Cytosine-Rich Oligonucleotide and Electrochemically Reduced Graphene Oxide Nanocomposite for Ultrasensitive Electrochemical Ag+ Sensing.

Silver ions (Ag+) are crucial in various fields, but pose environmental and health risks at high concentrations. This study presents a straightforward approach for the ultra-trace detection of Ag+, utilizing a composite of a cytosine-rich oligonucleotide (CRO) and an electrochemically reduced graphene oxide (ERGO). Initially, ERGO was synthesized on a glassy carbon electrode (GCE) through the reduction of graphene oxide (GO) via cyclic voltammetry. A methylene blue-tagged CRO (MB-CRO) was then anchored to the ERGO surface through π-π interactions, resulting in the formation of an MB-CRO-modified ERGO electrode (MB-CRO/ERGO-GCE). The interaction with Ag+ ions induced the formation of silver-mediated C-Ag+-C coordination, prompting the MB-CRO to adopt a hairpin structure. This conformational change led to the desorption of the MB-CRO from the ERGO-GCE, causing a variation in the redox current of the methylene blue associated with the MB-CRO. Electrochemical assays revealed that the sensor exhibits extraordinary sensitivity to Ag+ ions, with a linear detection range from 1 femtomolar (fM) to 100 nanomolars (nM) and a detection limit of 0.83 fM. Moreover, the sensor demonstrated high selectivity for Ag+ ions and several other benefits, including stability, reproducibility, and straightforward fabrication and operational procedures. Additionally, real sample analyses were performed using the modified electrode to detect Ag+ in tap and pond water samples, yielding satisfactory recovery rates.

Structural peculiarities of tandem repeats and their clinical significance.

While it is well established that a mere 2% of human DNA nucleotides are involved in protein coding, the remainder of the DNA plays a vital role in the preservation of normal cellular genetic function. A significant proportion of tandem repeats (TRs) are present in non-coding DNA. TRs - specific sequences of nucleotides that entail numerous repetitions of a given fragment. In this study, we employed our novel algorithm grounded in finite automata theory, which we refer to as Dafna, to investigate for the first time the likelihood of these nucleotide sequences forming non-canonical DNA structures (NS). Such structures include G-quadruplexes, i-motifs, hairpins, and triplexes. The tandem repeats under consideration in our research encompassed sequences containing 1 to 6 nucleotides per repeated fragment. For comparison, we employed a set of randomly generated sequences of the same length (60 nucleotides) as a benchmark. The outcomes of our research exposed a disparity between the potential for NS formation in random sequences and tandem repeats. Our findings affirm that the propensity of DNA and RNA to form NS is closely tied to various genetic disorders, including Huntington's disease, Fragile X syndrome, and Friedreich's ataxia. In the concluding discussion, we present a proposal for a new therapeutic mechanism to address these diseases. This novel approach revolves around the ability of specific nucleic acid fragments to form multiple types of NS.

All-Covalent Nuclease-Resistant and Hydrogel-Tethered DNA Hairpin Probes Map pN Cell Traction Forces.

Cells sense and respond to the physical properties of their environment through receptor-mediated signaling, a process known as mechanotransduction, which can modulate critical cellular functions such as proliferation, differentiation, and survival. At the molecular level, cell adhesion receptors, such as integrins, transmit piconewton (pN)-scale forces to the extracellular matrix, and the magnitude of the force plays a critical role in cell signaling. The most sensitive approach to measuring integrin forces involves DNA hairpin-based sensors, which are used to quantify and map forces in living cells. Despite the broad use of DNA hairpin sensors to study a variety of mechanotransduction processes, these sensors are typically anchored to rigid glass slides, which are orders of magnitude stiffer than the extracellular matrix and hence modulate native biological responses. Here, we have developed nuclease-resistant DNA hairpin probes that are all covalently tethered to PEG hydrogels to image cell traction forces on physiologically relevant substrate stiffness. Using HeLa cells as a model cell line, we show that the molecular forces transmitted by integrins are highly sensitive to the bulk modulus of the substrate, and cells cultured on the 6 and 13 kPa gels produced a greater number of hairpin unfolding events compared to the 2 kPa substrates. Tension signals are spatially colocalized with pY118-paxillin, confirming focal adhesion-mediated probe opening. Additionally, we found that integrin forces are greater than 5.8 pN but less than 19 pN on 13 kPa gels. This work provides a general strategy to integrate molecular tension probes into hydrogels, which can better mimic in vivo mechanotransduction.

Artemis inhibition as a therapeutic strategy for acute lymphoblastic leukemia.

As effective therapies for relapse and refractory B-cell acute lymphoblastic leukemia (B-ALL) remain problematic, novel therapeutic strategies are needed. Artemis is a key endonuclease in V(D)J recombination and nonhomologous end joining (NHEJ) of DNA double-strand break (DSB) repair. Inhibition of Artemis would cause chromosome breaks during maturation of RAG-expressing T- and B-cells. Though this would block generation of new B- and T-cells temporarily, it could be oncologically beneficial for reducing the proliferation of B-ALL and T-ALL cells by causing chromosome breaks in these RAG-expressing tumor cells. Currently, pharmacological inhibition is not available for Artemis. According to gene expression analyses from 207 children with high-risk pre-B acute lymphoblastic leukemias high Artemis expression is correlated with poor outcome. Therefore, we evaluated four compounds (827171, 827032, 826941, and 825226), previously generated from a large Artemis targeted drug screen. A biochemical assay using a purified Artemis:DNA-PKcs complex shows that the Artemis inhibitors 827171, 827032, 826941, 825226 have nanomolar IC50 values for Artemis inhibition. We compared these 4 compounds to a DNA-PK inhibitor (AZD7648) in three patient-derived B-ALL cell lines (LAX56, BLQ5 and LAX7R) and in two mature B-cell lines (3301015 and 5680001) as controls. We found that pharmacological Artemis inhibition substantially decreases proliferation of B-ALL cell lines while normal mature B-cell lines are not markedly affected. Inhibition of DNA-PKcs (which regulates Artemis) using the DNA-PK inhibitor AZD7648 had minor effects on these same primary patient-derived ALL lines, indicating that inhibition of V(D)J hairpin opening requires direct inhibition of Artemis, rather than indirect suppression of the kinase that regulates Artemis. Our data provides a basis for further evaluation of pharmacological Artemis inhibition of proliferation of B- and T-ALL.

Purification of DNA oligonucleotides to improve hybridization chain reaction performance.

Hybridization Chain Reaction (HCR) is a technique to generate a linear polymerization of oligonucleotide hairpins, used in multiple molecular biology methods. The HCR reaction is dependent on every hairpin being metastable in the absence of a triggering oligonucleotide and that every hairpin can continue the polymerization, which puts a strong demand on oligonucleotide quality. We show how further purification can greatly increase polymerization potential. It was found that a single extra PAGE-purification could greatly enhance hairpin polymerization both in solution and in situ. Purification using a ligation-based method further improved polymerization, yielding in situ immunoHCR stains at least 3.4-times stronger than a non-purified control. This demonstrates the importance of not only good sequence design of the oligonucleotide hairpins, but also the demand for high quality oligonucleotides to accomplish a potent and specific HCR.

Detection of E.coli 23S rRNA by electrocatalytic "off-on" DNA beacon assay with femtomolar sensitivity.

Prevention of food spoilage, environmental bio-contamination, and pathogenic infections requires rapid and sensitive bacterial detection systems. Among microbial communities, the bacterial strain of Escherichia coli is most widespread, with pathogenic and non-pathogenic strains being biomarkers of bacterial contamination. Here, we have developed a fM-sensitive, simple, and robust electrocatalytically-amplified assay facilitating specific detection of E.coli 23S ribosomal rRNA, in the total RNA sample, after its site-specific cleavage by RNase H enzyme. Gold screen-printed electrodes (SPE) were electrochemically pre-treated to be productively modified with a methylene-blue (MB) - labelled hairpin DNA probes, which hybridization with the E. coli-specific DNA placed MB in the top region of the DNA duplex. The formed duplex acted as an electrical wire, mediating electron transfer from the gold electrode to the DNA-intercalated MB, and further to ferricyanide in solution, enabling its electrocatalytic reduction otherwise impeded on the hairpin-modified SPEs. The assay facilitated 20 min 1 fM detection of both synthetic E. coli DNA and 23S rRNA isolated from E.coli (equivalent to 15 CFU mL-1), and can be extended to fM analysis of nucleic acids isolated from any other bacteria.

Nanopore-Based Fingerprint Immunoassay Based on Rolling Circle Amplification and DNA Fragmentation.

In recent years, nanopore-based sequencers have become robust tools with unique advantages for genomics applications. However, progress toward applying nanopores as highly sensitive, quantitative diagnostic tools has been impeded by several challenges. One major limitation is the insufficient sensitivity of nanopores in detecting disease biomarkers, which are typically present at pM or lower concentrations in biological fluids, while a second limitation is the general absence of unique nanopore signals for different analytes. To bridge this gap, we have developed a strategy for nanopore-based biomarker detection that utilizes immunocapture, isothermal rolling circle amplification, and sequence-specific fragmentation of the product to release multiple DNA reporter molecules for nanopore detection. These DNA fragment reporters produce sets of nanopore signals that form distinctive fingerprints, or clusters. This fingerprint signature therefore allows the identification and quantification of biomarker analytes. As a proof of concept, we quantify human epididymis protein 4 (HE4) at low pM levels in a few hours. Future improvement of this method by integration with a nanopore array and microfluidics-based chemistry can further reduce the limit of detection, allow multiplexed biomarker detection, and further reduce the footprint and cost of existing laboratory and point-of-care devices.

Stability of DNA and RNA hairpins: a comparative study based on ox-DNA.

Advances in single-molecule experiments on macromolecular crowding urgently need an efficient simulation method to resolve their discrepancies quantitatively. Ox-DNA model has been since reworked to treat the thermodynamics and mechanical properties of DNA/RNA hairpin at a stretching force. In hopping experiments, the critical forces of RNA hairpins at different temperatures are greater than those of DNA hairpins, in addition, the Gibbs free energy at a fixed temperature required to convert an RNA hairpin into a single-stranded molecule at zero force is obviously greater than that of DNA hairpin and gradually decreases by increasing the temperature. As far as force-ramping experiments are concerned, the first-rupture forces of RNA/DNA hairpins corresponding to the maximum probability density linearly pertain to the force-loading rate, with those of RNA hairpins being greater. The extended ox-DNA model could potentially identify the interaction between biologically inert polymer and RNA/DNA hairpins in crowded environments.

Long-term continuous monitoring of microRNA in living cells using modified gold nanoprobe.

Long-term and continuous monitoring of the microRNA (miRNA) expression in living cells is essential in biomedical research, but it is currently limited by fast consumption and easy digestion of probes in the intracellular environment. Herein, we report polydopamine-modified gold nanoparticles (AuNPs@PDA) as protective and efficient nanocarriers for DNA hairpin probes (hpDNA), achieving long-term monitoring (48 h) of the miRNA (let-7a) levels in living cells after drug treatments. This method enabled excellent sensitivity and high selectivity toward let-7a with a limit of detection of 0.51 nM (n = 3) and a linear range from 1 to 100 nM. More importantly, AuNPs@PDA can not only efficiently improve the loading of hpDNA on each nanoparticle, but also effectively protect hpDNA from hydrolysis in the cell microenvironment, finally realizing the continuous monitoring of let-7a in living cells for 48 h. This simple method would be of great significance for drug screening and precision medicine.

Structural Responses of Nucleic Acids to Mars-Relevant Salts at Deep Subsurface Conditions.

High pressure deep subsurface environments of Mars may harbor high concentrations of dissolved salts, such as perchlorates, yet we know little about how these salts influence the conditions for life, particularly in combination with high hydrostatic pressure. We investigated the effects of high magnesium perchlorate concentrations compared to sodium and magnesium chloride salts and high pressure on the conformational dynamics and stability of double-stranded B-DNA and, as a representative of a non-canonical DNA structure, a DNA-hairpin (HP), whose structure is known to be rather pressure-sensitive. To this end, fluorescence spectroscopies including single-molecule FRET methodology were applied. Our results show that the stability both of the B-DNA as well as the DNA-HP is largely preserved at high pressures and high salt concentrations, including the presence of chaotropic perchlorates. The perchlorate anion has a small destabilizing effect compared to chloride, however. These results show that high pressures at the kbar level and perchlorate anions can modify the stability of nucleic acids, but that they do not represent a barrier to the gross stability of such molecules in conditions associated with the deep subsurface of Mars.

Equivalence of the transition heat capacities of proteins and DNA.

It has been reported for many globular proteins that the native heat capacity at 25 °C, per gram, is the same. This has been interpreted to indicate that heat capacity is a fundamental property of native proteins that provides important information on molecular structure and stability. Heat capacities for both proteins and DNA has been suggested to be related to universal effects of hydration/solvation on native structures. Here we report on results from thermal denaturation analysis of two well-known proteins, human serum albumin and lysozyme, and a short DNA hairpin. The transition heat capacities at the Tm for the three molecules were quantitatively evaluated by differential scanning calorimetry. When normalized per gram rather than per mol the transition heat capacities were found to be precisely equivalent. This observation for the transition heat capacities of the proteins is consistent with previous reports. However, an identical transition heat capacity for DNA has not been reported and was unexpected. Further analysis of the collected data suggested a mass dependence of hydration effects on thermal denaturation that is preserved at the individual protein amino acid and DNA base levels. Equivalence of transition heat capacities suggests the possibility of a universal role of hydration effects on the thermal stability of both proteins and DNA.

Electrochemical biosensor for detecting pathogenic bacteria based on a hybridization chain reaction and CRISPR-Cas12a.

In this study, Lba Cas12a (Cpf1) as one of the CRISPR systems from Lachnospiraceae bacterium was coupled with a hybridization chain reaction (HCR) to develop an electrochemical biosensor for detecting the pathogenic bacterium, Salmonella typhimurium. Autonomous cross-opening of functional DNA hairpin structures of HCR yielded polymer double-stranded DNA wires consisting of numerous single-stranded DNAs, which initiated the trans-cleavage activity of CRISPR-Cas12a to indiscriminately cleave random single-stranded DNA labeling electrochemical tags on the surface of the electrode. It led to a variation in the electron transfer of electrochemical tags. The polymer double-stranded DNA of HCR was immobilized on dynabeads (DBs) via the S. typhimurium aptamer and released from DBs. The established method could selectively and sensitively quantify S. typhimurium in samples with detection limits of 20 CFU/mL. Our study provides a novel insight for exploring universal analytical methods for pathogenic bacteria based on CRISPR-Cas12a coupled with HCR.

Decoding the Structural Dynamics and Conformational Alternations of DNA Secondary Structures by Single-Molecule FRET Microspectroscopy.

In addition to the canonical double helix form, DNA is known to be extrapolated into several other secondary structural patterns involving themselves in inter- and intramolecular type hydrogen bonding. The secondary structures of nucleic acids go through several stages of multiple, complex, and interconvertible heterogeneous conformations. The journey of DNA through these conformers has significant importance and has been monitored thoroughly to establish qualitative and quantitative information about the transition between the unfolded, folded, misfolded, and partially folded states. During this structural interconversion, there always exist specific populations of intermediates, which are short-lived or sometimes even do not accumulate within a heterogeneous population and are challenging to characterize using conventional ensemble techniques. The single-molecule FRET(sm-FRET) microspectroscopic method has the advantages to overcome these limitations and monitors biological phenomena transpiring at a measurable high rate and balanced stochastically over time. Thus, tracing the time trajectory of a particular molecule enables direct measurement of the rate constant of each transition step, including the intermediates that are hidden in the ensemble level due to their low concentrations. This review is focused on the advantages of the employment of single-molecule Forster's resonance energy transfer (sm-FRET), which is worthwhile to access the dynamic architecture and structural transition of various secondary structures that DNA adopts, without letting the donor of one molecule to cross-talk with the acceptor of any other. We have emphasized the studies performed to explore the states of folding and unfolding of several nucleic acid secondary structures, for example, the DNA hairpin, Holliday junction, G-quadruplex, and i-motif.

DNA polymerase θ promotes CAG•CTG repeat expansions in Huntington's disease via insertion sequences of its catalytic domain.

Huntington's disease (HD), a neurodegenerative disease characterized by progressive dementia, psychiatric problems, and chorea, is known to be caused by CAG repeat expansions in the HD gene HTT. However, the mechanism of this pathology is not fully understood. The translesion DNA polymerase θ (Polθ) carries a large insertion sequence in its catalytic domain, which has been shown to allow DNA loop-outs in the primer strand. As a result of high levels of oxidative DNA damage in neural cells and Polθ's subsequent involvement in base excision repair of oxidative DNA damage, we hypothesized that Polθ contributes to CAG repeat expansion while repairing oxidative damage within HTT. Here, we performed Polθ-catalyzed in vitro DNA synthesis using various CAG•CTG repeat DNA substrates that are similar to base excision repair intermediates. We show that Polθ efficiently extends (CAG)n•(CTG)n hairpin primers, resulting in hairpin retention and repeat expansion. Polθ also triggers repeat expansions to pass the threshold for HD when the DNA template contains 35 repeats upward. Strikingly, Polθ depleted of the catalytic insertion fails to induce repeat expansions regardless of primers and templates used, indicating that the insertion sequence is responsible for Polθ's error-causing activity. In addition, the level of chromatin-bound Polθ in HD cells is significantly higher than in non-HD cells and exactly correlates with the degree of CAG repeat expansion, implying Polθ's involvement in triplet repeat instability. Therefore, we have identified Polθ as a potent factor that promotes CAG•CTG repeat expansions in HD and other neurodegenerative disorders.

Quantify the combined effects of temperature and force on the stability of DNA hairpin.

OxDNA, as a successful coarse-grain model, has been applied to reproduce the thermodynamic and mechanical properties of both single- and double-stranded DNA. In current simulation, oxDNA is extended to explore the combined effects of temperature and force on the stability of DNA hairpin and its free energy landscape. Simulations were carried out at different forces and temperatures, at each temperature, a 18-base-pair DNA hairpin dynamically transited between folded state and unfolded state, and the separation between two states is consistent with the full contour length of single-stranded DNA in the unfolded state. Two methods were used to identify the critical force of DNA hairpin at each temperature and the critical forces obtained from two methods were consistent with each other and gradually decreased with the increasing temperature from 300 K to 326 K. The critical force at 300 K is reasonably consistent with the single molecule result of DNA hairpin with the same stem length. The two-state free energy landscape can be elucidated from the probability distribution of DNA hairpin extension and its dependence on the force and temperature is totally different. The increasing temperature not only reduces the free energy barrier, but also alters the position of transition point along the extension coordinate, resulting in the reduction of folding distance and the extension of unfolding distance, but their sum is not obviously dependent on the temperature. Generally, an assumption that the location of transition state in two-state energy landscape is independent of the stretching force is used to analyze the data of the single molecule experiment, but current simulation results indicate that effects of stretching forces on the location of transition state in two-state energy landscape are dependent on temperature. At relatively high temperature, stretching force can also change the location of transition state in the free energy landscape.

Nanosensors for single cell mechanical interrogation.

The occurrence and development of many diseases are accompanied and sometimes dictated by the destruction of biomechanical homeostasis. For instance, cancer cells and normal cells show different cellular mechanical forces phenotypes, as the proliferation and invasion ability of cancer cells is often related to the changes in mechanical force in the tumor. With single cell analysis, variations in mechanics within a cell population can be detected and analyzed, opening new dimensions in the study of cancer. Nanosensor design for interrogation of cell mechanics is an interdisciplinary area bridging over cell biology, mechanics, and micro/nanotechnology. In this tutorial review, we give insight into the background and technical innovation of currently available methods for mechanical analysis of cells. First, we discuss the mechanism of mechanical changes in the development and progression of cancer that shows the feasibility of mechanical sensors in cancer cell detection. Next, we summarize the principle, progress, and essential problems of common technologies for cell force measurement, including single molecule force spectroscopy and elastic substrate-sensors. Following that, we discuss novel micro and nano-scale mechanical sensors and their applications in single cell level biological analysis. At last, we elaborate on the remaining issues and trends of the cellular mechanical sensors.

Alternative DNA Structures In Vivo: Molecular Evidence and Remaining Questions.

Duplex DNA naturally folds into a right-handed double helix in physiological conditions. Some sequences of unusual base composition may nevertheless form alternative structures, as was shown for many repeated sequences in vitro However, evidence for the formation of noncanonical structures in living cells is difficult to gather. It mainly relies on genetic assays demonstrating their function in vivo or through genetic instability reflecting particular properties of such structures. Efforts were made to reveal their existence directly in a living cell, mainly by generating antibodies specific to secondary structures or using chemical ligands selected for their affinity to these structures. Among secondary structure-forming DNAs are G-quadruplexes, human fragile sites containing minisatellites, AT-rich regions, inverted repeats able to form cruciform structures, hairpin-forming CAG/CTG triplet repeats, and triple helices formed by homopurine-homopyrimidine GAA/TTC trinucleotide repeats. Many of these alternative structures are involved in human pathologies, such as neurological or developmental disorders, as in the case of trinucleotide repeats, or cancers triggered by translocations linked to fragile sites. This review will discuss and highlight evidence supporting the formation of alternative DNA structures in vivo and will emphasize the role of the mismatch repair machinery in binding mispaired DNA duplexes, triggering genetic instability.

The assessment of three dimensional modelling design for single strand DNA aptamers for computational chemistry application.

Aptamers are oligonucleotides and peptides around 15-100 bases in length and are suitable as detection probes or as therapeutics molecules. There are growing interests in the aptamer screening approach through computational simulation methods. DNA and RNA modelling lacks of validation on their predicted 3D structures due to less number of validation tools, unlike protein structures. We suggest an approach to design the stem-loop/hairpin for the three dimensional structure of DNA aptamers through serial applications of computational prediction methods by comparing the simulated structures with the experimental data deposited in PDB Data bank, followed by MD simulations. The result shows minimal structural differences were observed between the designed and the original NMR aptamers, and the stem-loop conformational structures were also retained during the MD thus suggesting the proposed aptamers designing methods are able to synthesize a high quality molecular structure of hairpin aptamers, comparable to the NMR structures.

Interaction of a photosensitizer methylene blue with various structural forms (cruciform, bulge duplex and hairpin) of designed DNA sequences.

Functionally important, local structural transitions in DNA generate various alternative conformations. Cruciform is one of such alternative DNA structures, usually targeted in genomes by various proteins. Symmetry elements in sequence as inverted repeats are the key factor for cruciform formation, facilitated by the presence of the AT-rich regions. Here, we used biophysical and biochemical techniques such as Gel electrophoresis, Circular dichroism (CD), and UV-thermal melting analysis to explore the structural status of the designed DNA sequences, which had potential to form cruciform structures under physiological conditions. The gel electrophoresis analysis revealed that the designed 53-mer DNA oligonucleotide sequence CR forms an intermolecular bulge duplex with flanking ends, while another sequence CRC adopts an intramolecular hairpin structure with flanking ends. Their equimolar complex (CRCRC) bestowed much-retarded migration due to the formation of a quite intriguing cruciform structure. CD studies confirmed that all the alternative structures (cruciform, bulge duplex, and hairpin with flanking ends) exhibit characteristics of B-DNA type conformation. A triphasic UV-thermal melting curve displayed by the complex formed by the equimolar ratio (CRCRC) is also suggestive of the formation of the cruciform structure. The interaction studies of CR, CRC, and their equimolar complex (1:1) with a photosensitizer methylene blue (MB) indicated that MB could not stabilize the discrete structures formed by CR and CRC sequences, however, the cruciform structure showed a quite significant increment in the melting temperature. Such studies facilitate our understanding of various secondary structures possibly present inside the cell and their interactions with drug/dye molecules.

Alteration of the Conformational Dynamics of a DNA Hairpin by α-Synuclein in the Presence of Aqueous Two-Phase Systems.

The effect of an amyloidogenic intrinsically disordered protein, α-synuclein, which is associated with Parkinson's disease (PD), on the conformational dynamics of a DNA hairpin (DNA-HP) was studied by employing the single-molecule Förster resonance energy transfer method. The open-to-closed conformational equilibrium of the DNA-HP is drastically affected by binding of monomeric α-synuclein to the loop region of the DNA-HP. Formation of a protein-bound intermediate conformation is fostered in the presence of an aqueous two-phase system mimicking intracellular liquid-liquid phase separation. Using pressure modulation, additional mechanistic information about the binding complex could be retrieved. Hence, in addition to toxic amyloid formation, α-synuclein may alter expression profiles of disease-modifying genes in PD. Furthermore, these findings might also have significant bearings on the understanding of the physiology of organisms thriving at high pressures in the deep sea.