NAR Molecular Medicine

Background: Biallelic variants in GFM2, encoding mitochondrial elongation factor G2 (mtEFG2), a GTPase involved in the termination stage of mitochondrial translation, cause autosomal recessive combined oxidative phosphorylation deficiency. Noncoding structural variants may be missed by exome sequencing but can disrupt splicing and provide opportunities for variant-specific therapeutic rescue. We investigated the molecular mechanism underlying suspected Leigh syndrome in an infant with mitochondrial disease and evaluated whether splice-switching oligonucleotide (SSO) treatment could correct the pathogenic splicing defect. Methods: The proband underwent exome sequencing followed by short-read and long-read whole genome sequencing. RNA sequencing, reverse-transcription PCR, quantitative PCR, and cycloheximide treatment were used to characterize the effect of the identified intronic duplication on GFM2 splicing and transcript stability. Patient-derived fibroblasts were treated with SSOs targeting the aberrant splice junction. Rescue was assessed by RNA studies, western blotting, and spectrophotometric measurement of cytochrome c oxidase (COX). Results: Whole genome sequencing identified a paternally-inherited GFM2 missense variant, NM_032380.5:c.2195C>T p.(Pro732Leu), in trans to a maternally-inherited 221-nucleotide intronic duplication, NM_032380.5:c.2029-741_2029-521dup. RNA studies revealed a 87-nucleotide pseudoexon, generated by activation of a cryptic acceptor splice site within the duplicated sequence. The resulting transcript harbored a premature termination codon (PTC) and underwent nonsense-mediated decay, as confirmed by cycloheximide rescue. Together with reduced mtEFG2 protein levels on western blot, the findings supported a loss-of-function mechanism. Enzymatic analysis of affected fibroblasts showed reduced activity of the mtDNA-dependent complex IV subunit COX, with preservation of the nuclear-encoded complex II enzyme succinate dehydrogenase and the control enzyme citrate synthase, consistent with impaired mitochondrial translation. A SSO targeting the aberrant intron-pseudoexon junction nearly abolished pseudoexon inclusion, restored correctly spliced GFM2 transcript from the duplication-containing allele, increased mtEFG2 protein levels, and significantly improved COX activity. Conclusions: This study identifies a pathogenic intronic GFM2 duplication that causes mitochondrial disease through pseudoexon activation and nonsense-mediated decay. The findings demonstrate the value of integrated genome and transcriptome analysis for exome-negative mitochondrial disease and provide in-vitro proof of concept that SSOs can restore transcript processing, protein expression, and mitochondrial respiratory-chain function in patient-derived cells.

I.Understanding the mechanisms governing neuronal differentiation is essential for elucidating neurodevelopmental processes and identifying therapeutic targets for neurological disorders. In this study, we optimized serum-dependent induction conditions and benchmarked multiple RNA-seq pipelines to establish a robust in-vitro model of neurogenesis using P19 embryonal carcinoma cells. Retinoic acid (RA, 0.5 {micro}M) was used to induce neuronal differentiation under varying concentrations (1%, 2%, and 5%) of fetal bovine serum (FBS) obtained from three suppliers. Morphological observation and marker gene analysis (MAP2, OCT4) revealed that serum concentration strongly influenced aggregation, survival, and neuronal commitment, with 2-5% FBS yielding optimal neurogenic differentiation. Total RNA extracted on day 10 of differentiation was subjected to RNA sequencing, and the resulting datasets were analyzed using four independent bioinformatics workflows: a Linux-based R pipeline (HISAT2 + featureCounts + DESeq2), nf-core, Galaxy, and BGIs Dr. Tom platform. Differential gene expression analysis identified 9,943 differentially expressed genes (DEGs) (FDR < 0.05, |log2FC| > 1), enriched in synaptic assembly and axon development among upregulated genes, and in ribosome biogenesis and RNA processing among downregulated genes. Comparison across all pipelines revealed 62 consistently upregulated and 63 downregulated genes, representing a robust core signature of P19 neurogenesis. Together, these findings establish an optimized and reproducible framework for in-vitro neuronal differentiation and transcriptomic analysis, providing a foundation for mechanistic and disease-modeling studies in neurodevelopmental biology.

CAG repeat expansions in ATXN2 are implicated as risk factors for neurological diseases, including amyotrophic lateral sclerosis (ALS) when 27-33 CAG (intermediate) repeats are present. However, how haplotypes around the repeats and CAA interruptions within the repeats are associated with diseases remains poorly understood. Here, we used long-read sequencing on the Oxford Nanopore technologies (ONT) platform to simultaneously infer haplotypes around ATXN2, the number of CAG repeats, and the number of CAA interruptions. We found that haplotypes around ATXN2 and the number of interruptions show ethnicity-specific and ALS-specific distribution. Three CAA interruptions are present at low prevalence ([~]1%) in control populations in multiple ancestry groups, but high prevalence ([~]55%) in ALS individuals with intermediate repeats. Furthermore, we examined 159 individuals with ALS ([~]90% European ancestry) with intermediate ATXN2 repeats and found a unique haplotype in ALS individuals with three CAA interruptions, which can be tagged by an SNV, rs148019457. We further sequenced 41 individuals (EUR = 39) with neurological diseases with intermediate repeats by ONT, and validated that the rs148019457-G allele is only present in haplotypes with three CAA interruptions. Our study shows that 3 CAA interruptions are rare in healthy controls but are common in individuals with intermediate ATXN2 CAG repeats and neurological disorders, and that rs148019457 tags a specific haplotype with 3 CAA interruptions in individuals of European ancestry. These results have implications for the development of precision genomic medicine for neurological disorders, and the tag SNV may help identify those with interruptions from existing microarray genotyping data.

We present a novel integrative analysis of transposable elements (TEs) in 4 single cell RNA-seq (scRNA-seq) datasets of postmortem substantia nigra pars compacta samples of Parkinson Disease (PD) patients matched healthy controls, with the objective of building a cell-type specific trustworthy atlas of TEs that may clarify the role of TEs in sex differences in PD. We have used the soloTE tool to evaluate the TEs expression changes across all snRNA-seq studies identified in our previous systematic review, and then integrated the results using meta-analysis techniques. Finally, we evaluated the possible associations between TEs and protein coding genes by integrating our previous results in this matter with the information of TEs obtained, in order to propose the possible action mechanism by which some of the TEs contribute to PD.

Pathogenic variants in ATAD3A cause a spectrum of multisystem disorders, with a recurrent dominant-negative variant (c.1582C>T; p.Arg528Trp) associated with neurodevelopmental disease. Given the tolerance of ATAD3A to heterozygous loss of function variants, allele-specific transcript reduction represents a promising therapeutic strategy. We designed and optimized allele-specific antisense oligonucleotides (ASOs) targeting the c.1582C>T transcript and evaluated their efficacy and specificity in affected fibroblasts using allele-specific primers and amplicon-based next generation sequencing. Therapeutic potential was further assessed in vivo in zebrafish embryos expressing human wild-type or mutant ATAD3A transcripts. An optimized gapmer ASO selectively reduced mutant ATAD3A transcripts while relatively sparing the wild-type allele. In addition to RNase H-mediated degradation, the ASO induced exon skipping, leading to degradation of the aberrant transcript without production of a truncated protein. In zebrafish, expression of mutant human ATAD3A in embryos caused developmental abnormalities including reduced eye size, which were robustly rescued by co-injection of the optimized ASO. Our findings provide proof of concept for allele-targeted ASO therapy for dominant-negative ATAD3A variants. This work highlights the therapeutic potential of ASOs for rare dominant disorders involving genes tolerant to heterozygous loss-of-function, and establishes zebrafish as a versatile platform for in vivo ASO optimization.

Biallelic disease-causing variants in IGHMBP2 cause spinal muscular atrophy with respiratory distress type I (SMARD1) and Charcot-Marie-Tooth type 2S (CMT2S). We present 12 unrelated patients with clinically suspected IGHMBP2-related-disease, each carrying a variant deep in intron 8 of IGHMBP2 (c.1235+1076G>A (n=6), c.1235+450G>A (n=5), and c.1235+894C>A (n=1)), along with a known deleterious variant in trans. To assess aberrant pathogenic splicing induced by these deep intronic variants in a relevant model, patient-derived induced pluripotent stem cells were differentiated into motor neurons (iMNs). Long-read RNA sequencing revealed introduction of different pseudoexons by each variant: c.1235+450G>A (626bp), c.1235+1076G>A (112bp and 77bp) and c.1235+894C>A (182bp). Although each variant utilizes a unique splice acceptor site, they all activate the same cryptic donor site, enabling a therapeutic approach to redirect aberrant splicing for all the variants using a single shared antisense oligonucleotide (ASO). Treatment of iMNs with this single ASO restored full-length IGHMBP2 protein in c.1235+894G>A and c.1235+1076G>A by decreasing the use of the novel acceptor site. In contrast, ASO treatment did not correct the splicing in c.1235+450G>A, suggesting that additional splice correction will be needed for this specific variant. A CRISPR interference screen of IGHMBP2 loss-of-function in iMNs identified ribonucleoprotein complex biogenesis (RNP), and rRNA and tRNA processing as top pathways implicated in motor neuron vulnerability. Proteomics and transcriptomics analysis of successfully treated patient iMNs revealed correction of RNP biogenesis and rRNA processing defects. This study highlights the importance of characterizing deep intronic variants in disease-relevant cells to assist the diagnostic process and inform therapeutics development. One Sentence SummaryIntron 8 of IGHMBP2 is a hotspot for splice activating pathogenic variants causing SMARD1 and CMT2S, which can be targeted with a single antisense oligonucleotide to correct the aberrant splicing, increase protein and restore cellular function in patient derived motor neurons.

Mitochondrial DNA (mtDNA) heteroplasmy, which is the coexistence of wild-type and mutant mtDNA variants within the same cell, plays a critical role in modulating cellular phenotype as well as disease severity and penetrance. Bulk RNA sequencing is not able to detect cell-to-cell variability in heteroplasmy, limiting our understanding of mitochondrial pathological mechanisms. In this study, we leverage single-cell RNA sequencing (scRNA-seq) combined with a robust bioinformatics pipeline to characterize mtDNA heteroplasmy. We employed four fibroblast lines from patients harboring heteroplasmic mtDNA pathogenic variants in genes encoding respiratory complex I subunits. While RNA heteroplasmy corresponds to DNA-based measurements at the bulk-level, single-cell analysis uncovers a diverged distribution: most cells have near-homoplasmic (wild-type or mutant) mtDNA, with few cells showing intermediate levels. Furthermore, we find that high mutation levels correlate with transcriptional profile changes, though these responses are highly sample-specific, suggesting that nuclear background and cellular context critically influence mitochondrial dysfunction and compensatory mechanisms. Our findings highlight the power of single-cell technologies to better understand the complex link between mtDNA genetic diversity and mitochondrial phenotypic variability, and to study crucial aspects in mitochondrial biology and pathology, such as clonal dynamics, at single-cell resolution.

The spinocerebellar ataxias (SCAs) are a clinically heterogenous group of neurodegenerative disorders that affect movement, vision, speech and balance. Here, we reassign the linkage of SCA30 to 14q32.13 based on a cumulative LOD score >12. Within this interval we identified a 331 kb duplication, absent in population controls and not observed in >800 unrelated individuals with genetically unresolved cerebellar ataxia. RNA-Seq analysis of patient-derived lymphoblastoid cell lines revealed a splice-mediated chimeric transcript resulting from the duplication event. This transcript joined exon 1 of CLMN to exon 2 of SYNE3. In silico translation predicted that this chimeric transcript would produce a short N-terminal peptide corresponding to exon 1 of CLMN and the usually untranslated region of exon 2 of SYNE3 fused to the complete and in-frame SYNE3 protein. Transient overexpression of SYNE3 or the CLMN::SYNE3 fusion protein, in both HeLa cells and mouse primary cortical neurons, resulted in equivalent cellular outcomes including altered nuclear morphology and chromosomal DNA fragmentation. SYNE3 forms part of the linker of nucleoskeleton and cytoskeleton complex and is not usually expressed in cerebellar Purkyn[e] neurons while, CLMN has a Purkyn[e] specific expression pattern within the brain. Our data suggests that ectopic expression of SYNE3 in cerebellar Purkyn[e] neurons, mediated by the CLMN promoter, leads to cerebellar atrophy and causes spinocerebellar ataxia in the SCA30 family. This is an example of Mendelian disease arising from a novel, chimeric transcript with a likely dominant negative effect. Chimeric transcripts are commonly associated with cancers, but they are not often associated with monogenic disorders. Detection of chimeric transcripts as part of structural variant analysis could increase the genetic diagnostic yield of Mendelian disorders.

The role of the epigenome in age-related neurodegenerative disorders remains understudied. Here, we analyzed circulating cell-free DNA (cfDNA) from blood to detect methylation changes as a liquid-biopsy for Amyotrophic Lateral Sclerosis (ALS). Our study included 20 patients with sporadic ALS, 10 patients with C9orf72-associated ALS, 10 asymptomatic carriers of the C9orf72 repeat expansion mutation, and 21 non-disease controls. Following targeted enzymatic methyl-sequencing (EM-seq) of [~]4 million CpG sites, we detected numerous differentially methylated genes, including several implicated in ALS disease risk and pathogenesis. By integrating multiple epigenetic features, we delineated a distinct epigenetic signature, which achieved an average area under the curve (AUC) of 0.91 {+/-} 0.10 upon receiver operator characteristic (ROC) analysis, which enabled detection of [~]70% of ALS patients with close to 100% specificity. Furthermore, we also identified a set of genes whose methylation status significantly correlated with clinical disease progression and cerebrospinal fluid (CSF) neurofilament levels. Our results reveal the potential of cfDNA-based biomarkers to accurately diagnose ALS and potentially predict disease progression.

Multiple sclerosis (MS) is a debilitating disease affecting more than 1 million Americans, and today is assessed primarily through magnetic resonance imaging (MRI) and observational clinical symptoms. Given the autoimmune nature of MS, we hypothesized that high-dimensional gene expression data from peripheral blood mononuclear cells (PBMCs), when analyzed with the assistance of AI, may collectively serve as valuable biomarkers for the real-time risk and progression of MS. Here, we present PBMC RNA sequencing (RNAseq) results from N=997 samples, including 540 MS, 221 neuromyelitis optica (NMO), and 149 healthy controls. We constructed and optimized ensemble models for three clinical outcomes: (1) discrimination of early MS (EDSS [&le;] 2.0) from healthy individuals with 74% AUC at 100% coverage, (2) differential diagnosis of MS from NMO with 91% AUC at 80% coverage, and (3) subtyping RRMS from progressive MS with 79% AUC at 80% coverage. To our knowledge, no prior molecular test has been reported for any of these three MS clinical tasks, and these results may have immediate impact on clinical management of MS patients. Two innovations that improved the stratification accuracy of our models: selection of gene sets based on expression variance in disease states, and use of non-linear rank sort and conviction weighting in the ensemble score calculation.

BackgroundSmall non-coding RNAs (sncRNAs) play central roles in post-transcriptional gene regulation. In addition to canonical microRNAs (miRNAs), fragments derived from vault RNAs (vtRNAs), called small vault RNAs (svtRNAs), have been reported in human cells. However, the absence of a standardized annotation framework has hindered their systematic detection, quantification, and comparison across small RNA sequencing (small RNA-seq) studies. MethodsWe developed an expression-based annotation strategy to identify svtRNAs from human small RNA-seq datasets. Using FlaiMapper followed by structure and expression-based filtering, we generated two annotation sets: a stringent "miRNA-like" set enriched in Argonaute-associated datasets, and (ii) a broader "Total" set derived from total small RNA-seq libraries under relaxed structural constraints. We explored the expression of the annotated svtRNAs across the different datasets analyzed: multiple normal and tumor-derived human cell lines, including Argonaute immunoprecipitation datasets. ResultsWe identified a repertoire of svtRNAs that are detected across independent datasets and, in several cases, reach abundance levels comparable to canonical miRNAs. Several highly abundant svtRNAs correspond to molecules with experimental validation from prior studies, supporting the robustness of our annotation strategy. Importantly, the same "dominant" (in terms of gene expression) svtRNAs emerged independently from Argonaute-associated and total small RNA datasets, supporting the idea of enzymatically consistent, reproducible svtRNA processing. We further identified svtRNAs derived from distinct vtRNA precursors that could share identical seed sequences, suggesting the possibility of svtRNA families with potential miRNA-like regulatory properties. We provide a standardized annotation that enables reproducible svtRNA quantification. ConclusionsOur study establishes a comprehensive expression-based annotation resource for human svtRNAs. By enabling their systematic detection and reproducible quantification, we show that svtRNAs appear to represent an abundant component of the human small RNA landscape.

TAR DNA-binding protein 43 (TDP-43) is a multifunctional DNA/RNA-binding protein implicated in transcriptional and post-transcriptional regulation. Dysregulation of TDP-43 is closely correlated with human diseases such as cancer and neurodegenerative diseases. Although its roles in RNA metabolism are well characterized, its function in transcriptional regulation remains largely underexplored. DNA G-quadruplexes (dG4s) are non-canonical nucleic acid structures enriched at gene promoters and regulatory elements, where they facilitate chromatin looping and gene transcription. Here, we investigated the transcriptional regulatory role of TDP-43 by integrating multi-omics datasets, including Hi-C, dG4 ChIP-seq, TDP-43 ChIP-seq, RNA-seq and ATAC-seq from K562 and HepG2 cells. Our analyses demonstrate TDP-43 binding and dG4s formation are highly colocalized at chromatin loop anchors, particularly at promoter and enhancer regions. TDP-43 occupancy at these anchors correlates with increased dG4 stability, chromatin loop interaction frequency, elevated chromatin accessibility, and upregulated gene expression. Morover, TDP-43 knockdown in HepG2 cells revealed a significant reduction in dG4 formation and loop interaction strength, accompanied by widespread transcriptional dysregulation. Collectively, our findings highlight a novel regulatory role of TDP-43 in facilitating long-range chromatin interactions and transcriptional activation through binding to and stabilizing dG4 structures, providing a mechanistic basis for gene dysregulation driven by TDP-43 dysfunction in diseases.

Duchenne muscular dystrophy (DMD) is the most common, lethal X-linked neuromuscular disorder of childhood and is caused by mutations in the Dmd gene that disrupt dystrophin expression. Although adeno-associated virus-mediated gene therapies hold tremendous promise for DMD treatment, their clinical applications have been limited by dose-dependent vector and genome-level toxicities. Here, we developed and tested a single-vector adenine base editing strategy as a potentially safer genome editing approach to recode the pathogenic nonsense mutation into a benign missense mutation in mdx4cvDMD mouse model. Delivered using a muscle-tropic adeno-associated virus (MyoAAV) at a clinically-feasible dose (4E13 VG/kg), this strategy enabled detectable molecular recoding of the mdx4cv mutation in mice ranging in age from 3 days to 6 months. Yet, the overall efficiency and therapeutic impact of in vivo base editing with this system was highest in mice treated at the juvenile stage, with animals administered MyoAAV vectors at 3 weeks of age showing robust recovery of dystrophin expression and significant improvement in muscle contractile properties only one month later. Notably, introduction of adenine base editors either earlier in development, in neonatal mice, or later, in adulthood, yielded substantially lower editing efficiencies, particularly in muscle satellite cells whose editing is essential to ensure durable rescue of dystrophin expression in growing and regenerating muscle. Taken together, these results demonstrate the therapeutic potential of single-vector adenine base editing for DMD and underscore the importance of recipient age and disease stage in achieving optimal treatment outcomes for this and other genetic muscle disorders.

Spinal muscular atrophy (SMA) is caused by loss of SMN protein and is increasingly recognized as a multisystem disorder involving molecular pathology beyond motor neurons. Recently, we identified NRF2-KEAP1 signaling as dysregulated in SMA mice. Because NRF2 coordinates transcriptional programs that maintain cellular redox homeostasis and adaptive stress responses, we investigated whether NRF2 signaling is similarly altered in SMA type I patient-derived fibroblasts and whether it can be pharmacologically engaged. Compared with control fibroblasts, SMA fibroblasts displayed reduced basal expression of NRF2 target proteins, including NQO1 and xCT (SLC7A11), along with decreased levels of PGC1. Omaveloxolone (OMAV), a pharmacological NRF2 activator approved for the treatment of Friedreichs ataxia, increased cell viability and upregulated NRF2 target proteins in both control and SMA fibroblasts. Notably, OMAV produced a modest increase in SMN protein abundance and PGC1 levels selectively in SMA cells. Together, these findings support diminished NRF2 pathway output as a feature of SMA fibroblasts and demonstrate that OMAV induces NRF2 target proteins in this human SMA cellular model, consistent with enhanced cytoprotective signaling. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/712434v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1904bfeorg.highwire.dtl.DTLVardef@6d20e2org.highwire.dtl.DTLVardef@89f365org.highwire.dtl.DTLVardef@ca9638_HPS_FORMAT_FIGEXP M_FIG C_FIG

Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system affecting 2.8 million people worldwide. Both genetic and environmental factors contribute to MS risk, with Epstein-Barr virus (EBV) infection being an important environmental factor. To better clarify the role of EBV in MS, we examined its impact on gene expression, chromatin accessibility, and transcription factor binding in primary B cells and EBV-transformed B cells derived from patients with MS and healthy controls. RNA-seq and ATAC-seq analyses revealed extensive MS-dependent gene expression and chromatin accessibility differences in EBV-transformed, but not in primary B cells. These changes are largely accounted for by the expression levels of EBNA2, an EBV-encoded transcriptional regulator previously implicated in MS. ChIP-seq analysis revealed that EBNA2 binding with its interacting human partners RBPJ, EBF1, and PU.1 is highly enriched at MS genetic risk loci, with extensive EBNA2 allelic binding and increased enrichment at MS genetic risk loci in MS-derived cells. Our findings demonstrate that enhanced EBNA2 activity in MS alters human gene expression, chromatin accessibility, and transcription factor binding in an MS-dependent manner. Collectively, this study provides new insights into the molecular mechanisms through which EBV, particularly EBNA2, interacts with host genetic risk to contribute to MS pathogenesis.

BackgroundMetazoan adenosine-to-inosine (A-to-I) mRNA editing temporospatially diversifies the neuronal transcriptome and proteome. The limited read length from next-generation sequencing (NGS) constrains the quantification of the potentially differential editing levels across different splicing isoforms, restricting our understanding of the extent to which RNA editing contributes to molecular diversity and its interplay with splicing. MethodsWe employed reverse transcription nested PCR (RT-nPCR) and developed a novel interfering-Primer PCR (iPrimer PCR) technique to distinguish different transcripts of any gene. We selected multiple essential genes exhibiting RNA editing in coding sequences (CDSs) or untranslated regions (UTRs) for isoform-specific amplification and Sanger sequencing. ResultsNine different Adar isoforms together with pre-mRNA had distinct editing levels at the S>G auto-recoding site, which was predicted to have isoform-specific effects on catalytic activities. Although pre-mRNA editing might exert isoform-dependent promotion/suppression of splicing, closely located editing sites, such as those in neuronal genes qvr and stj, still exhibited high correlation in editing levels due to co-editing. iPrimer strategy further discovered differential recoding levels between the long/short 3UTR isoforms of gene jef. ConclusionsWe provide the first comprehensive solution for isoform-specific PCR amplification of any gene, enabling quantification of RNA editing level of different isoforms. Our results offer insights into how RNA editing interplays with splicing, and highlight its complicated role in expanding molecular diversity. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/725286v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1ebc82org.highwire.dtl.DTLVardef@1ea365dorg.highwire.dtl.DTLVardef@1971aceorg.highwire.dtl.DTLVardef@160d053_HPS_FORMAT_FIGEXP M_FIG C_FIG We developed isoform-specific PCR followed by Sanger sequencing, and achieved the quantification of differential RNA editing levels in different transcripts of a gene.

Long-read RNA sequencing (lrRNA-seq) provides advantages for transcript discovery and quantification through the sequencing of full-length transcripts. Although recent benchmarks have evaluated long-read technologies and quantification tools, to the best of our knowledge, no study to date has jointly compared sequencing technology, quantification choice, and depth across both bulk and single-cell platforms. Here, we generate a matched dataset using NGN2-induced neurons derived from Fragile X syndrome and isogenic rescue lines, profiled with bulk and single-cell Illumina, Oxford Nanopore Technologies (ONT), and Pacific Biosciences (PB) Kinnex technologies. All platforms and technologies capture the expected FMR1 reactivation signal. We find that PB bulk under-detects and under-quantifies short transcripts (less than 1.25 kb), ONT bulk under-detects and under-quantifies long transcripts (greater than 5 kb), and single-cell long-read technologies a large number of single-cell specific transcripts associated with truncations. Across six bulk and four single-cell long-read quantification tools, Isosceles, Miniquant, and Oarfish provide the best compromise between computational efficiency and performance in terms of quantification accuracy as measured by spike-ins, comparisons to Illumina, and on consensus based down-stream tasks such as differential transcript expression (DTE). Depth-equivalency analyses reveal that PB single-cell sequencing requires approximately three- to four-fold greater depth than bulk to reach comparable power for transcript discovery and differential transcript expression. Our work aims to offer practical guidance for study design, including the choice of technology, sequencing depth, and quantification method. In addition, we hope our data may serve a reference dataset to evaluate emerging long-read transcriptomic protocols and methods as well as more closely investigate FMR1 biology.

Glioblastoma multiforme (GBM) is among the most aggressive malignant brain tumors originating from glial cells and characterized by severe infiltration into surrounding brain tissue, rendering early detection difficult with current diagnostic imaging methods. S100A4 has been identified as a biomarker protein associated with glioblastoma invasiveness due to its role in cell motility and tumor metastasis. Similarly, midkine (MDK) poses an optimal biomedical target for identifying GBM invasive phenotypes because of its connection to the tumor microenvironment and infiltrative proliferation. Both proteins notably possess a positive charge that interacts electrostatically with the negatively charged phosphate backbone of DNA. It has been established that early molecular detection remains a critical unmet need. This study investigates a promising strategy for GBM diagnosis based on how S100A4 and MDK can selectively bind with DNA tweezer nanostructures. Computationally predicting eight distinct nucleotide sequences yielded three-stranded, hinge-scaffolded tweezer conformations for each candidate. The target protein and DNA structures, derived from AlphaFold, were paired together by molecular docking simulations conducted with HDOCK. Docking analyses evaluated binding affinity, structural complementarity, and conformational stability of the complexes formed. Among the evaluated candidates, DT3_8 computationally established the most biochemically robust interaction with both biomarker proteins. Selectivity is especially important because many S100 proteins share similar electrostatic profiles, yet DT3_8 indicates stronger selectivity for S100A4 and MDK over other S100 family proteins. These findings establish a biomechanical basis for the development of nanoscale DNA biosensors, which suggests the potential for detecting invasive GBM phenotypes, preceding radiographic manifestation and pending experimental validation.

Accurate detection of RNA splice variants is often hindered when transcripts lack large distinguishable exonic regions, making conventional PCR strategies challenging. We developed a simple melting temperature (Tm)-guided exon-exon junction (EEJ) RT-PCR method to enable variant-specific detection under these conditions. Uni-directional primers spanning exon-exon junctions were designed so that approximately each half anneals to adjacent exons. The Tm of each half-site was set >7{degrees}C below the annealing temperature, preventing stable binding to individual exons and enforcing junction-dependent amplification. The method was evaluated using HTRA1-AS1 long noncoding RNA variants that share overlapping exon sequences but differ in splice connectivity. HTRA1-AS1 comprises five variants, only one with a large distinguishable exon. Tm-guided EEJ primers robustly discriminated the remaining four variants. After optimization, amplification yielded sharp, single bands with minimal cross-reactivity. Compared with conventional designs, this approach reduced heteroduplex and heteroquadruplex formation, improving band clarity. Sanger sequencing confirmed junction specificity, and the method performed well in multiplex settings. Overall, Tm-guided EEJ RT-PCR is a cost-effective, high-resolution approach for detecting RNA variants lacking easily distinguishable exonic regions, readily compatible with standard RT-PCR and qPCR workflows.

The CRISPR-Cas9 system has revolutionized genome engineering, yet its full therapeutic potential remains constrained by challenges in precisely modulating its activity and specificity. Here we report a fully computational end-to-end pipeline for the de novo design of a single-domain VHH nanobody (NbSpCas9-v1) targeting a structurally conserved, non-catalytic epitope at the PAM-interacting (PI) and RuvC-III interface of Streptococcus pyogenes Cas9 (SpCas9; PDB: 4UN3). Nanobody sequences were generated using BoltzGen, a generative diffusion binder design framework, and co-folded with SpCas9 using Boltz-2 to evaluate structural confidence and binding affinity. The top-ranked model (SpCas9_4UN3_Bivalent_Hub_v1) achieved a complex pLDDT of 0.8406, an aggregate score of 0.8016, and an ipTM of >0.8, indicating high confidence in the nanobody-antigen interface. The designed 1,616-residue quaternary complex (SpCas9 + sgRNA + DNA + nanobody) was subjected to 10 ns of all-atom molecular dynamics (MD) simulation using the AMBER14SB force field within the GROMACS/OpenMM framework. The complex stabilized at RMSD [~]6 [A] with a radius of gyration of 39-44 [A], confirming thermodynamic stability under physiological conditions (310 K, 0.15 M NaCl). A conserved 96.3 [A] inter-molecular distance between the nanobody centroid and the HNH catalytic residue H840 establishes NbSpCas9-v1 as a distal, non-inhibitory binder -- ideally suited for a Bivalent Hub architecture recruiting secondary effectors to the Cas9 ribonucleoprotein (RNP). The nanobody-Cas9 interface is stabilized by 8 hydrogen bonds, 4 salt bridges, and [~]1,850 [A]2 of buried solvent-accessible surface area. These results provide a rigorous structural and dynamic foundation for experimental validation of VHH-based CRISPR-Cas9 enhancers and modulators. GRAPHICAL ABSTRACTThe computational workflow proceeds from SpCas9 crystal structure acquisition (PDB: 4UN3) through BoltzGen nanobody design, Boltz-2 structural co-folding, 10 ns explicit-solvent MD validation, and Bivalent Hub functional characterization. The PyMOL rendering below shows the full quaternary complex at atomistic resolution.