The chromatin fiber, formed by nucleosomes attached together by linker DNA, organizes into higher-order structures such as chromatin domains and chromosomes. The domains’ compaction level plays a...Show moreThe chromatin fiber, formed by nucleosomes attached together by linker DNA, organizes into higher-order structures such as chromatin domains and chromosomes. The domains’ compaction level plays a role in the regulation of transcription and depends on chromatin folding and aggregation mechanisms. However, the relation between conformational changes of the fibers and their aggregation has not been clearly resolved yet. Here, we test in vitro conditions that favor or prevent the aggregation of chromatin fibers with linker DNA lengths of 20 bp, 25 bp, 50 bp, and 55 bp and use fluorescent correlation spectroscopy to analyze the mobility of the aggregates and their constituents. Our results show that chromatin fibers with 25, 50 and 55 bp linker DNA form micron-sized aggregates in the presence of mono and divalent cation salts. The auto-correlation and cross-correlation models yielded diffusion times in the order of seconds for early-stage aggregation. The protocols and results obtained can provide a starting point for further research on the aggregation mechanism of chromatin and its impact on DNA transcription.Show less
Doxorubicine and Aclarubicine are widely used anti-tumor medicine that intercalate in betweenDNA basepairs. Exactly how these medicines get their anti-tumor behaviour is still a subject fordebate....Show moreDoxorubicine and Aclarubicine are widely used anti-tumor medicine that intercalate in betweenDNA basepairs. Exactly how these medicines get their anti-tumor behaviour is still a subject fordebate. In an effort to understand the anti-tumor behaviour of these medicines better, the effectsof intercalation on the intrinsic twist and the twist modulus of DNA was investigated. Changesto these two factors have effects on the way the DNA compacted through supercoiling whichcould have effects on the accessibility of genes. It was found that both medicines cause a similarunwinding of the double helix namely, 10.7°±0.8°for Doxorubicine and 14.1°±0.6°for Aclaru-bicine. Furthermore, the twist modulus was decreased significantly from 100 nm in naked DNAto 40 nm±2 nm with Doxorubicine bound and 44 nm±3 nm with Aclarubicine bound. Ad-ditionally, bound intercalators increase the force necessary to melt DNA, which could inhibitcellular processes such as DNA replication. Therefore, to investigate the binding strength ofDoxorubicine and Aclarubicine a force spectroscopy experiment was prepared with a hairpinon a custom made flow cell. While this hairpin construction is still a work in progress, workon creating a hairpin out of sequenced DNA on a Miseq flow cell was started. A hairpin de-sign was tested that altered the linear sequenced DNA into a hairpin construct by restrictingthe DNA and ligating a small hairpin oligo. An azide-nucleotide was built in and throughstrain-promoted azide-alkyne cycloaddition a long tether with a paramagnetic bead was at-tached. With such new hairpins generated from sequenced DNA, the sequence specific bindingstrength of intercalators could be investigated. On top of this, the hairpins could be used toinvestigate the sequence specificity of a wide range of DNA binders.Show less
