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.

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.

Ionic Current-Based Mapping of Short Sequence Motifs in Single DNA Molecules Using Solid-State Nanopores.

Nanopore sensors show great potential for rapid, single-molecule determination of DNA sequence information. Here, we develop an ionic current-based method for determining the positions of short sequence motifs in double-stranded DNA molecules with solid-state nanopores. Using the DNA-methyltransferase M.TaqI and a biotinylated S-adenosyl-l-methionine cofactor analogue we create covalently attached biotin labels at 5'-TCGA-3' sequence motifs. Monovalent streptavidin is then added to bind to the biotinylated sites giving rise to additional current blockade signals when the DNA passes through a conical quartz nanopore. We determine the relationship between translocation time and position along the DNA contour and find a minimum resolvable distance between two labeled sites of ∼200 bp. We then characterize a variety of DNA molecules by determining the positions of bound streptavidin and show that two short genomes can be simultaneously detected in a mixture. Our method provides a simple, generic single-molecule detection platform enabling DNA characterization in an electrical format suited for portable devices for potential diagnostic applications.

The N6-Position of Adenine Is a Blind Spot for TAL-Effectors That Enables Effective Binding of Methylated and Fluorophore-Labeled DNA.

Transcription-activator-like effectors (TALEs) are programmable DNA binding proteins widely used for genome targeting. TALEs consist of multiple concatenated repeats, each selectively recognizing one nucleobase via a defined repeat variable diresidue (RVD). Effective use of TALEs requires knowledge about their binding ability to epigenetic and other modified nucleobases occurring in target DNA. However, aside from epigenetic cytosine-5 modifications, the binding ability of TALEs to modified DNA is unknown. We here study the binding of TALEs to the epigenetic nucleobase N6-methyladenine (6mA) found in prokaryotic and recently also eukaryotic genomes. We find that the natural, adenine (A)-binding RVD NI is insensitive to 6mA. Model-assisted structure-function studies reveal accommodation of 6mA by RVDs with altered hydrophobic surfaces and abilities of hydrogen bonding to the N6-amino group or N7 atom of A. Surprisingly, this tolerance of N6 substitution was transferrable to bulky N6-alkynyl substituents usable for click chemistry and even to a large rhodamine dye, establishing the N6 position of A as the first site of DNA that offers label introduction within TALE target sites without interference. These findings will guide future in vivo studies with TALEs and expand their applicability as DNA capture probes for analytical applications in vitro.

Light-Enhancing Plasmonic-Nanopore Biosensor for Superior Single-Molecule Detection.

A stacked plasmonic nanowell-nanopore biosensor strongly suppresses the background fluorescence from the bulk and yields net more than tenfold enhancement of the fluorescence intensity. The device offers extremely high signal-to-background (S/B) ratio for single-molecule detection at ultralow excitation laser intensities, while maintaining extremely high temporal bandwidth for single-DNA sensing.

Single-Molecule DNA Methylation Quantification Using Electro-optical Sensing in Solid-State Nanopores.

Detection of epigenetic markers, including 5-methylcytosine, is crucial due to their role in gene expression regulation and due to the mounting evidence of aberrant DNA methylation patterns in cancer biogenesis. Single-molecule methods to date have primarily been focused on hypermethylation detection; however, many oncogenes are hypomethylated during cancer development, presenting an important unmet biosensing challenge. To this end, we have developed a labeling and single-molecule quantification method for multiple unmethylated cytosine-guanine dinucleotides (CpGs). Our method involves a single-step covalent coupling of DNA with synthetic cofactor analogues using DNA methyltransferases (MTases) followed by molecule-by-molecule electro-optical nanopore detection and quantification with single or multiple colors. This sensing method yields a calibrated scale to directly quantify the number of unmethylated CpGs in the target sequences of each DNA molecule. Importantly, our method can be used to analyze ∼10 kbp long double-stranded DNA while circumventing PCR amplification or bisulfite conversion. Expanding this technique to use two colors, as demonstrated here, would enable sensing of multiple DNA MTases through orthogonal labeling/sensing of unmethylated CpGs (or other epigenetic modifications) associated with specific recognition sites. Our proof-of-principle study may permit sequence-specific, direct targeting of clinically relevant hypomethylated sites in the genome.

Sequence-specific labeling of nucleic acids and proteins with methyltransferases and cofactor analogues.

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5'-ATCGAT-3' sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.