eoe sub articles

EoE Sub Articles

1. Working concentration of the reaction components
<ol>
	<li>Prepare the working concentrations of buffers for CD freshly and keep them at 4 &#176;C before setting up the reactions. 
	NOTE: For the CD reactions described in this paper, the working concentrations of components are as follows: Sodium phosphate buffer (pH 7.0) 1 mM, ATP 2 mM, DNA 500 nM, Protein 1 &#181;M, MgCl2 10 mM, EDTA 50 mM, ADAADiN 5 &#181;M.</li>
</ol>
2. Choosing and...

the circular dichroism spectroscopy technique to study dna-protein interactions

Source: Arya, V., et al. <a href="https://app.jove.com/v/63147">CD Spectroscopy to Study DNA-Protein Interactions.</a> J. Vis. Exp. (2022).
This video demonstrates the circular dichroism (CD) spectroscopy technique to study conformational changes occurring in DNA in the presence of ATP-dependent chromatin remodeling protein. The changes in the CD spectra measured in the presence and absence of ATP hydrolysis indicate the importance of ATP in the ability of the remodeler to induce conformational changes in the bound DNA.

The Circular Dichroism Spectroscopy Technique to Study DNA-Protein Interactions

Source: Arya, V., et al. CD Spectroscopy to Study DNA-Protein Interactions. J. Vis. Exp. (2022).
This video demonstrates the circular dichroism (CD) ...

ATP-dependent chromatin remodeling proteins regulate gene expression in tightly-packed chromatin by altering the DNA conformation in an ATP-dependent manner.
To study chromatin remodeler-DNA interactions using circular dichroism, or CD, spectroscopy, take a solution of double-stranded DNA oligonucleotides. Heat to denature the oligonucleotides, and place on ice.
Fast cooling causes the DNA to renature while forming secondary structures. The double-to-single-strand transition region provides an optimal binding site for the chromatin remodelers.
Add the heat-cooled DNA into a cuvette. Add a buffer containing ATP-dependent chromatin remodeler, ATP, and magnesium ions.
The remodeler binds to the DNA, followed by the binding of magnesium ion-complexed ATP to the protein &#8212; activating it. The activated remodeler hydrolyzes ATP &#8212; inducing a stem-loop conformation in the DNA.
Pass left and right circularly-polarized light &#8212; with a 90&#176; phase difference &#8212; through the sample. Optically chiral DNA absorbs the left and right circularly-polarized light to different extents.
The differential absorption, termed circular dichroism, results in the transmitted light being elliptically polarized, which is measured as ellipticity. Two positive peaks at specific wavelengths in the CD spectra confirm the stem-loop structure.
Add EDTA to chelate the magnesium ions, inhibiting the ATPase activity.
A change in the CD spectra upon chelation &#8212; showing a negative peak and a broad positive peak at specific wavelengths &#8212; indicates the disruption of the stem-loop conformation in the absence of ATP hydrolysis.

the-circular-dichroism-spectroscopy-technique-to-study-dna-protein

Research

JoVE Encyclopedia of Experiments

Biological Techniques

Spectroscopy Techniques

Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. Therefore, DNA-protein interactions regulate multiple physiological, pathophysiological, and biological functions, such as cell differentiation, cell proliferation, cell cycle control, chromosome stability, epigenetic gene regulation, and cell transformation. In eukaryotic cells, the DNA interacts with histone and nonhistone proteins and is condensed into chromatin. Several technical tools can be used to analyze DNA-protein interactions, such as the Electrophoresis (gel) Mobility Shift Assay (EMSA) and DNase I footprinting. However, these techniques analyze the protein-DNA interaction in vitro, not within the cellular context. Chromatin immunoprecipitation (ChIP) is a technique that captures proteins at their specific DNA binding sites, thereby allowing for the identification of DNA-protein interactions within their chromatin context. It is done by fixation&#160;of the DNA-protein interaction, followed by&#160;immunoprecipitation of the protein of interest. Subsequently, the genomic site that the protein was bound to is characterized. Here, we describe and discuss ChIP and demonstrate its analytical value for the identification of the Transforming Growth Factor-&#946; (TGF-&#946;)-induced binding of the transcription factor SMAD2 to SMAD Binding Elements (SBE) within the promoter region of the tyrosine-protein kinase Kit (c-KIT) receptor ligand Stem Cell Factor (SCF).

DNA-protein interactions are essential for multiple biological processes. During the evaluation of cellular functions, the analysis of DNA-protein interactions is indispensable for understanding gene regulation. Chromatin immunoprecipitation (ChIP) is a powerful tool to analyze such interactions in vivo.

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. ...

JoVE Journal

Genetics

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular methodology for applications in various fields and for different research purposes. The procedure described herein focuses on the synthesis, purification, and characterization of dodecamers of RNA 5&#39;-[CUA CGG AAU CAU]-3&#39; containing zero, one, or two modifications located at the C2&#39;-O-position. The probes are based on 2-thiophenylmethyl groups, incorporated into RNA nucleotides via standard organic synthesis and introduced into the corresponding oligonucleotides via their respective phosphoramidites. This report makes use of phosphoramidite chemistry via the four canonical nucleobases (Uridine (U), Cytosine (C), Guanosine (G), Adenosine (A)), as well as 2-thiophenylmethyl functionalized nucleotides modified at the 2&#39;-O-position; however, the methodology is amenable for a large variety of modifications that have been developed over the years. The oligonucleotides were synthesized on a controlled-pore glass (CPG) support followed by cleavage from the resin and deprotection under standard conditions, i.e., a mixture of ammonia and methylamine (AMA) followed by hydrogen fluoride/triethylamine/N-methylpyrrolidinone. The corresponding oligonucleotides were purified via polyacrylamide electrophoresis (20% denaturing) followed by elution, desalting, and isolation via reversed-phase chromatography (Sep-pak, C18-column). Quantification and structural parameters were assessed via ultraviolet-visible (UV-vis) and circular dichroism (CD) photometric analysis, respectively. This report aims to serve as a resource and guide for beginner and expert researchers interested in embarking in this field. It is expected to serve as a work-in-progress as new technologies and methodologies are developed. The description of the methodologies and techniques within this document correspond to a DNA/RNA synthesizer (refurbished and purchased in 2013) that uses phosphoramidite chemistry.

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

This article provides a detailed procedure on the solid-phase synthesis, purification, and characterization of dodecamers of RNA modified at the C2&#39;-O-position. UV-vis and circular dichroism photometric analyses are used to quantify and characterize structural aspects, i.e., single-strands or double-strands.

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular ...

Chemistry

Synthesis and Characterization of 1,2-Dithiolane Modified Self-Assembling Peptides

This report focuses on the synthesis of an N-terminus 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting self-assembled supramolecular structures. The synthetic route takes advantage of solid-phase peptide synthesis with the on-resin coupling of the dithiolane precursor molecule, 3-(acetylthio)-2-(acetylthiomethyl)propanoic acid, and the microwave-assisted thioacetate deprotection of the peptide N-terminus before final cleavage from the resin to yield the 1,2-dithiolane modified peptide. After the high-performance liquid chromatography (HPLC) purification of the 1,2-dithiolane peptide, derived from the nucleating core of the A&#946; peptide associated with Alzheimer&#39;s disease, the peptide is shown to self-assemble into cross-&#946; amyloid fibers. Protocols to characterize the amyloid fibers by Fourier-transform infrared spectroscopy (FT-IR), circular dichroism spectroscopy (CD) and transmission electron microscopy (TEM) are presented. The methods of N-terminal modification with a 1,2-dithiolane moiety to well-characterized self-assembling peptides can now be explored as model systems to develop post-assembly modification strategies and explore dynamic covalent chemistry on supramolecular peptide nanofiber surfaces.

A protocol for the synthesis of a 1,2-dithiolane modified peptide and the characterization of the supramolecular structures resulting from the peptide self-assembly.

The Circular Dichroism Spectroscopy Technique to Study DNA-Protein Interactions

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