Topological constraints strongly affect chromatin reconstitution in silico

The fundamental building block of chromatin, and of chromosomes, is the nucleosome, a composite material made up from DNA wrapped around a his-tone octamer. In this study we provide the first com-puter simulations of chromatin self-assembly, start-ing from DNA and histone proteins, and use these to understand the constraints which are imposed by the topology of DNA molecules on the creation of a polynucleosome chain. We take inspiration from the in vitro chromatin reconstitution protocols which are used in many experimental studies. Our simulations indicate that during self-assembly, nucleosomes can fall into a number of topological traps (or local folding defects), and this may eventually lead to the forma-tion of disordered structures, characterised by nu-cleosome clustering. Remarkably though, by intro-ducing the action of topological enzymes such as type I and II topoisomerase, most of these defects can be avoided and the result is an ordered 10-nm chromatin fibre. These findings provide new insight into the biophysics of chromatin formation, both in the context of reconstitution in vitro and in terms of the topological constraints which must be overcome during de novo nucleosome formation in vivo, e.g. following DNA replication or repair

Base pair mismatch identification with DNA nanoswitch and long lifetime acridine fluorophore

Detection of single base pair mutations in an unlabelled nucleic acid target sequence is demonstrated using the time-resolved measurement of the Förster Resonance Energy Transfer (FRET) between a long lifetime acridine-based fluorophore and a non-fluorescent quencher molecule covalently bound to a DNA Holliday junction-based nanoswitch

Bait-and-switch molecular recognition in nucleic acid sensors: Time-resolved fluorescence, single nucleotide polymorphism detection

We investigate the properties of a simple DNA-based nanodevice capable of detecting single nucleotide polymorphisms (SNPs) in unlabelled nucleic acid target sequences. Detection is achieved by a two-stage bait-and-switch process combining complementary-base hybridization and switching as molecular recognition criteria. A probe molecule is constructed from a single DNA strand designed to adopt a partial cruciform structure with a pair of exposed (unhybridized) strands. Upon target binding, a switchable cloverleaf construct (similar to a Holliday junction) is formed where the states are the open and closed junction conformations. Switching between these occurs by junction folding in the presence of divalent ions. A combination of steady-state and time-resolved fluorescence spectroscopy is used to measure Forster resonance energy transfer

Detection of single nucleotide polymorphisms using a DNA Holliday junction nanoswitch?a high-throughput fluorescence lifetime assay

We report a simple DNA sensor device, using a combination of binding and conformational switching, capable of rapid detection of specific single nucleotide polymorphisms in an unlabelled nucleic acid target sequence. The detection is demonstrated using fluorescence lifetime measurements in a high-throughput micro plate reader instrument based on the time-correlated single-photon counting technique. The sensor design and instrumental architecture are capable of detecting perturbations in the molecular structure of the probe–target complex (which is similar to that of a Holliday junction), due to a single base pair mismatch in a synthetic target. Structural information, including fluorophore separations, is obtained using time-resolved Förster resonance energy transfer between two fluorophores covalently bound to the probe molecule. The two probes required are designed to detect a single nucleotide polymorphism from a sequence present on each of the two copies of human chromosome 1