Evaluation by Experimentation and Simulation of a FRET Pair Comprising Fluorescent Nucleobase Analogs in Nucleosomes

Abstract

Förster resonance energy transfer (FRET) is an attractive tool for understanding biomolecular dynamics. FRET‐based analysis of nucleosomes has the potential to fill the knowledge gaps between static structures and dynamic cellular behaviors. Compared with typical FRET pairs using bulky fluorophores introduced by flexible linkers, fluorescent nucleoside‐based FRET pair has great potential since it can be fitted within the helical structures of nucleic acids. Herein we report on the construction of nucleosomes containing a nucleobase FRET pair and the investigation of experimental and theoretical FRET efficiencies through steady‐state fluorescence spectroscopy and calculation based on molecular dynamics simulations, respectively. Distinguishable experimental FRET efficiencies were observed depending on the positions of FRET pairs in nucleosomal DNA. The tendency could be supported by theoretical study. This work suggests the possibility of our approach to analyze structural changes of nucleosomes by epigenetic modifications or internucleosomal interactions.

Template-dependent DNA ligation for the synthesis of modified oligonucleotides

“…Importantly, this method is not only flexible in terms of the nature of chemical modifications but also in terms of size of the sequences that can be achieved since we were capable of producing shorter oligonucleotides similar to that of clinically relevant ASOs and siRNAs, but also larger fragments (> 100 nt) which might be directly amenable to address biological questions such as the three-dimensional structure and dynamics of nucleosomes. 49 Additionally, our ligation method demonstrated great flexibility in terms of the length and sequence combinations of the shortmers, which is an additional important perquisite for the construction of literally any given sequence of interest. This variant synthesis protocol also displays a high atom and enzyme economy as fewer functional groups per nucleotide are involved in the ligation reactions.…”

Template-dependent DNA ligation for the synthesis of modified oligonucleotides

“…Importantly, this method is not only flexible in terms of the nature of chemical modifications but also in terms of size of the sequences that can be achieved since we were capable of producing shorter oligonucleotides similar to that of clinically relevant ASOs and siRNAs, but also much larger fragments (> 150 nt) which might be directly amenable to address biological questions such as the three-dimensional structure and dynamics of nucleosomes. 48 Additionally, our ligation method demonstrated great flexibility in terms of the length and sequence combinations of the shortmers, which is an additional important perquisite for the construction of literally any given sequence of interest. This variant synthesis protocol also displays a high atom and enzyme economy as fewer functional groups per nucleotide are involved in the ligation reactions.…”

Currently Used Methods to Evaluate the Efficacy of Therapeutic Drugs and Kidney Safety

“…Importantly, these simulations can also forecast how biomolecules react to alterations like mutations, phosphorylation, protonation, or the addition or removal of ligands at the atomic level. A wide range of experimental structural biology techniques, such as x-ray crystallography [120], cryo-electron microscopy (cryo-EM) [121], nuclear magnetic resonance (NMR) [122], electron paramagnetic resonance (EPR) [123], and fluorescence resonance energy transfer (FRET) [124] are frequently utilized in conjunction with MD simulations. To study the treatments for kidney failure, MD simulations can applied to investigate several innovative drugs for treating kidney diseases.…”