Electrochemical Sensing of Idarubicin—DNA Interaction Using Electropolymerized Azure B and Methylene Blue Mediation

A highly sensitive electrochemical DNA sensor for detection of the chemotherapeutic drug idarubicin mediated by Methylene blue (MB) has been developed. DNA from fish sperm has been immobilized at the electropolymerized layers of Azure B. The incorporation of MB into the DNA layers substantially increased the sensor sensitivity. The concentration range for idarubicin determination by cyclic voltammetry was from 1 fM to 0.1 nM, with a limit of detection (LOD) of 0.3 fM. Electrochemical impedance spectroscopy (EIS) in the presence of a redox probe ([Fe(CN)6]3−/4−) allowed for the widening of a linear range of idarubicin detection from 1 fM to 100 nM, retaining LOD 0.3 fM. The DNA sensor has been tested in various real and artificial biological fluids with good recovery ranging between 90–110%. The sensor has been successfully used for impedimetric idarubicin detection in medical preparation Zavedos®. The developed DNA biosensor could be useful for the control of the level of idarubicin during cancer therapy as well as for pharmacokinetics studies.

DNA Flow-Stretch Assays for Studies of Protein-DNA Interactions at the Single-Molecule Level

Protein-DNA interactions are the core of the cell’s molecular machinery. For a long time, conventional biochemical methods served as a powerful investigatory basis of protein-DNA interactions and target search mechanisms. Currently single-molecule (SM) techniques have emerged as a complementary tool for studying these interactions and have revealed plenty of previously obscured mechanistic details. In comparison to the traditional ones, SM methods allow direct monitoring of individual biomolecules. Therefore, SM methods reveal reactions that are otherwise hidden by the ensemble averaging observed in conventional bulk-type methods. SM biophysical techniques employing various nanobiotechnology methods for immobilization of studied molecules grant the possibility to monitor individual reaction trajectories of biomolecules. Next-generation in vitro SM biophysics approaches enabling high-throughput studies are characterized by much greater complexity than the ones developed previously. Currently, several high-throughput DNA flow-stretch assays have been published and have shown many benefits for mechanistic target search studies of various DNA-binding proteins, such as CRISPR-Cas, Argonaute, various ATP-fueled helicases and translocases, and others. This review focuses on SM techniques employing surface-immobilized and relatively long DNA molecules for studying protein-DNA interaction mechanisms.

A Comprehensive In Silico Perspective For Discovery of Novel Inhibitory Candidates Targeting Versatile Transcriptional Repressor MBD2

Genome methylation is a key epigenetic mechanism in various biological events such as development, cellular differentiation, cancer progression, aging, and iPSC reprogramming. Crosstalk between DNA methylation and regulation in gene expression is employed through MBD2, known as reader of DNA methylation and suggested as a drug target. Despite its magnitude of significance and rationale of nomination, a scarcely limited number of druggable ligands has been detected so far. Hence, we screened a comprehensive compound library, and then certain of them were subjected to computational docking analysis by targeting the methylated DNA-binding domain of human MBD2. We could detect reasonable binding energies and docking residues presumably located in druggable pockets. Docking results were also validated via MD simulation and per-residue energy decomposition calculation. Drug-likeness of tested ligands was assessed through ADMET prediction in order to foresee off-target side effects for future studies. Herein, on the basis of collaborating approaches such as molecular docking, MD simulation, energy decomposition, and ADMET prediction, notably two compounds named CID3100583 and 8,8-Ethylenebistheophylline, have become prominent as novel candidates, possibly disrupting MBD2MBD–DNA interaction. Hereby, these compounds exhibit a promising usage potential in a wide range of implementations from cancer treatment to somatic cell reprogramming protocols.

DNA-Based Electrodes and Computational Approaches on the Intercalation Study of Antitumoral Drugs

The binding between anticancer drugs and double-stranded DNA (dsDNA) is a key issue to understand their mechanism of action, and many chemical methods have been explored on this task. Molecular docking techniques successfully predict the affinity of small molecules into the DNA binding sites. In turn, various DNA-targeted drugs are electroactive; in this regard, their electrochemical behavior may change according to the nature and strength of interaction with DNA. A carbon paste electrode (CPE) modified with calf thymus ds-DNA (CPDE) and computational methods were used to evaluate the drug–DNA intercalation of doxorubicin (DOX), daunorubicin (DAU), idarubicin (IDA), dacarbazine (DAR), mitoxantrone (MIT), and methotrexate (MTX), aiming to evaluate eventual correlations. CPE and CPDE were immersed in pH 7 0.1 mM solutions of each drug with different incubation times. As expected, the CPDE response for all DNA-targeted drugs was higher than that of CPE, evidencing the drug–DNA interaction. A peak current increase of up to 10-fold was observed; the lowest increase was seen for MTX, and the highest increase for MIT. Although this increase in the sensitivity is certainly tied to preconcentration effects of DNA, the data did not agree entirely with docking studies, evidencing the participation of other factors, such as viscosity, interfacial electrostatic interactions, and coefficient of diffusion.

DiMeLo-seq: Directed Methylation with Long-read sequencing v2

Directed Methylation and Long-read sequencing (DiMeLo-seq) is a powerful method to map protein-DNA interactions at a single-molecule level across the genome (including repetitive regions). It can be multiplexed to analyze multiple base modifications at once (e.g. endogenous CpG methylation and directed pA-Hia5 adenine methylation). Additionally, PCR amplification is not necessary for this protocol, which means that sequencing readout is proportional to protein-DNA interaction frequency. Finally, DiMeLo-seq can be used to map multiple protein interactions across a long single molecule.