Merkel Cell Polyomavirus (MCPyV) is the etiological agent of the majority of Merkel Cell Carcinomas (MCC). MCPyV positive MCCs harbor integrated, defective viral genomes that constitutively express viral oncogenes. Which molecular mechanisms promote viral integration, if distinct integration patterns exist, and if integration occurs preferentially at loci with specific chromatin states is unknown. We here combined short and long-read (nanopore) next-generation sequencing and present the first high-resolution analysis of integration site structure in MCC cell lines as well as primary tumor material. We find two main types of integration site structure: Linear patterns with chromosomal breakpoints that map closely together, and complex integration loci that exhibit local amplification of genomic sequences flanking the viral DNA. Sequence analysis suggests that linear patterns are produced during viral replication by integration of defective/linear genomes into host DNA double strand breaks via non-homologous end joining, NHEJ. In contrast, our data strongly suggest that complex integration patterns are mediated by microhomology-mediated break-induced replication, MMBIR. Furthermore, we show by ChIP-Seq and RNA-Seq analysis that MCPyV preferably integrates in open chromatin and provide evidence that viral oncogene expression is driven by the viral promoter region, rather than transcription from juxtaposed host promoters. Taken together, our data explain the characteristics of MCPyV integration and may also provide a model for integration of other oncogenic DNA viruses such as papillomaviruses.

Imprinted fluidic devices with nanochannels and transient inlets with smooth, gradually decreasing dimensions are used to analyze the flow of DNA single molecules.

No abstract

We present the fabrication of three-dimensional inlets with gradually decreasing widths and depths and with nanopillars on top, all defined in just one lithography step. In addition, as an application, we show how these micro-and nanostructures can be used for micro and nanofluidics and lab-on-a-chip devices to facilitate the flow and analyze single molecules of DNA. For the fabrication of 3D inlets in a single layer process, dose-modulated electron beam lithography was used, producing depths between 750 nm and 100 nm along a 30 µm long inlet, which is additionally structured with nanometer-scale pillars as a result of incomplete exposure and underdevelopment of the resist. Here, we show how we can fabricate and control the slope of the inlet, the nanopillar density, and coverage with the fabrication parameters. The key parameters are the dose used for the electron beam exposure and the development conditions, like the developer's dilution, stirring and development time. After patterning these structures in silicon, they can be replicated in polymer by UV-imprinting. The 3D inlets with nanostructured pillars act as a transition between micro and nanofluidic structures for pre-stretching and unfolding DNA molecules, avoiding the intrusion of folded molecules and clogging the analysis channel. Furthermore, the inlets with pillars also slow down the molecules before they enter the nanochannels, resulting in a 3-fold decrease in speed, which would translate to an improvement in the resolution for DNA optical mapping.