An efficient electrochemical biosensor has been developed for the simultaneous evaluation of DNA bases using AgNPs-embedded covalent organic framework (COF). The COF (p-Phenylenediamine and terephthalaldehyde) was synthesized by reflux (DMF; 150 °C; 12 h) and the nanoparticles were embedded from the aqueous solutions of AgNO3 and NaBH4. The nanocomposite-modified COF was confirmed by spectral, microscopic, and electrochemical techniques. The nanocomposite material was deposited on a glassy carbon electrode (GCE) and the redox behavior of AgNPs was confirmed by cyclic voltammetry. The electrocatalytic activities of DNA bases were analyzed by differential pulse voltammetry (DPV) in a physiological environment (PBS; pH = 7.0) based on simple and easy-to-use electrocatalyst. The AgNPs-COF/GCE showed well-defined anodic peak currents for the bases guanine (+ 0.63 V vs. Ag/AgCl), adenine (+ 0.89 V vs. Ag/AgCl), thymine (+ 1.10 V vs. Ag/AgCl), and cytosine (+ 1.26 V vs. Ag/AgCl) in a mixture as well as individuals with respect to the conventional, COF, and AgNPs/GCEs. The AgNPs-COF/GCE showed linear concentration range of DNA bases from 0.2-1000 µM (guanine; (G)), 0.1-500 µM (adenine (A)), 0.25-250 µM (thymine (T)) and 0.15-500 µM (cytosine (C)) and LOD of 0.043, 0.056, 0.062, and 0.051 µM (S/N = 3), respectively. The developed sensor showed reasonable selectivity, reproducibility (RSD = 1.53 ± 0.04%-2.58 ± 0.02% (n = 3)), and stability (RSD = 1.22 ± 0.06%-2.15 ± 0.04%; n = 3) over 5 days of storage) for DNA bases. Finally, AgNPs-COF/GCE was used for the determination of DNA bases in human blood serum, urine and saliva samples with good recoveries (98.60-99.11%, 97.80-99.21%, and 98.69-99.74%, respectively).
DNA methylation in a CpG context can be recognised by methyl-CpG binding protein 2 (MeCP2) via its methyl-CpG binding domain (MBD). An A/T run next to a methyl-CpG maximises the binding of MeCP2 to the methylated DNA. The A/T run characteristics are reported here with an X-ray structure of MBD A140V in complex with methylated DNA. The A/T run geometry was found to be strongly stabilised by a string of conserved water molecules regardless of its flanking nucleotide sequences, DNA methylation and bound MBD. New water molecules were found to stabilise the Rett syndrome-related E137, whose carboxylate group is salt bridged to R133. A structural comparison showed no difference between the wild type and MBD A140V. However, differential scanning calorimetry showed that the melting temperature of A140V constructs in complex with methylated DNA was reduced by ~7 °C, although circular dichroism showed no changes in the secondary structure content for A140V. A band shift analysis demonstrated that the larger fragment of MeCP2 (A140V) containing the transcriptional repression domain (TRD) destabilises the DNA binding. These results suggest that the solution structure of MBD A140V may differ from the wild-type MBD although no changes in the biochemical properties of X-ray A140V were observed.