Hermans, Nicolaas and Huisman, Jurien Jori and Brouwer, Thomas Bauke and Schachner, Christopher and van Heusden, G. Paul H. and Griesenbeck, Joachim and van Noort, John (2017) Toehold-enhanced LNA probes for selective pull down and single-molecule analysis of native chromatin. SCIENTIFIC REPORTS, 7: 16721. ISSN 2045-2322,
Full text not available from this repository. (Request a copy)Abstract
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.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | LOCKED NUCLEIC-ACIDS; FORCE SPECTROSCOPY; THERMODYNAMIC PARAMETERS; INDIVIDUAL NUCLEOSOMES; DNA-MOLECULES; REVEALS; LOCI; TRANSCRIPTION; PURIFICATION; COMPLEXES; |
| Subjects: | 500 Science > 570 Life sciences |
| Divisions: | Biology, Preclinical Medicine > Institut für Biochemie, Genetik und Mikrobiologie > Lehrstuhl für Biochemie III > Dr. Joachim Griesenbeck |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 14 Dec 2018 13:19 |
| Last Modified: | 25 Feb 2019 14:36 |
| URI: | https://pred.uni-regensburg.de/id/eprint/1779 |
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