Orientation-Guided Immobilization of Probe DNA on swCNT-FET for Enhancing Sensitivity of EcoRV Detection.

We present a novel approach that integrates electrical measurements with molecular dynamics (MD) simulations to assess the activity of type-II restriction endonucleases, specifically EcoRV. Our approach employs a single-walled carbon nanotube field-effect transistor (swCNT-FET) functionalized with the EcoRV substrate DNA, enabling the detection of enzymatic cleavage events. Notably, we leveraged the methylene blue (MB) tag as an "orientation guide" to immobilize the EcoRV substrate DNA in a specific direction, thereby enhancing the proximity of the DNA cleavage reaction to the swCNT surface and consequently improving the sensitivity in EcoRV detection. We conducted computational modeling to compare the conformations and electrostatic potential (ESP) of MB-tagged DNA with its MB-free counterpart, providing strong support for our electrical measurements. Both conformational and ESP simulations exhibited robust agreement with our experimental data. The inhibitory efficacy of the EcoRV inhibitor aurintricarboxylic acid (ATA) was also evaluated, and the selectivity of the sensing device was examined.

Relationship between CDX2 rs11568820 and EcoRV rs4516035 polymorphisms on the vitamin D receptor gene with susceptibility to different SARS-CoV-2 variants.

Several studies have revealed that vitamin D deficiency is linked to an increased risk of developing coronavirus disease 19 (COVID-19). In individuals with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, vitamin D receptor activation is required to decrease acute respiratory distress syndrome. The purpose of this study was to examine the genotypic distribution and allelic frequencies of CDX2 rs11568820 and EcoRV rs4516035 polymorphisms in COVID-19 patients with various SARS-CoV-2 variants. For genotyping of CDX2 rs11568820 and EcoRV rs4516035 polymorphisms, we used the polymerase chain reaction-restriction fragment length polymorphism technique in 1734 and 1450 recovered and deceased patients, respectively. The results indicated the rate of COVID-19 mortality was associated with CDX2 rs11568820 AA and GA genotypes in the Delta variant and with CDX2 rs11568820 AA in the Omicron BA.5 variant, while no association was shown in the Alpha variant. Therefore, the rate of COVID-19 mortality was associated with EcoRV rs4516035 TC and CC genotypes in the Delta variant, while no association was shown in the Alpha and Omicron BA.5 variants. According to our analysis, the T-G haplotype was more common in all SARS-CoV-2 variants. The C-A haplotype was associated with COVID-19 mortality in the Delta and Omicron BA.5 variants, and the T-A haplotype was related to the Alpha variant. In conclusion, the genotype frequencies of the CDX2 rs11568820 and EcoRV rs4516035 polymorphisms between SARS-CoV-2 variants were significantly different between the deceased patients and recovered patients. However, more studies should be done to confirm the results.

Quantum biology.π-πentanglement signatures in protein-DNA interactions.

The biological functions of DNA are carried out by individual proteins that interact with specific sequences along the DNA in order to prime the molecular processes required by the cellular metabolism. Protein-DNA interactions include DNA replication, gene expression and its regulation, DNA repair, DNA restriction and modification by endonucleases, generally classified as enzymatic functions, or transcription factors functions. To find specific binding target sequences and achieve their aims, in less than one second proteins operate in symbiosis with a crowded cellular environment, identifying extremely small cognate sequences along the DNA chain, which range from 15-20 bps for repressors to 4-6 bps for restriction enzymes. In a previous work, we proposed that the extraordinary ability of proteins to identify consensus sequences on DNA in a short time appears to be dependent on specific quantum signatures such as the entanglement ofπ-πelectrons between DNA nucleotides and protein amino acids, where the couple ofπelectrons function as a radical pair, oneπelectron is located on a specific site of sequence to be identified and the other one performs a quantum walk to identify possible sites of consensus sequence. In this paper, we use the restriction endonucleases enzymes, EcoRV and EcoRI as a case study. These enzymes are able to recognize 3'-GATACT-5' or 3'-GAATCT-5' sequences, respectively. We exploit the analogy of a coin operator with a Bloch sphere to demonstrate that the entanglement betweenπ-πelectrons generated at the contacts on specific GA dimers between proteins and DNA relies on the spin of the electrons that form an initial singlet state. The latter is a maximally entangled state so that the identification of specific nucleotides is associated with the formation of singlet states. On the other hand, during the identification of subsequent GA dimers, the spin-orbit interaction on walkingπelectron induces triplet transitions so that singlet-triplet transitions should manifest an experimentally measurable effect. We propose that the possible experimental evidence of entanglement betweenπ-πelectrons may be due to the phosphorescence signal correspondence to triplet decay processes.

Microscopic insight to specificity of metal ion cofactor in DNA cleavage by restriction endonuclease EcoRV.

Restriction endonucleases protect bacterial cells against bacteriophage infection by cleaving the incoming foreign DNA into fragments. In presence of Mg2+ ions, EcoRV is able to cleave the DNA but not in presence of Ca2+ , although the protein binds to DNA in presence of both metal ions. We make an attempt to understand this difference using conformational thermodynamics. We calculate the changes in conformational free energy and entropy of conformational degrees of freedom, like DNA base pair steps and dihedral angles of protein residues in Mg2+ (A)-EcoRV-DNA complex compared to Ca2+ (S)-EcoRV-DNA complex using all-atom molecular dynamics (MD) trajectories of the complexes. We find that despite conformational stability and order in both complexes, the individual degrees of freedom behave differently in the presence of two different metal ions. The base pairs in cleavage region are highly disordered in Ca2+ (S)-EcoRV-DNA compared to Mg2+ (A)-EcoRV-DNA. One of the acidic residues ASP90, coordinating to the metal ion in the vicinity of the cleavage site, is conformationally destabilized and disordered, while basic residue LYS92 gets conformational stability and order in Ca2+ (S) bound complex than in Mg2+ (A) bound complex. The enhanced fluctuations hinder placement of the metal ion in the vicinity of the scissile phosphate of DNA. Similar loss of conformational stability and order in the cleavage region is observed by the replacement of the metal ion. Considering the placement of the metal ion near scissile phosphate as requirement for cleavage action, our results suggest that the changes in conformational stability and order of the base pair steps and the protein residues lead to cofactor sensitivity of the enzyme. Our method based on fluctuations of microscopic conformational variables can be applied to understand enzyme activities in other protein-DNA systems.

Electrochemical DNA Cleavage Sensing for EcoRV Activity and Inhibition with an ERGO Electrode.

An electrochemically reduced graphene oxide (ERGO) electrode-based electrochemical assay was developed for rapid, sensitive, and straightforward analysis of both activity and inhibition of the endonuclease EcoRV. The procedure uses a DNA substrate designed for EcoRV, featuring a double-stranded DNA (dsDNA) region labeled with methylene blue (MB) and a single-stranded DNA (ssDNA) region immobilized on the ERGO surface. The ERGO electrode, immobilized with the DNA substrate, was subsequently exposed to a sample containing EcoRV. Upon enzymatic hydrolysis, the cleaved dsDNA fragments were detached from the ERGO surface, leading to a decrease in the MB concentration near the electrode. This diminished the electron transfer efficiency for MB reduction, resulting in a decreased reduction current. This assay demonstrates excellent specificity and high sensitivity, with a limit of detection (LOD) of 9.5 × 10-3 U mL-1. Importantly, it can also measure EcoRV activity in the presence of aurintricarboxylic acid, a known inhibitor, highlighting its potential for drug discovery and clinical diagnostic applications.

Roles of Hostility and Depression in the Association between the MAOA Gene Polymorphism and Internet Gaming Disorder.

The metabolism of bioamine in the central nervous system contributes to the development of addiction. We examined the roles of hostility and depression in the association between internet gaming disorder (IGD) and monoamine oxidase-A (MAOA) EcoRV polymorphism (rs1137070). A total of 69 adults with IGD and 138 without IGD were recruited through diagnostic interviewing. We evaluated participants for rs1137070, depression, and hostility. The participants with the TT genotype of rs1137070 had a higher odds ratio of 2.52 (1.37-4.64) for IGD compared with the C carriers. Expressive hostility behavior and hostility cognition mediated the association between rs1137070 and IGD. Indicating lower MAOA activity, the TT genotype predicted IGD and higher expressive hostility behavior and hostility cognition. Expressive hostility behavior and hostility cognition may underline the association between rs1137070 and IGD. Assessment of and intervention for hostility behavior and cognition should be provided to attenuate the risk of IGD, particularly in those with the TT genotype. Further brain imaging or neurobiological studies are required to elucidate the possible mechanism underlying the association between MAOA activity and IGD.

Protein-DNA target search relies on quantum walk.

Protein-DNA interactions play a fundamental role in all life systems. A critical issue of such interactions is given by the strategy of protein search for specific targets on DNA. The mechanisms by which the protein are able to find relatively small cognate sequences, typically 15-20 base pairs (bps) for repressors, and 4-6 bps for restriction enzymes among the millions of bp of non-specific chromosomal DNA have hardly engaged researchers for decades. Recent experimental studies have generated new insights on the basic processes of protein-DNA interactions evidencing the underlying complex dynamic phenomena involved, which combine three-dimensional and one-dimensional motion along the DNA chain. It has been demonstrated that protein molecules have an extraordinary ability to find the target very quickly on the DNA chain, in some cases, with two orders of magnitude faster than the diffusion limit. This unique property of protein-DNA search mechanism is known as facilitated diffusion. Several theoretical mechanisms have been suggested to describe the origin of facilitated diffusion. However, none of such models currently has the ability to fully describe the protein search strategy. In this paper, we suggest that the ability of proteins to identify consensus sequences on DNA is based on the entanglement of π-π electrons between DNA nucleotides and protein amino acids. The π-π entanglement is based on Quantum Walk (QW), through Coin-position entanglement (CPE). First, the protein identifies a dimer belonging to the consensus sequence, and localize a π on such dimer, hence, the other π electron scans the DNA chain until the sequence is identified. Focusing on the example of recognition of consensus sequences of EcoRV or EcoRI, we will describe the quantum features of QW on protein-DNA complexes during the search strategy, such as walker quadratic spreading on a coherent superposition of different vertices and environment-supported long-time survival probability of the walker. We will employ both discrete- or continuous-time versions of QW. Biased and unbiased classical Random Walk (CRW) have been used for a long time to describe the Protein-DNA search strategy. QW, the quantum version of CRW, has been widely studied for its applications in quantum information applications. In our biological application, the walker (the protein) resides at a vertex in a graph (the DNA structural topology). Differently to CRW, where the walker moves randomly, the quantum walker can hop along the edges in the graph to reach other vertices entering coherently a superposition across different vertices spreading quadratically faster than CRW analogous evidencing the typical speed up features of the QW. When applied to a protein-DNA target search problem, QW gives the possibility to achieve the experimental diffusional motion of proteins over diffusion classical limits experienced along DNA chains exploiting quantum features such as CPE and long-time survival probability supported by the environment. In turn, we come to the conclusion that, under quantum picture, the protein search strategy does not distinguish between one-dimensional (1D) and three-dimensional (3D) cases.