Toehold-enhanced LNA probes for selective pull down and single-molecule analysis of native chromatin

The organization of DNA into chromatin is thought to regulate gene expression in eukaryotes. To study its structure in vitro , there is a need for techniques that can isolate specific chromosomal loci of natively assembled chromatin. Current purification methods often involve chemical cross-linking...

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Bibliographic Details
Published in:Scientific reports 2017-12, Vol.7 (1), p.16721-9, Article 16721
Main Authors: Hermans, Nicolaas, Huisman, Juriën Jori, Brouwer, Thomas Bauke, Schächner, Christopher, van Heusden, G. Paul H., Griesenbeck, Joachim, van Noort, John
Format: Article
Language:English
Subjects:
45/22
631/1647/2230
631/337/100/101
631/337/2265
631/57/2265
639/766/747
Biotin
Chromatin
Chromatin - metabolism
Deoxyribonucleic acid
DNA
DNA - metabolism
DNA probes
Gene expression
Heterogeneity
Humanities and Social Sciences
multidisciplinary
Nucleic Acid Hybridization
Nucleic acids
Nucleotide sequence
Nucleotides
Oligonucleotides - metabolism
Probes
Purification
Ribosomal DNA
RNA, Ribosomal, 18S - chemistry
RNA, Ribosomal, 18S - metabolism
Saccharomyces cerevisiae - genetics
Science
Science (multidisciplinary)
Spectroscopy
Yeasts
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Summary:The organization of DNA into chromatin is thought to regulate gene expression in eukaryotes. To study its structure in vitro , there is a need for techniques that can isolate specific chromosomal loci of natively assembled chromatin. Current purification methods often involve chemical cross-linking to preserve the chromatin composition. However, such cross-linking may affect the native structure. It also impedes single molecule force spectroscopy experiments, which have been instrumental to probe chromatin folding. Here we present a method for the incorporation of affinity tags, such as biotin, into native nucleoprotein fragments based on their DNA sequence, and subsequent single molecule analysis by magnetic tweezers. DNA oligos with several Locked Nucleic Acid (LNA) nucleotides are shown to selectively bind to target DNA at room temperature, mediated by a toehold end in the target, allowing for selective purification of DNA fragments. The stability of the probe-target hybrid is sufficient to withstand over 65 pN of force. We employ these probes to obtain force-extension curves of native chromatin fragments of the 18S ribosomal DNA from the yeast Saccharomyces cerevisiae . These experiments yield valuable insights in the heterogeneity in structure and composition of natively assembled chromatin at the single-molecule level.
ISSN:2045-2322
2045-2322
DOI:10.1038/s41598-017-16864-7