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.
Highlights
The activity of a gene is regulated by a plethora of proteins that contribute to structural changes in chromatin, which in turn provide access to the transcription machinery
It was shown that Locked Nucleic Acids (LNA) probes have a higher efficiency when invading DNA ends[20]
We show that LNA-toehold probes can be used to introduce affinity tags into DNA fragments in a sequence specific manner, without the need for temperature-induced melting of the DNA
Summary
The activity of a gene is regulated by a plethora of proteins that contribute to structural changes in chromatin, which in turn provide access to the transcription machinery. To elucidate chromatin-related mechanisms of transcription regulation, the structural changes and the precise stoichiometry of the proteins involved need to be resolved This is an arduous challenge because large variations in chromatin composition have been reported within a single locus[2,3]. A notable exception was the pioneering study by Cui and Bustamante, who probed nucleosome interactions in natively assembled chromatin fibers from chicken erythrocytes[9] These experiments were done on random fragments, impeding any insight into the local structure of a specific locus. Rather than elevated temperatures and crosslinking, we use restriction enzymes to create a 4-base toehold on the target DNA which enhances both hybridization efficiency and specificity We used these LNA probes to insert a digoxigenin (Dig) tag on the 18S fragment of yeast rDNA, and subsequently to tether the molecules between a paramagnetic bead and an antibody coated glass coverslip
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