Detection of G-Quadruplex DNA Structures in Macrophages.

In addition to the canonical B-DNA conformation, DNA can fold into different secondary structures. Among them are G-quadruplex structures (G4s). G4 structures are very stable and can fold in specific guanine-rich regions in DNA and RNA. Different in silico, in vitro, and in cellulo experiments have shown that G4 structures form so far in all tested organisms. There are over 700,000 predicted G4s in higher eukaryotes, but it is so far assumed that not all will form at the same time. Their formation is dynamically regulated by proteins and is cell type-specific and even changes during the cell cycle or during different exogenous or endogenous stimuli (e.g., infection or developmental stages) can alter the G4 level. G4s have been shown to accumulate in cancer cells where they contribute to gene expression changes and the mutagenic burden of the tumor. Specific targeting of G4 structures to impact the expression of oncogenes is currently discussed as an anti-cancer treatment. In a tumor microenvironment, not only the tumor cells will be targeted by G4 stabilization but also immune cells such as macrophages. Although G4s were detected in multiple organisms and different cell types, only little is known about their role in immune cells. Here, we provide a detailed protocol to detect G4 formation in the nucleus of macrophages of vertebrates and invertebrates by microscopic imaging.

Abridged solid-phase extraction with alkaline Poly(ethylene) glycol lysis (ASAP) for direct DNA amplification.

Complexity of sample preparation decelerate the development of sample-in-answer-out devices for point-of-need nucleic acid amplification testing. Here, we present the consolidation of alkaline poly(ethylene) glycol-based lysis and solid-phase extraction for rapid and simple sample preparation compatible with direct on-bead amplification. Simultaneous cell lysis and binding of DNA were achieved using an optimised reagent comprising 15% PEG8000, 0.5 M NaCl, and 3.5 mM KOH. This was combined with direct, on-bead amplification using 1.5 µg beads per 20 µL PCR reaction mix. The novel single reagent, 5-min method improved the detection limit by 10 and 100-fold compared with commercial DNA extraction kits and the original alkaline PEG lysis method, respectively. The sensitivity can be further enhanced by one amplification cycle with an ethanol wash or by extending the incubation to 10 min before collecting the magnetic particles. Both methods successfully detected a single copy of Escherichia coli DNA. In biological fluids (saliva, sweat, and urine), the 5-min method was delayed by about one cycle compared to the 15-min method. The proposed methods are attractive for incorporation in the workflow for point-of-need testing of biological samples by providing a practical and chemical method for simple alternative DNA sample preparation.

Target-triggered stochastic DNAzyme motors on spherical nucleic acids for simultaneous fluorescence assay of double miRNAs.

Simultaneous quantifications of multiple miRNAs in the single-sampling system would be conducive to the accurate diagnosis of diseases in contrast with single miRNA analysis. In this work, a stochastic DNAzyme motor on spherical nucleic acids (SNAs) for simultaneous fluorescence assay of double miRNAs was established. Hairpin 1 (H1)-FAM-7a and H1-TAMRA-133a-functionalized magnetic beads (MBs) as SNAs were mixed. Targets (let-7a and miRNA-133a) reacted with two different S1 and S2, triggering the formation of two types of metal DNAzymes. The DNAzymes can further react with H1 stem-loop DNA on SNAs to release the two fluorescent DNA-FAM and DNA-TAMRA fragments in the presence of Mg2+. Meanwhile, the DNAzyme as DNA motors were separated from the previous H1 probe to participate the next cycling operations, resulting in the signal amplification toward the simultaneous and sensitive detection of let-7a and miRNA-133a. SNAs with three dimensional nanostructures provided enough space for the operation of DNAzyme walker, promoting the sensitivity of this proposed analytical system. The two mixed SNAs enable one-step and specific quantification of miRNA let-7a and miRNA-133a with lower detection limits of 90.5 fM and 74.9 fM, respectively. Finally, this proposed strategy was employed to simultaneously detect double miRNAs in practical applicability.

Signal-on electrogenerated chemiluminescence detection of gonyautoxin 1/4 based on proximity ligation-induced an electrode-bound pseudoknot DNA.

A "signal on" electrogenerated chemiluminescence (electrochemiluminescence, ECL) aptasensor based on proximity ligation-induced an electrode-bound pseudoknot DNA for sensitive detection of gonyautoxin 1/4 (GTX1/4) was developed on basis of the competitive type reaction mode. Aptamer was adopted as recognition element. Ru(bpy)32+ as ECL signal, was attached on the glassy carbon electrode (GCE) surface modified with nafion and gold nanoparticles (AuNPs) by electrostatic attraction to obtain the ECL platform. The pseudoknot DNA as capture probe, was immobilized onto the ECL platform via Au-S bond to obtain the ECL aptasensor. In the absence of GTX1/4, Y-shape proximate cooperative complex among aptamer, pseudoknot DNA and DNA1 was formed, drawing the ferrocene groups Fc, as ECL quencher) of both pseudoknot DNA and DNA1 near the electrode surface and resulting in low ECL signal. In the presence of GTX1/4, GTX1/4 competed with pseudoknot DNA and DNA1 for aptamer in homogeneous solution, preventing the formation of proximate cooperative complex and keeping the capture DNA in the pseudoknot conformation with Fc groups far away from the electrode surface, generating a high ECL signal. The recovery of ECL intensity increased with the GTX1/4 concentration and allowed the detection of GTX1/4 in the range of 0.01 ng/mL to 10 ng/mL with a detection of limit as low as 6.56 pg/mL. Additionally, the accuracy of this method was validated for analysis of spiked sea water samples with good recoveries, which indicates great potential in commercial application.

DNA induced CTAB-caped gold bipyramidal nanoparticles self-assembly using for Raman detection of DNA molecules.

DNA is an indispensable part of metabolism, which affects many important processes in the body, including gene expression, protein synthesis, and drug delivery. Surface-enhanced Raman spectroscopy (SERS) is one of the most important methods used to study the structure and function of DNA and can obtain rich DNA molecular fingerprints. However, it is still a great challenge to use SERS to directly analyze the characteristic Raman signals of the DNA molecule and achieve rapid and simple detection. Hence, a detection platform based on gold bipyramidal nanoparticles (AuNBs) self-assembly that can be directly used for the detection of DNA molecules without the need for additional aggregators and cleaning agents was designed in this study. The original hexadecyltrimethylammonium bromide (CTAB) of AuNBs can be used as the internal standard for DNA quantification without an additional standard. This is the first time that the Raman signals of the analyte molecule can be obtained directly without labels by using the interaction between the molecule and the enhanced substrate. We used this method to capture the original DNA molecules in methylated DNA, serum, and cell metabolites and obtained spectral data processing results using linear discriminant analysis (LDA). This provides new ideas for the digitization of disease treatment and the study of the metabolic processes of life.

An electrochemical biosensor for detection of T4 polynucleotide kinase activity based on host-guest recognition between phosphate pillar[5]arene and methylene blue.

T4 polynucleotide kinase (T4 PNK) is an important DNA repair-related enzyme that plays a crucial role in DNA recombination, replication and damage repair. Herein, an electrochemical biosensor was developed for detection of T4 PNK activity and inhibitor screening based on supramolecular host-guest recognition between phosphate pillar (Dumitrache and McKinnon, 2017) [5] arene (PP5) and methylene blue (MB). The water-soluble PP5 employed as the host for complexation of MB guest molecules. The substrate DNA with 5'-hydroxyl group was first self-assembled on the gold electrode surface through the chemical adsorption of the thiol group, which was phosphorylated in the presence of T4 PNK and adenosine triphosphate (ATP). TiO2 served as a bridge to link phosphorylated DNA and PP5 via the robust phosphate-Ti4+-phosphate chemistry. The immobilized PP5 captured the MB on electrode surface via the supramolecular host-guest recognition interaction, resulting in an enhanced electrochemical response signal. The electrochemical signal is proportional to the T4 PNK concentration in the range of 2 × 10-4 to 5 U mL-1 with a detection limit of 1 × 10-4 U mL-1. It was also successfully used for PNK inhibitor screening and PNK activity assay in HeLa cell lysates sample. The proposed strategy provides a novel sensing platform for kinase activity assay and inhibitor screening, holding a great potential in clinical diagnostics, inhibitor screening, and nucleotide kinase-target drug discovery.

Large-scale assembly of geometrically diverse metal nanoparticles-based 3D plasmonic DNA nanostructures for SERS detection of PNK in cancer cells.

The organization of geometrically diverse metal nanoparticles into a core/satellite structure at a large scale is a promising strategy for improve SERS performance due to hot spots localized enrichment and signal increase. However, due to the lack of extensional frames and strong electrostatic repulsion between plasma NPs, the fabrication of such 3D architectures with a high-density periodic hotspot in the focus volume has proven exceedingly difficult. Herein, we demonstrate a facile large-scale assembly of geometrically diverse metal nanoparticles strategy for constructing spatially extended 3D plasmonic nanostructures resembling "signal towers" based on RCA-mediated periodic organization of gold nanospheres (GNS) surrounding gold nanorods (GNRs). Using cancer cell T4 PNK as a model, a padlock probe with 5'- hydroxyl (P-circle) was designed as the T4 PNK substrate. The center Au nanorod was coated with P1 and served as a "pedestal" to allow substantial loading of P-circle after target phosphorylation to initiate the rolling ring amplification reaction (RCA). The resultant DNA nanowire serves as an "antenna" to successively lock numerous Raman reporter P2 (Cy3-P2-SH) through base pairing at regular intervals. Finally, the 3D plasma DNA nanostructures that resemble "signal towers" could be obtained by placing a large number of GNS with a strong affinity for Au-S. The proposed 3D SERS sensor exhibited a sensitivity of LOD as low as 0.274 mU/mL, which was attributed to a substantial electromagnetic field enhancement at the inter-nanoparticle gaps between the adjacent pedestal and antenna. Moreover, by exploiting the synergistic effect of the periodically extended DNA scaffold generated by RCA amplification and the co-assembly of thiol ligand, the loaded GNS can be extended to three-dimensional space, forming a high-density periodic hotspot in the focal volume, thereby ensuring high enhancement and high reproducibility of Raman signals. In addition, this method can be used to quantify T4 PNK in HeLa cells, demonstrating its applicability in diagnosing and estimating PNK-related diseases in complex fluids.

A triple-aptamer tetrahedral DNA nanostructures based carbon-nanotube-array transistor biosensor for rapid virus detection.

Outbreaks of infectious viruses cause enormous challenges to global public health. Recently, the coronavirus disease 2019 (COVID-19) induced by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has severely threatened human health and resulted in the global pandemic. A strategy to detect SARS-CoV-2 with both fast sensing speed and high accuracy is urgently required. Here, rapid detection of SARS-CoV-2 antigen using carbon-nanotube-array-based thin-film transistor (CNT-array-based TFT) biosensors merged with tetrahedral DNA nanostructures (TDNs) and triple aptamers is demonstrated for the first time. Compared with CNT-network-based TFT biosensors and metal-electrode-based CNT-TFT biosensors, the response of CNT-array-based TFT biosensors can be enhanced up to 102% for SARS-CoV-2 receptor-binding domain (RBD) detection, which is supported by its sensing mechanism. By combining TDNs with triple aptamers, the biosensor has realized the wildtype SARS-CoV-2 RBD detection in a broad detection range spanning eight orders of magnitude with a low limit of detection (LOD) of 10 aM (6 copies/µL) owing to the improved protein capture efficiency. Moreover, the triple-aptamer biosensor platform has achieved the detection of SARS-CoV-2 Omicron RBD in a low LOD of 6 aM (3.6 copies/µL). Additionally, the CNT-array-based TFT biosensors have exhibited excellent specificity, enabling identification among SARS-CoV-2 antigen, SARS-CoV antigen and MERS-CoV antigen. The platform of CNT-array-based TFT biosensors combined with TDNs and triple aptamers provides a high-performance and rapid approach for SARS-CoV-2 detection, and its versatility by altering specific aptamers enables the possibility for rapid virus detection.

Tandem CRISPR nucleases-based lateral flow assay for amplification-free miRNA detection via the designed "locked RNA/DNA" as fuels.

Currently, available biosensors based on CRISPR/Cas typically depend on coupling with nucleic acid amplification technologies to enhance their sensitivity. However, this approach often involves intricate amplification processes, which could be time-consuming and susceptible to contamination. In addition, signal readouts often require sophisticated and cumbersome equipment, obstructing the applicability of CRISPR/Cas assays in resource-limited regions. Herein, a tandem CRISPR/Cas13a/Cas12a mechanism (tanCRISPR) has been developed via the designed "Locked RNA/DNA" probe as fuels for the trans-cleavage nucleic acid of Cas13a and activated nucleic acid of Cas12a. Meanwhile, a lateral flow assay (LFA) is designed to combine with this tandem CRISPR/Cas13a/Cas12a mechanism (termed tanCRISPR-LFA), realizing the portable monitoring of miRNA-21. The tanCRISPR could realize the limit of detection at pM levels (266 folds lower than Cas13a-based miRNA testing alone) without the resort to target amplification procedures. Furthermore, the miRNA-21 levels of MDA-MB-231 cell extracts are sensed by tanCRISPR-LFA, which is comparable to qRT-PCR. With the virtues of portability, rapidity, sensitivity, and low cost, tanCRISPR-LFA is amenable for CRISPR/Cas-based biosensing and potential applications in the clinical diagnosis of miRNA-associated diseases.

Development of the viscosity biosensor for the detection of DNase I based on the flow distance on the paper with DNA mucus.

Deoxyribonuclease I (DNase I) is a biomarker which has important applications in various biological processes. Thus, it is highly important to develop a user-friendly method for the detection of DNase I. Here, we present a paper-based distance sensor for the rapid detection of DNase I based on changes in the viscosity of DNA mucus. The viscosity of DNA mucus varies with different concentrations of DNase I, showing different water flow lengths on the pH test papers, this makes the quantification of DNase I possible. This method has a wide linear range (0.01-10 U/mL), excellent sensitivity, remarkable specificity and excellent reproducibility. The detection limit reaches 0.003 U/mL. Additionally, it can be well applied to detection of DNase I inhibitors, assay of DNase I in human serum and quality evaluation of nucleic acid scavengers. In general, this study offers a brief, convenient, label-free, and economical method to construct paper-based distance sensors using DNA mucus, which is very promising in the detection of DNase I in various applications.

A highly sensitive and robust electrochemical biosensor for microRNA detection based on PNA-DNA hetero-three-way junction formation and target-recycling catalytic hairpin assembly amplification.

Robust and sensitive methods for the detection of microRNAs (miRNAs) are crucial in the clinical diagnosis of cancers. In this study, a novel electrochemical biosensor with high sensitivity for miRNA-21 detection is developed, which relies on the formation of a peptide nucleic acid (PNA)-DNA hetero-three-way junction (H3WJ) and target-recycling catalytic hairpin assembly (CHA) amplification. The electroneutral PNA probes are initially immobilized onto a gold electrode to construct the sensor. Upon introduction of miRNA-21, target-recycling CHA is initiated, resulting in abundant double-stranded CHA products. Subsequently, association between the PNA probes and these products leads to the formation of PNA-DNA H3WJs. Consequently, the electrode surface is densely populated with numerous electroactive Ferrocene (Fc) groups, resulting in a significantly amplified current response for highly sensitive detection of miRNA-21 at concentrations as low as 0.15 fM. This approach demonstrates remarkable specificity towards target miRNAs and can be utilized for quantitative monitoring of miRNA-21 expression in human cancer cells. More importantly, the sensor exhibits exceptional stability and shows a significant reduction in background noise during miRNA detection, making this method a highly promising sensing platform for monitoring various miRNA biomarkers to facilitate the diagnosis of diverse cancers.

Enzyme-assisted amplification of target cycle triggers the unlocking of locked hairpin probes for let-7a detection.

The detection of miRNA in cells is difficult owing to its substantially low cellular content. Therefore, developing a highly sensitive sensor to detect cellular miRNA remains a significant challenge. Herein, we report an enzyme-assisted biosensor with target cycle amplification that can trigger the unlocking of locked hairpin probes for sensitive and robust let-7a gene detection. In the research, three kinds of hairpin probes were skillfully designed. The hairpin probe comprises a complementary sequence of a target, primer, and recognition site of Nt. BbvCI restriction endonucleases. In addition, the alternating synergistic impact of polymerase and the nicking enzyme generates considerable triggers to unlock the locked hairpin probe LH1, consequently triggering a subsequent circulating strand displacement reaction to form a stable H1-H2 double strand to ensure sufficient distance between a fluorophore on H1 and a quenching group on bolt DNA (bDNA), and resulting in the recovery of fluorescence. Furthermore, this process does not require complicated operation procedures and instruments, and the target gene let-7a can be sensitively detected. Specifically, the detection limit of the biosensor is as low as 160 fM, and its linear range is 0.5 pM-250 nM. Moreover, this biosensor can be employed to detect let-7a in human serum with good selectivity.

opp-Dibenzoporphyrin Pyridinium Derivatives as Potential G-Quadruplex DNA Ligands.

Since the occurrence of tumours is closely associated with the telomerase function and oncogene expression, the structure of such enzymes and genes are being recognized as targets for new anticancer drugs. The efficacy of several ligands in telomerase inhibition and in the regulation of genes expression, by an effective stabilisation of G-quadruplexes (G4) DNA structures, is being considered as a promising strategy in cancer therapies. When evaluating the potential of a ligand for telomerase inhibition, the selectivity towards quadruplex versus duplex DNA is a fundamental attribute due to the large amount of double-stranded DNA in the cellular nucleus. This study reports the evaluated efficacy of three tetracationic opp-dibenzoporphyrins, a free base, and the corresponding zinc(II) and nickel(II) complexes, to stabilise G4 structures, namely the telomeric DNA sequence (AG3(T2AG3)3). In order to evaluate the selectivity of these ligands towards G4 structures, their interaction towards DNA calf thymus, as a double-strand DNA sequence, were also studied. The data obtained by using different spectroscopic techniques, such as ultraviolet-visible, fluorescence, and circular dichroism, suggested good affinity of the free-base porphyrin and of its zinc(II) complex for the considered DNA structures, both showing a pattern of selectivity for the telomeric G4 structure. A pattern of aggregation in aqueous solution was detected for both Zn(II) and Ni(II) metallo dibenzoporphyrins and the ability of DNA sequences to induce ligand disaggregation was observed.

Microfluidic delivery of cutting enzymes for fragmentation of surface-adsorbed DNA molecules.

We describe a method for fragmenting, in-situ, surface-adsorbed and immobilized DNAs on polymethylmethacrylate(PMMA)-coated silicon substrates using microfluidic delivery of the cutting enzyme DNase I. Soft lithography is used to produce silicone elastomer (Sylgard 184) gratings which form microfluidic channels for delivery of the enzyme. Bovine serum albumin (BSA) is used to reduce DNase I adsorption to the walls of the microchannels and enable diffusion of the cutting enzyme to a distance of 10mm. Due to the DNAs being immobilized, the fragment order is maintained on the surface. Possible methods of preserving the order for application to sequencing are discussed.

DNA barcoding of marine teleost fishes (Teleostei) in Cebu, the Philippines, a biodiversity hotspot of the coral triangle.

A morphology-based barcoding library of market teleost fishes (Teleostei) in Cebu is built based on cytochrome c oxidase subunit I (COI) sequences and voucher specimens which aimed to establish a reliable reference of frequently traded fishes in the province, a biodiversity hotspot at the center of the Philippine archipelago. A total of 1721 specimens were collected from 18 fish markets and landing sites around the province, in which 538 specimens were sequenced belonging to 393 species from 229 genera, 86 families, and 37 orders. Most speciose families are coral reef or reef-related shallow-water species. Twelve species from 11 families are newly recorded in the Philippine waters, among which 7 species are deep-sea inhabitants, while 3 species have expanded their distribution range. Only 20 taxa could not be identified to the species level due to the difficulty in morphological examinations, absence of matched reference sequences in online databases, and/or problematic species awaiting further studies. This first comprehensive DNA barcoding survey of Cebu fishes can facilitate further taxonomic research as well as the conservation and management of fisheries in the Philippines.

Spectral analysis of DNA superhelical dynamics from molecular minicircle simulations.

Torsional and bending deformations of DNA molecules often occur in vivo and are important for biological functions. DNA "under stress" is a conformational state, which is by far the most frequent state during DNA-protein and gene regulation. In DNA minicircles of length <100 base pairs (bp), the combined effect of torsional and bending stresses can cause local unusual conformations, with certain base pair steps often absorbing most of the stress, leaving other steps close to their relaxed conformation. To better understand the superhelical dynamics of DNA under stress, molecular simulations of 94 bp minicircles with different torsional linking numbers were interpreted using Fourier analyses and principal component analyses. Sharp localized bends of nearly 90° in the helical axis were observed, which in turn decreased fluctuations of the rotational register and helped redistribute the torsional stress into writhe, i.e., superhelical turn up to 360°. In these kinked minicircles, only two-thirds of the DNA molecule bends and writhes and the remaining segment stays close to straight and preserves a conformational flexibility typical of canonical B-DNA (bending of 39° ± 17° distributed parsimoniously across 36 bp), which was confirmed and visualized by principal component analysis. These results confirm that stressed DNA molecules are highly heterogeneous along their sequence, with segments designed to locally store and release stress so that nearby segments can stay relaxed.

Comparative genomics and DNA methylation analysis of Pseudomonas aeruginosa clinical isolate PA3 by single-molecule real-time sequencing reveals new targets for antimicrobials.

Introduction: Pseudomonas aeruginosa (P.aeruginosa) is an important opportunistic pathogen with broad environmental adaptability and complex drug resistance. Single-molecule real-time (SMRT) sequencing technique has longer read-length sequences, more accuracy, and the ability to identify epigenetic DNA alterations. Methods: This study applied SMRT technology to sequence a clinical strain P. aeruginosa PA3 to obtain its genome sequence and methylation modification information. Genomic, comparative, pan-genomic, and epigenetic analyses of PA3 were conducted. Results: General genome annotations of PA3 were discovered, as well as information about virulence factors, regulatory proteins (RPs), secreted proteins, type II toxin-antitoxin (TA) pairs, and genomic islands. A genome-wide comparison revealed that PA3 was comparable to other P. aeruginosa strains in terms of identity, but varied in areas of horizontal gene transfer (HGT). Phylogenetic analysis showed that PA3 was closely related to P. aeruginosa 60503 and P. aeruginosa 8380. P. aeruginosa's pan-genome consists of a core genome of roughly 4,300 genes and an accessory genome of at least 5,500 genes. The results of the epigenetic analysis identified one main methylation sites, N6-methyladenosine (m6A) and 1 motif (CATNNNNNNNTCCT/AGGANNNNNNNATG). 16 meaningful methylated sites were picked. Among these, purH, phaZ, and lexA are of great significance playing an important role in the drug resistance and biological environment adaptability of PA3, and the targeting of these genes may benefit further antibacterial studies. Disucssion: This study provided a detailed visualization and DNA methylation information of the PA3 genome and set a foundation for subsequent research into the molecular mechanism of DNA methyltransferase-controlled P. aeruginosa pathogenicity.

DNA nanopores as artificial membrane channels for bioprotonics.

Biological membrane channels mediate information exchange between cells and facilitate molecular recognition. While tuning the shape and function of membrane channels for precision molecular sensing via de-novo routes is complex, an even more significant challenge is interfacing membrane channels with electronic devices for signal readout, which results in low efficiency of information transfer - one of the major barriers to the continued development of high-performance bioelectronic devices. To this end, we integrate membrane spanning DNA nanopores with bioprotonic contacts to create programmable, modular, and efficient artificial ion-channel interfaces. Here we show that cholesterol modified DNA nanopores spontaneously and with remarkable affinity span the lipid bilayer formed over the planar bio-protonic electrode surface and mediate proton transport across the bilayer. Using the ability to easily modify DNA nanostructures, we illustrate that this bioprotonic device can be programmed for electronic recognition of biomolecular signals such as presence of Streptavidin and the cardiac biomarker B-type natriuretic peptide, without modifying the biomolecules. We anticipate this robust interface will allow facile electronic measurement and quantification of biomolecules in a multiplexed manner.

The COMPASS subunit Spp1 protects nascent DNA at the Tus/Ter replication fork barrier by limiting DNA availability to nucleases.

Homologous recombination factors play a crucial role in protecting nascent DNA during DNA replication, but the role of chromatin in this process is largely unknown. Here, we used the bacterial Tus/Ter barrier known to induce a site-specific replication fork stalling in S. cerevisiae. We report that the Set1C subunit Spp1 is recruited behind the stalled replication fork independently of its interaction with Set1. Spp1 chromatin recruitment depends on the interaction of its PHD domain with H3K4me3 parental histones deposited behind the stalled fork. Its recruitment prevents the accumulation of ssDNA at the stalled fork by restricting the access of Exo1. We further show that deleting SPP1 increases the mutation rate upstream of the barrier favoring the accumulation of microdeletions. Finally, we report that Spp1 protects nascent DNA at the Tus/Ter stalled replication fork. We propose that Spp1 limits the remodeling of the fork, which ultimately limits nascent DNA availability to nucleases.

A quantum physics layer of epigenetics: a hypothesis deduced from charge transfer and chirality-induced spin selectivity of DNA.

BACKGROUND: Epigenetic mechanisms are informational cellular processes instructing normal and diseased phenotypes. They are associated with DNA but without altering the DNA sequence. Whereas chemical processes like DNA methylation or histone modifications are well-accepted epigenetic mechanisms, we herein propose the existence of an additional quantum physics layer of epigenetics. RESULTS: We base our hypothesis on theoretical and experimental studies showing quantum phenomena to be active in double-stranded DNA, even under ambient conditions. These phenomena include coherent charge transfer along overlapping pi-orbitals of DNA bases and chirality-induced spin selectivity. Charge transfer via quantum tunneling mediated by overlapping orbitals results in charge delocalization along several neighboring bases, which can even be extended by classical (non-quantum) electron hopping. Such charge transfer is interrupted by flipping base(s) out of the double-strand e.g., by DNA modifying enzymes. Charge delocalization can directly alter DNA recognition by proteins or indirectly by DNA structural changes e.g., kinking. Regarding sequence dependency, charge localization, shown to favor guanines, could influence or even direct epigenetic changes, e.g., modification of cytosines in CpG dinucleotides. Chirality-induced spin selectivity filters electrons for their spin along DNA and, thus, is not only an indicator for quantum coherence but can potentially affect DNA binding properties. CONCLUSIONS: Quantum effects in DNA are prone to triggering and manipulation by external means. By the hypothesis put forward here, we would like to foster research on "Quantum Epigenetics" at the interface of medicine, biology, biochemistry, and physics to investigate the potential epigenetic impact of quantum physical principles on (human) life.