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.

One-Pot Chemoenzymatic Cascade for Labeling of the Epigenetic Marker 5-Hydroxymethylcytosine.

The epigenetic DNA modification 5-hydroxymethylcytosine (5-hmC) is important for the regulation of gene expression during development and in tumorigenesis. 5-hmC can be selectively glycosylated by T4 ß-glucosyltransferase (ß-GT); introduction of an azide on the attached sugar provides a chemical handle for isolation or fluorescent tagging of 5-hmC residues by click chemistry. This approach has not been broadly adopted because of the challenging synthesis and limited commercial availability of the glycosylation substrate, 6-deoxy-6-azido-α-D-glucopyranoside. We report the enzyme-assisted synthesis of this precursor by the uridylyltransferase from Pasteurella multocida (PmGlmU). We were able to directly label 5-hmC in genomic DNA by an enzymatic cascade involving successive action of PmGlmU and ß-GT. This is a facile and cost-effective one-pot chemoenzymatic methodology for 5-hmC analysis.

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.

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.

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.

Epigenetic Optical Mapping of 5-Hydroxymethylcytosine in Nanochannel Arrays.

The epigenetic mark 5-hydroxymethylcytosine (5-hmC) is a distinct product of active DNA demethylation that is linked to gene regulation, development, and disease. In particular, 5-hmC levels dramatically decline in many cancers, potentially serving as an epigenetic biomarker. The noise associated with next-generation 5-hmC sequencing hinders reliable analysis of low 5-hmC containing tissues such as blood and malignant tumors. Additionally, genome-wide 5-hmC profiles generated by short-read sequencing are limited in providing long-range epigenetic information relevant to highly variable genomic regions, such as the 3.7 Mbp disease-related Human Leukocyte Antigen (HLA) region. We present a long-read, highly sensitive single-molecule mapping technology that generates hybrid genetic/epigenetic profiles of native chromosomal DNA. The genome-wide distribution of 5-hmC in human peripheral blood cells correlates well with 5-hmC DNA immunoprecipitation (hMeDIP) sequencing. However, the long single-molecule read-length of 100 kbp to 1 Mbp produces 5-hmC profiles across variable genomic regions that failed to show up in the sequencing data. In addition, optical 5-hmC mapping shows a strong correlation between the 5-hmC density in gene bodies and the corresponding level of gene expression. The single-molecule concept provides information on the distribution and coexistence of 5-hmC signals at multiple genomic loci on the same genomic DNA molecule, revealing long-range correlations and cell-to-cell epigenetic variation.

Single-molecule quantification of 5-hydroxymethylcytosine for diagnosis of blood and colon cancers.

BACKGROUND: The DNA modification 5-hydroxymethylcytosine (5hmC) is now referred to as the sixth base of DNA with evidence of tissue-specific patterns and correlation with gene regulation and expression. This epigenetic mark was recently reported as a potential biomarker for multiple types of cancer, but its application in the clinic is limited by the utility of recent 5hmC quantification assays. We use a recently developed, ultra-sensitive, fluorescence-based single-molecule method for global quantification of 5hmC in genomic DNA. The high sensitivity of the method gives access to precise quantification of extremely low 5hmC levels common in many cancers. METHODS: We assessed 5hmC levels in DNA extracted from a set of colon and blood cancer samples and compared 5hmC levels with healthy controls, in a single-molecule approach. RESULTS: Using our method, we observed a significantly reduced level of 5hmC in blood and colon cancers and could distinguish between colon tumor and colon tissue adjacent to the tumor based on the global levels of this molecular biomarker. CONCLUSIONS: Single-molecule detection of 5hmC allows distinguishing between malignant and healthy tissue in clinically relevant and accessible tissue such as blood and colon. The presented method outperforms current commercially available quantification kits and may potentially be developed into a widely used, 5hmC quantification assay for research and clinical diagnostics. Furthermore, using this method, we confirm that 5hmC is a good molecular biomarker for diagnosing colon and various types of blood cancer.