Biology subjects

Weinhold, E.

Publications and source records attributed to Weinhold, E..

6 recordsLinked to original sources

bioRxiv2023

A genetic circuit on a single DNA molecule as autonomous dissipative nanodevice

Realizing genetic circuits on single DNA molecules as self-encoded dissipative nanodevices is a major step toward miniaturization of autonomous biological systems. A circuit operating on a single DNA implies that genetically encoded proteins localize during coupled transcriptiontranslation to DNA, but a single-molecule measurement demonstrating this has remained a challenge. Here, we used a genetically encoded fluorescent reporter system with improved spatiotemporal resolution and observed the synthesis of individual proteins tethered to a DNA molecule by transient complexes of RNA polymerase, messenger RNA, and ribosome. Against expectations in dilute cell-free conditions where equilibrium considerations favor dispersion, these nascent proteins linger long enough to regulate cascaded reactions on the same DNA. We rationally designed a pulsatile genetic circuit by encoding an activator and repressor in feedback on the same DNA molecule. Driven by the local synthesis of only several proteins per hour and gene, the circuit dynamics exhibited enhanced variability between individual DNA molecules, and fluctuations with a broad power spectrum. Our results demonstrate that coexpressional localization, as a nonequilibrium process, facilitates single-DNA genetic circuits as dissipative nanodevices, with implications for nanobiotechnology applications and artificial cell design.

synthetic biology↗

bioRxiv2022

Simultaneous global labeling (SiGL) of 5-methylcytosine and 5-hydroxymethylcytosine by DNA alkylation with a synthetic cofactor and engineered methyltransferase

5-methylcytosine and 5-hydroxymethylcytosine are epigenetic modifications involved in gene regulation and cancer. Here, we describe a new, simple, and high-throughput platform for multi-colour 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 labelling 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 reduction in 5-hydroxymethylcytosine levels, but no change in DNA methylation levels between sick and healthy individuals. We also measured epigenetic modifications in chronic lymphocytic leukaemia and observed a decrease in both modification levels. Our results indicate that this assay may be used for the epigenetic characterization of clinical samples for research and patient management.

genetics↗

bioRxiv2022

Optical Genome and Epigenome Mapping of Clear Cell Renal Cell Carcinoma

Cancer cells display complex genomic aberrations that include large-scale genetic rearrangements and epigenetic modulation that are not easily characterized by short-read sequencing. We present a method for simultaneous profiling of long-range genetic/epigenetic changes in matched cancer samples. Clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Most ccRCC cases demonstrate somatic genomic alterations involving the short arm of chromosome 3 (3p), most often targeting the von Hippel-Lindau (VHL) gene. Aiming to identify somatic alterations that characterize early stage ccRCC, we performed comprehensive genetic, cytogenetic and epigenetic analyses comparing ccRCC tumor to adjacent non-tumorous tissue. Optical genome mapping identified genomic aberrations such as structural and copy number variations, complementing exome-sequencing results. Single-molecule methylome and hydroxymethylome mapping revealed multiple differential regions, some of them known to be associated with ccRCC pathogenesis. Among them, metabolic pathways were significantly enriched. Moreover, significant global epigenetic differences were detected between the tumor and the adjacent non-tumorous tissue, and a correlation between epigenetic signals and gene expression was found. This is the first reported comparison of a human tumor and a matched tissue by optical genome/epigenome mapping, revealing well-established and novel somatic aberrations.

genomics↗

bioRxiv2021

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 among the most significant epigenetic modifications. Here we developed a chemoenzymatic procedure to fluorescently label non-methylated cytosines in the CpG context allowing epigenetic profiling of single DNA molecules spanning hundreds of thousands of base pairs. For this method, a CpG methyltransferase was used to transfer an azide to cytosines from a synthetic S-adenosyl-l-methionine cofactor analog. 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 two-fold by the addition of a nucleosidase that degrades the inactive by-product of the azide-cofactor after labeling, and prevents its inhibitory effect. We first used the method to determine the decline in global DNA methylation in chronic lymphocytic leukemia patients and then performed whole genome methylation mapping of the model plant Arabidopsis thaliana. Our genome maps show high concordance with published methylation maps produced by bisulfite sequencing. Although mapping resolution is limited by optical detection to 500-1000 base pairs, 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.

genomics↗

bioRxiv2017

Reduced representation optical methylation mapping (R2OM2).

Reduced representation methylation profiling is a method of analysis in which a subset of CpGs is used to report the overall methylation status of the probed genomic regions. This approach has been widely adopted for genome-scale bisulfite sequencing since it requires fewer sequencing reads and uses significantly less starting material than whole-genome analysis. Consequently, this method is suitable for profiling medical samples and single cells at high throughput and reduced costs. Here, we use this concept in order to create a pattern of fluorescent optical methylation profiles along individual DNA molecules. Reduced representation optical methylation mapping (R2OM2) in combination with Bionano Genomics next generation genome mapping (NGM) technology provides a hybrid genetic/epigenetic genome map of individual chromosome segments spanning hundreds of kilobase pairs (kbp). These long reads, along with the single-molecule resolution, allow for epigenetic variation calling and methylation analysis of large structural aberrations such as pathogenic macrosatellite arrays not accessible to single-cell next generation sequencing (NGS). We apply this method to facioscapulohumeral dystrophy (FSHD) showing both structural variation and hypomethylation status of a disease-associated, highly repetitive locus on chromosome 4q.

genetics↗

bioRxiv2017

Reduced representation optical methylation mapping (R2OM2).

Reduced representation methylation analysis utilizes a subset of CpGs in order to report the overall methylation status of the probed genomic regions. Here, we use this concept in order to create fluorescent optical methylation profiles along chromosomal DNA molecules for epigenetic profiling. Reduced representation optical methylation mapping (R2OM2) in combination with Bionano Genomics next generation genome mapping (NGM) technology provides a hybrid genetic/epigenetic genome map of individual chromosome segments spanning hundreds of kilobase pairs (kbp). These long reads, along with the single-molecule resolution, allow for epigenetic variation calling and methylation analysis of large structural aberrations such as pathogenic macrosatellite arrays not accessible to single-cell next generation sequencing (NGS). We show that in addition to the inherent long-read benefits of R2OM2, it provides genomic methylation patterns comparable to whole genome bisulfite sequencing (WGBS) while retaining single-molecule information. The method is applied here to detect methylation along genes, around regulatory histone marks and to study facioscapulohumeral muscular dystrophy (FSHD), simultaneously recording the haplotype, copy number and methylation status of the disease-associated, highly repetitive locus onchromosome 4q.

genetics↗