Chemoenzymatic labeling of DNA methylation patterns for single-molecule epigenetic mapping.

DNA methylation, specifically, methylation of cytosine (C) nucleotides at the 5-carbon position (5-mC), is the most studied and significant epigenetic modification. Here we developed a chemoenzymatic procedure to fluorescently label non-methylated cytosines in CpG context, allowing epigenetic profiling of single DNA molecules spanning hundreds of thousands of base pairs. We used a CpG methyltransferase with a synthetic S-adenosyl-l-methionine cofactor analog to transfer an azide to cytosines instead of the natural methyl group. A fluorophore was then clicked onto the DNA, reporting on the amount and position of non-methylated CpGs. We found that labeling efficiency was increased up to 2-fold by the addition of a nucleosidase, presumably by degrading the inactive by-product of the cofactor after labeling, preventing its inhibitory effect. We used the method to determine the decline in global DNA methylation in a chronic lymphocytic leukemia patient and then performed whole-genome methylation mapping of the model plant Arabidopsis thaliana. Our genome maps show high concordance with published bisulfite sequencing methylation maps. Although mapping resolution is limited by optical detection to 500-1000 bp, the labeled DNA molecules produced by this approach are hundreds of thousands of base pairs long, allowing access to long repetitive and structurally variable genomic regions.

Chemo-Enzymatic Fluorescence Labeling Of Genomic DNA For Simultaneous Detection Of Global 5-Methylcytosine And 5-Hydroxymethylcytosine.

5-Methylcytosine and 5-hydroxymethylcytosine are epigenetic modifications involved in gene regulation and cancer. We present a new, simple, and high-throughput platform for multi-color epigenetic analysis. The novelty of our approach is the ability to multiplex methylation and de-methylation signals in the same assay. We utilize an engineered methyltransferase enzyme that recognizes and labels all unmodified CpG sites with a fluorescent cofactor. In combination with the already established labeling of the de-methylation mark 5-hydroxymethylcytosine via enzymatic glycosylation, we obtained a robust platform for simultaneous epigenetic analysis of these marks. We assessed the global epigenetic levels in multiple samples of colorectal cancer and observed a 3.5-fold reduction in 5hmC levels but no change in DNA methylation levels between sick and healthy individuals. We also measured epigenetic modifications in chronic lymphocytic leukemia and observed a decrease in both modification levels (5-hydroxymethylcytosine: whole blood 30 %; peripheral blood mononuclear cells (PBMCs) 40 %. 5-methylcytosine: whole blood 53 %; PBMCs 48 %). Our findings propose using a simple blood test as a viable method for analysis, simplifying sample handling in diagnostics. Importantly, our results highlight the assay's potential for epigenetic evaluation of clinical samples, benefiting research and patient management.

Long-read single-molecule maps of the functional methylome.

We report on the development of a methylation analysis workflow for optical detection of fluorescent methylation profiles along chromosomal DNA molecules. In combination with Bionano Genomics genome mapping technology, these profiles provide a hybrid genetic/epigenetic genome-wide map composed of DNA molecules spanning hundreds of kilobase pairs. The method provides kilobase pair-scale genomic methylation patterns comparable to whole-genome bisulfite sequencing (WGBS) along genes and regulatory elements. These long single-molecule reads allow for methylation variation calling and analysis of large structural aberrations such as pathogenic macrosatellite arrays not accessible to single-cell second-generation sequencing. The method is applied here to study facioscapulohumeral muscular dystrophy (FSHD), simultaneously recording the haplotype, copy number, and methylation status of the disease-associated, highly repetitive locus on Chromosome 4q.

5-Hydroxymethylcytosine as a clinical biomarker: Fluorescence-based assay for high-throughput epigenetic quantification in human tissues.

Epigenetic transformations may provide early indicators for cancer and other disease. Specifically, the amount of genomic 5-hydroxymethylcytosine (5-hmC) was shown to be globally reduced in a wide range of cancers. The integration of this global biomarker into diagnostic workflows is hampered by the limitations of current 5-hmC quantification methods. Here we present and validate a fluorescence-based platform for high-throughput and cost-effective quantification of global genomic 5-hmC levels. We utilized the assay to characterize cancerous tissues based on their 5-hmC content, and observed a pronounced reduction in 5-hmC level in various cancer types. We present data for glioblastoma, colorectal cancer, multiple myeloma, chronic lymphocytic leukemia and pancreatic cancer, compared to corresponding controls. Potentially, the technique could also be used to follow response to treatment for personalized treatment selection. We present initial proof-of-concept data for treatment of familial adenomatous polyposis.

Rapid Quantification of Oxidation and UV Induced DNA Damage by Repair Assisted Damage Detection-(Rapid RADD).

Knowing the amount and type of DNA damage is of great significance for a broad range of clinical and research applications. However, existing methods are either lacking in their ability to distinguish between types of DNA damage or limited in their sensitivity and reproducibility. The method described herein enables rapid and robust quantification of type-specific single-strand DNA damage. The method is based on repair-assisted damage detection (RADD) by which fluorescent nucleotides are incorporated into DNA damage sites using type-specific repair enzymes. Up to 90 DNA samples are then deposited on a multiwell glass slide, and analyzed by a conventional slide scanner for quantification of DNA damage levels. Accurate and sensitive measurements of oxidative or UV-induced DNA damage levels and repair kinetics are presented for both in vitro and in vivo models.

Selective nanopore sequencing of human BRCA1 by Cas9-assisted targeting of chromosome segments (CATCH).

Next generation sequencing (NGS) is challenged by structural and copy number variations larger than the typical read length of several hundred bases. Third-generation sequencing platforms such as single-molecule real-time (SMRT) and nanopore sequencing provide longer reads and are able to characterize variations that are undetected in NGS data. Nevertheless, these technologies suffer from inherent low throughput which prohibits deep sequencing at reasonable cost without target enrichment. Here, we optimized Cas9-Assisted Targeting of CHromosome segments (CATCH) for nanopore sequencing of the breast cancer gene BRCA1. A 200 kb target containing the 80 kb BRCA1 gene body and its flanking regions was isolated intact from primary human peripheral blood cells, allowing long-range amplification and long-read nanopore sequencing. The target was enriched 237-fold and sequenced at up to 70× coverage on a single flow-cell. Overall performance and single-nucleotide polymorphism (SNP) calling were directly compared to Illumina sequencing of the same enriched sample, highlighting the benefits of CATCH for targeted sequencing. The CATCH enrichment scheme only requires knowledge of the target flanking sequence for Cas9 cleavage while providing contiguous data across both coding and non-coding sequence and holds promise for characterization of complex disease-related or highly variable genomic regions.

Lighting up individual DNA damage sites by in vitro repair synthesis.

DNA damage and repair are linked to fundamental biological processes such as metabolism, disease, and aging. Single-strand lesions are the most abundant form of DNA damage; however, methods for characterizing these damage lesions are lacking. To avoid double-strand breaks and genomic instability, DNA damage is constantly repaired by efficient enzymatic machinery. We take advantage of this natural process and harness the repair capacity of a bacterial enzymatic cocktail to repair damaged DNA in vitro and incorporate fluorescent nucleotides into damage sites as part of the repair process. We use single-molecule imaging to detect individual damage sites in genomic DNA samples. When the labeled DNA is extended on a microscope slide, damage sites are visualized as fluorescent spots along the DNA contour, and the extent of damage is easily quantified. We demonstrate the ability to quantitatively follow the damage dose response to different damaging agents as well as repair dynamics in response to UV irradiation in several cell types. Finally, we show the modularity of this single-molecule approach by labeling DNA damage in conjunction with 5-hydroxymethylcytosine in genomic DNA extracted from mouse brain tissue.

Spectroscopic quantification of 5-hydroxymethylcytosine in genomic DNA.

5-Hydroxymethylcytosine (5hmC), a modified form of the DNA base cytosine, is an important epigenetic mark linked to regulation of gene expression in development, and tumorigenesis. We have developed a spectroscopic method for a global quantification of 5hmC in genomic DNA. The assay is performed within a multiwell plate, which allows simultaneous recording of up to 350 samples. Our quantification procedure of 5hmC is direct, simple, and rapid. It relies on a two-step protocol that consists of enzymatic glucosylation of 5hmC with an azide-modified glucose, followed by a "click reaction" with an alkyne-fluorescent tag. The fluorescence intensity recorded from the DNA sample is proportional to its 5hmC content and can be quantified by a simple plate reader measurement. This labeling technique is specific and highly sensitive, allowing detection of 5hmC down to 0.002% of the total nucleotides. Our results reveal significant variations in the 5hmC content obtained from different mouse tissues, in agreement with previously reported data.

Effective training of nanopore callers for epigenetic marks with limited labelled data.

Nanopore sequencing platforms combined with supervised machine learning (ML) have been effective at detecting base modifications in DNA such as 5-methylcytosine (5mC) and N6-methyladenine (6mA). These ML-based nanopore callers have typically been trained on data that span all modifications on all possible DNA [Formula: see text]-mer backgrounds-a complete training dataset. However, as nanopore technology is pushed to more and more epigenetic modifications, such complete training data will not be feasible to obtain. Nanopore calling has historically been performed with hidden Markov models (HMMs) that cannot make successful calls for [Formula: see text]-mer contexts not seen during training because of their independent emission distributions. However, deep neural networks (DNNs), which share parameters across contexts, are increasingly being used as callers, often outperforming their HMM cousins. It stands to reason that a DNN approach should be able to better generalize to unseen [Formula: see text]-mer contexts. Indeed, herein we demonstrate that a common DNN approach (DeepSignal) outperforms a common HMM approach (Nanopolish) in the incomplete data setting. Furthermore, we propose a novel hybrid HMM-DNN approach, amortized-HMM, that outperforms both the pure HMM and DNN approaches on 5mC calling when the training data are incomplete. This type of approach is expected to be useful for calling other base modifications such as 5-hydroxymethylcytosine and for the simultaneous calling of different modifications, settings in which complete training data are not likely to be available.

Real-Life Experience With Purified Cannabidiol Treatment for Refractory Epilepsy: A Multicenter Retrospective Study.

BACKGROUND: Drug-resistant epilepsy (DRE) affects the development and quality of life of children and young adults. We analyzed the effectiveness and safety of purified CBD in this population. METHODS: A retrospective analysis of medical records of 139 children and young adults (54.7% female, median age 12.0 years) with DRE treated with purified CBD from 2018 to 2022 at five medical centers in Israel. RESULTS: The most common diagnosis was Lennox-Gastaut syndrome (37.4%) followed by Dravet syndrome (16.5%) and tuberous sclerosis complex (16.5%). Median purified CBD dose was 12.5 mg/kg (range 2.5 to 20.0), and median treatment duration was 9.0 months (range 0.5 to 48.0). Most patients (92.2%) had a reduced seizure frequency following treatment initiation; 41.1% had >50% reduction. Fifty-three patients (38.1%) had positive effects: improved alertness (31.7%), improved speech (10.1%), and achievement of new developmental milestones (2.2%). A multivariate linear model assessing predictive factors for seizure reduction demonstrated that patients previously treated with CBD oils, especially those with >50% seizure reduction on prior treatment, were also more likely to have a reduced seizure frequency while they were treated with purified CBD (P = 0.01, P < 0.0001). Development, diagnosis, age, purified CBD dose (0 to 10 mg/kg/day vs 10 to 20 mg/kg/day), and concomitant treatment with clobazam, valproic acid, or everolimus did not affect seizure reduction by purified CBD. The most common adverse events were irritability (20.9%) and drowsiness (12.9%). CONCLUSION: Purified CBD is well-tolerated and effective in reducing seizure frequency in children and young adults with DRE.

Long-Read Structural and Epigenetic Profiling of a Kidney Tumor-Matched Sample with Nanopore Sequencing and Optical Genome Mapping.

Carcinogenesis often involves significant alterations in the cancer genome architecture, marked by large structural and copy number variations (SVs and CNVs) that are difficult to capture with short-read sequencing. Traditionally, cytogenetic techniques are applied to detect such aberrations, but they are limited in resolution and do not cover features smaller than several hundred kilobases. Optical genome mapping and nanopore sequencing are attractive technologies that bridge this resolution gap and offer enhanced performance for cytogenetic applications. These methods profile native, individual DNA molecules, thus capturing epigenetic information. We applied both techniques to characterize a clear cell renal cell carcinoma (ccRCC) tumor's structural and copy number landscape, highlighting the relative strengths of each method in the context of variant size and average read length. Additionally, we assessed their utility for methylome and hydroxymethylome profiling, emphasizing differences in epigenetic analysis applicability.

Melanoma cells present high levels of HLA-A2-tyrosinase in association with instability and aberrant intracellular processing of tyrosinase.

Short-lived protein translation products are proposed to be a major source of substrates for major histocompatibility complex (MHC) class I antigen processing and presentation; however, a direct link between protein stability and the presentation level of MHC class I-peptide complexes has not been made. We have recently discovered that the peptide Tyr((369-377)) , derived from the tyrosinase protein is highly presented by HLA-A2 on the surface of melanoma cells. To examine the molecular mechanisms responsible for this presentation, we compared characteristics of tyrosinase in melanoma cells lines that present high or low levels of HLA-A2-Tyr((369-377)) complexes. We found no correlation between mRNA levels and the levels of HLA-A2-Tyr((369-377)) presentation. Co-localization experiments revealed that, in cell lines presenting low levels of HLA-A2-Tyr((369-377)) complexes, tyrosinase co-localizes with LAMP-1, a melanosome marker, whereas in cell lines presenting high HLA-A2-Tyr((369-377)) levels, tyrosinase localizes to the endoplasmic reticulum. We also observed differences in tyrosinase molecular weight and glycosylation composition as well as major differences in protein stability (t(1/2) ). By stabilizing the tyrosinase protein, we observed a dramatic decrease in HLA-A2-tyrosinase presentation. Our findings suggest that aberrant processing and instability of tyrosinase are responsible for the high presentation of HLA-A2-Tyr((369-377)) complexes and thus shed new light on the relationship between intracellular processing, stability of proteins, and MHC-restricted peptide presentation.

Dam Assisted Fluorescent Tagging of Chromatin Accessibility (DAFCA) for Optical Genome Mapping in Nanochannel Arrays.

Proteins and enzymes in the cell nucleus require physical access to their DNA target sites in order to perform genomic tasks such as gene activation and transcription. Hence, chromatin accessibility is a central regulator of gene expression, and its genomic profile holds essential information on the cell type and state. We utilized the E. coli Dam methyltransferase in combination with a fluorescent cofactor analogue to generate fluorescent tags in accessible DNA regions within the cell nucleus. The accessible portions of the genome are then detected by single-molecule optical genome mapping in nanochannel arrays. This method allowed us to characterize long-range structural variations and their associated chromatin structure. We show the ability to create whole-genome, allele-specific chromatin accessibility maps composed of long DNA molecules extended in silicon nanochannels.

Expression hierarchy of T cell epitopes from melanoma differentiation antigens: unexpected high level presentation of tyrosinase-HLA-A2 Complexes revealed by peptide-specific, MHC-restricted, TCR-like antibodies.

Peptide Ags presented by class I MHC molecules on human melanomas and that are recognized by CD8(+) T cells are the subjects of many studies of antitumor immunity and represent attractive candidates for therapeutic approaches. However, no direct quantitative measurements exist to reveal their expression hierarchy on the cell surface. Using novel recombinant Abs which bind these Ags with a peptide-specific, MHC-restricted manner, we demonstrate a defined pattern of expression hierarchy of peptide-HLA-A2 complexes derived from three major differentiation Ags: gp100, Melan-A/Mart-1, and tyrosinase. Studying melanoma cell lines derived from multiple patients, we reveal a surprisingly high level of presentation of tyrosinase-derived complexes and moderate to very low expression of complexes derived from other Ags. No correlation between Ag presentation and mRNA expression was found; however, protein stability may play a major role. These results provide new insights into the characteristics of Ag presentation and are particularly important when such targets are being considered for immunotherapy. These results may shed new light on relationships between Ag presentation and immune response to cancer Ags.

Single-molecule optical genome mapping in nanochannels: multidisciplinarity at the nanoscale.

The human genome contains multiple layers of information that extend beyond the genetic sequence. In fact, identical genetics do not necessarily yield identical phenotypes as evident for the case of two different cell types in the human body. The great variation in structure and function displayed by cells with identical genetic background is attributed to additional genomic information content. This includes large-scale genetic aberrations, as well as diverse epigenetic patterns that are crucial for regulating specific cell functions. These genetic and epigenetic patterns operate in concert in order to maintain specific cellular functions in health and disease. Single-molecule optical genome mapping is a high-throughput genome analysis method that is based on imaging long chromosomal fragments stretched in nanochannel arrays. The access to long DNA molecules coupled with fluorescent tagging of various genomic information presents a unique opportunity to study genetic and epigenetic patterns in the genome at a single-molecule level over large genomic distances. Optical mapping entwines synergistically chemical, physical, and computational advancements, to uncover invaluable biological insights, inaccessible by sequencing technologies. Here we describe the method's basic principles of operation, and review the various available mechanisms to fluorescently tag genomic information. We present some of the recent biological and clinical impact enabled by optical mapping and present recent approaches for increasing the method's resolution and accuracy. Finally, we discuss how multiple layers of genomic information may be mapped simultaneously on the same DNA molecule, thus paving the way for characterizing multiple genomic observables on individual DNA molecules.

From single-molecule to genome-wide mapping of DNA lesions: repair-assisted damage detection sequencing.

Mapping DNA damage and its repair has immense potential in understanding environmental exposures, their genotoxicity, and their impact on human health. Monitoring changes in genomic stability also aids in the diagnosis of numerous DNA-related diseases, such as cancer, and assists in monitoring their progression and prognosis. Developments in recent years have enabled unprecedented sensitivity in quantifying the global DNA damage dose in cells via fluorescence-based analysis down to the single-molecule level. However, genome-wide maps of DNA damage distribution are challenging to produce. Here, we describe the localization of DNA damage and repair loci by repair-assisted damage detection sequencing (RADD-seq). Based on the enrichment of damage lesions coupled with a pull-down assay and followed by next-generation sequencing, this method is easy to perform and can produce compelling results with minimal coverage. RADD-seq enables the localization of both DNA damage and repair sites for a wide range of single-strand damage types. Using this technique, we created a genome-wide map of the oxidation DNA damage lesion 8-oxo-7,8-dihydroguanine before and after repair. Oxidation lesions were heterogeneously distributed along the human genome, with less damage occurring in tight chromatin regions. Furthermore, we showed repair is prioritized for highly expressed, essential genes and in open chromatin regions. RADD-seq sheds light on cellular repair mechanisms and is capable of identifying genomic hotspots prone to mutation.

Multimodal single-molecule microscopy with continuously controlled spectral resolution.

Color is a fundamental contrast mechanism in fluorescence microscopy, providing the basis for numerous imaging and spectroscopy techniques. Building on spectral imaging schemes that encode color into a fixed spatial intensity distribution, here, we introduce continuously controlled spectral-resolution (CoCoS) microscopy, which allows the spectral resolution of the system to be adjusted in real-time. By optimizing the spectral resolution for each experiment, we achieve maximal sensitivity and throughput, allowing for single-frame acquisition of multiple color channels with single-molecule sensitivity and 140-fold larger fields of view compared with previous super-resolution spectral imaging techniques. Here, we demonstrate the utility of CoCoS in three experimental formats, single-molecule spectroscopy, single-molecule Förster resonance energy transfer, and multicolor single-particle tracking in live neurons, using a range of samples and 12 distinct fluorescent markers. A simple add-on allows CoCoS to be integrated into existing fluorescence microscopes, rendering spectral imaging accessible to the wider scientific community.

Label as you fold: methyltransferase-assisted functionalization of DNA nanostructures.

Non-DNA labels are key components for the construction of functional DNA nanostructures. Here, we present a method to graft covalent labels onto DNA origami nanostructures in an enzymatic one-pot reaction. The DNA methyltransferase M.TaqI labels the DNA nanostructures with azide groups, which serve as universal attachment points via click chemistry. Direct labeling with fluorescent dyes is also demonstrated. The procedure yields structures with high fluorescence intensities and narrow intensity distributions. In combination with UV crosslinking it enables the creation of temperature-stable, intense fluorescent beacons.

Simultaneous detection of multiple DNA damage types by multi-colour fluorescent labelling.

Herein we present an assay allowing concurrent detection of oxidative DNA damage and photoproducts. We apply DNA repair enzymes specific for each lesion type to incorporate spectrally distinct fluorescent nucleotides, enabling simultaneous quantification of the lesions on individual DNA molecules. We follow the repair of both damage types in skin cells exposed to artificial sunlight.

Analytical epigenetics: single-molecule optical detection of DNA and histone modifications.

The field of epigenetics describes the relationship between genotype and phenotype, by regulating gene expression without changing the canonical base sequence of DNA. It deals with molecular genomic information that is encoded by a rich repertoire of chemical modifications and molecular interactions. This regulation involves DNA, RNA and proteins that are enzymatically tagged with small molecular groups that alter their physical and chemical properties. It is now clear that epigenetic alterations are involved in development and disease, and thus, are the focus of intensive research. The ability to record epigenetic changes and quantify them in rare medical samples is critical for next generation diagnostics. Optical detection offers the ultimate single-molecule sensitivity and the potential for spectral multiplexing. Here we review recent progress in ultrasensitive optical detection of DNA and histone modifications.