Saccharomyces cerevisiae, and recovered 2.6% and 13.1% more aligned reads than BWA-MEM, respectively. tRNAZAP resolved these reads at an isodecoder-level and with consistently higher alignment identity. tRNAZAP is a powerful complement to sequence-based profiling and can contribute towards resolving the isodecoder landscape in more complex organisms including humans.

genomics↗

bioRxiv2025

Ancient DNA from shells reveals delayed genomic erosion and rapid immune adaptation in the critically endangered black abalone

Predicting the genetic consequences of population decline is a major problem in conservation genomics. Time lags following demographic bottlenecks can delay genomic erosion and make it difficult to determine a populations current and future risk, especially when pre-bottleneck genomic baselines are unavailable. Black abalone (Haliotis cracherodii) suffered a severe disease bottleneck in the 1980s, resulting in an estimated 99% population decline. However, recent work found surprisingly high genetic diversity and little population structure in current black abalone populations, raising questions of whether genomic erosion has been delayed. To investigate this, we applied ancient DNA methods to pre-bottleneck abalone shells, generating 59 whole genomes including one 34-fold coverage genome from a 1,500-year-old specimen. These data show that heterozygosity, runs of homozygosity, genetic load and population structure remained stable up to and following the bottleneck. Simulations reveal that this stability is consistent with even severe bottleneck scenarios because too few generations have lapsed since the decline. Projections suggest that future genomic erosion may be avoided even in limited recovery scenarios. Following the bottleneck we observe widespread balancing selection at genes with immune function, along with parallel increases of two inversions on separate chromosomes that are in linkage disequilibrium, where the disease bottleneck was most severe. Altogether, these findings explain why genomic change has thus far been limited, outline recovery scenarios that minimize genomic erosion, and identify loci likely that may harbor adaptive variation key to the success of future black abalone populations.

evolutionary biology↗

bioRxiv2025

Cytoplasmic localization of PUS7 facilitates a pseudouridine-dependent enhancement of cellular stress tolerance

Pseudouridine ({Psi}) is an abundant post-transcriptional modification found across all classes of RNA. It has been widely speculated that {Psi} 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 PUS7 promote {Psi}-incorporation into hundreds of mRNA targets. Furthermore, engineered PUS7 cytoplasmic localization increases cellular fitness under ROS and divalent metal ion stress. Consistent with this, transcripts modified upon PUS7 cytoplasmic localization are enriched within mRNAs encoding proteins involved in divalent metal metabolism and ROS stress pathways. In contrast, tRNA sites modified by PUS7 ({Psi}13 and {Psi}35 are unperturbed). Quantitative proteomics reveal a reshaping of the proteome upon PUS7 relocalization under stress, with proteins involved in metal and ROS homeostasis being particularly sensitive to PUS7 localization. Collectively, our data demonstrate that PUS7 localization alters mRNA pseudouridylation patterns to modulate protein production and enhance cellular fitness.

biochemistry↗

bioRxiv2025

Evaluation of Nanopore direct RNA sequencing updates for modification detection

Nanopore technology can directly sequence intact RNA molecules, offering a unique capability to read native modifications. Oxford Nanopore Technologies recently updated its direct RNA sequencing technology from RNA002 to RNA004 chemistry. This update included an improved basecaller (Dorado) for increased sequencing accuracy, and ionic current models for de novo detection of four RNA modifications. Using a single RNA extraction from GM12878 B-lymphocyte cell line, we compared RNA002 and RNA004 sequencing chemistries and evaluated Dorado modification calling accuracy. We computed U-to-C mismatches, previously used to identify putative pseudouridine sites, and ran m6anet for identifying putative N6-methyladenosine sites. Dorado results for each respective modification showed both global and site-specific differences when compared to RNA002 results. We used DRS data from in vitro transcription of GM12878 genomic DNA as well as synthetic oligonucleotides to evaluate Dorado modification calling performance. Dorados pseudouridine model achieved 96-98% for both accuracy and F1-score. Similarly, Dorados N6-methyladenosine model achieved 94-98% accuracy, 96-99% F1-score. Our results demonstrate that Nanopore Direct RNA sequencing could simultaneously detect pseudouridine, N6-methyladenosine, 5-methylcytosine, and inosine modifications on individual mRNA strands. It is critical to validate these calls using orthogonal methods (e.g., Liquid Chromatography with Tandem Mass Spectrometry) for increased confidence. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/651717v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1215534org.highwire.dtl.DTLVardef@160f38forg.highwire.dtl.DTLVardef@164a9corg.highwire.dtl.DTLVardef@17c72e5_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2025

Genomic in vitro transcription and Nanopore direct RNA sequencing of a human B-Lymphocyte cell line

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 development of RNA modification models. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=119 SRC="FIGDIR/small/650674v2_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@962feorg.highwire.dtl.DTLVardef@4237aborg.highwire.dtl.DTLVardef@154d346org.highwire.dtl.DTLVardef@1fad8f2_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗