Trigger DNA Research Articles

DNAR-07. ATR INHIBITORS IN COMBINATION WITH RADIATION AND TEMOZOLOMIDE INCREASE CELL DEATH OF PATIENT-DERIVED GLIOBLASTOMA CELLS

Abstract Glioblastoma is treated with surgery to remove as much as the tumour as possible then radiation therapy (RT) and temozolomide (TMZ) to cause extensive DNA damage in any remaining tumour cells, triggering cell death by apoptosis. However, glioblastoma cells respond by upregulating the DNA damage response (DDR) triggering DNA repair that contributes to treatment resistance. Progression then generally occurs rapidly leaving patients with a poor prognosis and survival. We previously showed that gartisertib, an inhibitor of ATM- and Rad3-Related protein (ATR), a serine/threonine kinase that triggers DNA repair, sensitises glioblastoma cells to TMZ and RT. We extend this investigation to brain-penetrant ATR inhibitors elimusertib (BAY1895344) and ceralasertib (AZD6738). Patient-derived glioblastoma cell lines were treated with a dose response of ATRi (elimusertib and ceralasertib) combined with a clinically relevant dose of TMZ (35uM) and/or RT (2Gy). A human astrocyte cell line was also treated with a dose response of single agent elimusertib and ceralasertib. Cells were incubated for 7 days and assessed for cell viability through an MTT assay or cell-cell Incucyte analysis. Both elimusertib and ceralasertib as single agents reduced glioblastoma cell growth, with elimusertib being more potent compared to ceralasertib. Elimusertib had greater potency in causing cell death of the glioblastoma cell lines than a human astrocyte cell line. However, there was less difference between glioblastoma and astrocyte cell death when treated with ceralasetib. When combined with RT and/or TMZ, elimusertib once again was more potent in reducing glioblastoma cell growth compared to ceralasertib. We have expanded our in vitro data showing that various ATR inhibitors (elimusertib, ceralasertib and previously gartisertib) are able to reduce the growth of glioblastoma cells in combination with RT and TMZ treatment. This provides further support for initiating a clinical trial of ATR inhibition in combination with standard treatment for patients with glioblastoma.

Facile bio-inspired fabrication of AgNPs from Salvia elegans leaf extract and determination of their cytotoxic DNA photocleavage potential

This study introduces a sustainable method for producing silver nanoparticles (AgNPs), focusing on sustainability and environmental protection. The technique includes the use of Salvia elegans aqueous leaf extract as a reducing agent. In addition, the research investigates the dose-dependent degradation of pUC19 DNA by silver nanoparticles, which is facilitated by the generation of singlet oxygen. The presence of S. elegans extract results in significant color changes, going from a colorless state to a dark brown hue, serving as an indication of the synthesis of AgNPs. A range of experimental methods were employed to analyze the biogenic silver nanoparticles, such as UV–visible absorption spectroscopy, X-ray diffraction (XRD), transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The meticulously produced AgNPs demonstrate a high level of uniformity, featuring a spherical shape and a particle size of 60 nm. DNA photo-cleavage studies show that singlet oxygen plays a crucial role in triggering DNA damage caused by AgNPs indicates their potential as powerful cytotoxic agents specifically aimed at cancer cells. Additional studies are required to clarify the effectiveness and specificity of AgNPs against various cancer cell types to assess their therapeutic potential. The cytotoxic effects of AgNPs, particularly in relation to DNA photocleavage, are of considerable interest in cancer research. This research holds promise for developing novel and sustainable cancer therapies based on the unique properties of biogenic silver nanoparticles.

Impacts of iodoacetic acid on reproduction: current evidence, underlying mechanisms, and future research directions.

In light of the undeniable and alarming fact that human fertility is declining, the harmful factors affecting reproductive health are garnering more and more attention. Iodoacetic acid (IAA), an emerging unregulated drinking water disinfection byproduct, derives from chlorine disinfection and is frequently detected in the environment and biological samples. Humans are ubiquitously exposed to IAA daily mainly through drinking water, consuming food and beverages made from disinfected water, contacting swimming pools and bath water, etc. Mounting evidence has indicated that IAA could act as a reproductive toxicant and bring about multifarious adverse reproductive damage. For instance, it can interfere with gonadal development, weaken ovarian function, impair sperm motility, trigger DNA damage to germ cells, perturb steroidogenesis, etc. The underlying mechanisms predominantly include cytotoxic and genotoxic effects on germ cells, disturbance of the hypothalamic-pituitary-gonadal axis, oxidative stress, inhibition of steroidogenic proteins or enzymes, and dysbiosis of gut microbiota. Nevertheless, there are still some knowledge gaps and limitations in studying the potential impact of IAA on reproduction, which urgently need to be addressed in the future. We suppose that necessary population epidemiological studies, more sensitive detection methods for internal exposure, and mechanism-based in-depth exploration will contribute to a more comprehensive understanding of characteristics and biological effects of IAA, thus providing an important scientific basis for revising sanitary standards for drinking water quality.

Synthesis and characterization of 7-diethylamino-4-Chloromethyl coumarin: Spectroscopic analysis, molecular docking, and anticancer activity on large intestine carcinoma cells

Cancer, characterized by uncontrolled cell growth and metastasis, poses a significant global health burden, ranking as a leading cause of mortality worldwide. Colorectal cancer (CRC) specifically accounts for a substantial portion of cancer cases, with increasing incidence projected over the coming decades. While conventional treatments exist, they often entail adverse effects and limited efficacy, driving interest in natural remedies like coumarin derivatives due to their diverse biological activities and perceived safety profile. This study focuses on the synthesis and characterization of a novel compound, 7-diethylamino-4-chloromethyl coumarin (referred to as 7D4C), derived from coumarin. Structural elucidation employed Fourier transform infrared spectroscopy (FT-IR), proton and carbon-13 nuclear magnetic resonance spectroscopy (1H and 13C NMR), and mass spectrometry (MALDI-TOF-MS). Molecular docking studies were conducted to explore potential biological interactions. Furthermore, the anti-cancer potential of 7D4C was assessed using human epithelial adenocarcinom (LoVo) and healthy fibroblast (CCD-18Co) cell lines. Viability analysis, comet assay for DNA damage, and evaluation of cancer biomarkers including apoptosis, intracellular reactive oxygen species (iROS) levels, mitochondrial membrane potential (MMP), intracellular glutathione (GSH) concentration, and intracellular calcium (iCa2+) levels were performed. The synthesis of 7D4C was successfully completed, and its structure was confirmed. Molecular docking results indicate that 7D4C exhibits strong binding affinity to the p53 protein, highlighting its potential as a novel modulator of p53 activity. Subsequent investigations revealed that the synthesized compound induced apoptosis in cancer cells by reducing MMP and triggering DNA damage through the production of iROS. The promising anti-cancer activity of 7D4C in the LoVo cell line highlights its importance in coumarin-based therapies. Introducing 7D4C could significantly enhance future research in this area, leveraging insights from in vitro coumarin studies.

Integrated OPECT and Smartphone Colorimetry Dual‐Mode Detection of Okadaic Acid Based on Ce‐MOF Modified MXene@SnO2 Z‐Scheme Heterostructure

AbstractThe organic photoelectrochemical transistor (OPECT) biosensing relies solely on a singular signal readout inherently, which restrains the precision and dependability nestled within pertinent biological measurements. Herein, a high‐precision magnetic assisted OPECT and smartphone colorimetric (SCL) dual‐mode biosensing platform is first established for detecting harmful algal toxin okadaic acid (OA) by biocatalytic reaction. MXene@SnO2‐Ce‐MOF (MXSnO/Ce‐MOF) Z‐scheme heterojunctions with abundant oxygen vacancies are prepared as photoactive materials. Initially, in the presence of OA, the coupling of trigger DNA (tDNA) to magnetic beads (MBs) via anchor DNA (aDNA) is released through the interaction of the target analyte with the aptamer. Subsequently, the carried tDNA triggers HCR between the two hairpin sequences, producing long double helix chains to capture glucose oxidase (GOx). The obtained GOx supernatant catalyzes glucose to produce H2O2, which can oxidize Ce‐MOF, leading to the alteration of electrode color and a significant decrease in the overall photocurrent of MXSnO/Ce‐MOF. Crucially, the novel OPECT‐SCL biosensor exhibits excellent sensitivity and precision, boasting detection thresholds as low as 42.9 pM and 1.2 nM, respectively, and accomplishes the automated detection of OA within real samples. The proposed OPECT‐SCL dual‐signal measurement model constitutes a sensitive, portable, and precise platform for the quantification of marine toxins.

Malate dehydrogenase-2 inhibition shields renal tubular epithelial cells from anoxia-reoxygenation injury by reducing reactive oxygen species.

Ischemia-reperfusion (I-R) injury is the most common cause of acute kidney injury. In experiments involving primary human renal proximal tubular epithelial cells (RPTECs) exposed to anoxia-reoxygenation, we explored the hypothesis that mitochondrial malate dehydrogenase-2 (MDH-2) inhibition redirects malate metabolism from the mitochondria to the cytoplasm, towards the malate-pyruvate cycle and reversed malate-aspartate shuttle. Colorimetry, fluorometry, and western blotting showed that MDH2 inhibition accelerates the malate-pyruvate cycle enhancing cytoplasmic NADPH, thereby regenerating the potent antioxidant reduced glutathione. It also reversed the malate-aspartate shuttle and potentially diminished mitochondrial reactive oxygen species (ROS) production by transferring electrons, in the form of NADH, from the mitochondria to the cytoplasm. The excessive ROS production induced by anoxia-reoxygenation led to DNA damage and protein modification, triggering DNA damage and unfolded protein response, ultimately resulting in apoptosis and senescence. Additionally, ROS induced lipid peroxidation, which may contribute to the process of ferroptosis. Inhibiting MDH-2 proved effective in mitigating ROS overproduction during anoxia-reoxygenation, thereby rescuing RPTECs from death or senescence. Thus, targeting MDH-2 holds promise as a pharmaceutical strategy against I-R injury.

Controlled mechanochemical coupling of anti-junctions in DNA origami arrays

Allostery is a hallmark of cellular function and important in every biological system. Still, we are only starting to mimic it in the laboratory. Here, we introduce an approach to study aspects of allostery in artificial systems. We use a DNA origami domino array structure which–upon binding of trigger DNA strands–undergoes a stepwise allosteric conformational change. Using two FRET probes placed at specific positions in the DNA origami, we zoom in into single steps of this reaction cascade. Most of the steps are strongly coupled temporally and occur simultaneously. Introduction of activation energy barriers between different intermediate states alters this coupling and induces a time delay. We then apply these approaches to release a cargo DNA strand at a predefined step in the reaction cascade to demonstrate the applicability of this concept in tunable cascades of mechanochemical coupling with both spatial and temporal control.

SARS-CoV-2 N protein-induced Dicer, XPO5, SRSF3, and hnRNPA3 downregulation causes pneumonia

Though RNAi and RNA-splicing machineries are involved in regulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication, their precise roles in coronavirus disease 2019 (COVID-19) pathogenesis remain unclear. Herein, we show that decreased RNAi component (Dicer and XPO5) and splicing factor (SRSF3 and hnRNPA3) expression correlate with increased COVID-19 severity. SARS-CoV-2 N protein induces the autophagic degradation of Dicer, XPO5, SRSF3, and hnRNPA3, inhibiting miRNA biogenesis and RNA splicing and triggering DNA damage, proteotoxic stress, and pneumonia. Dicer, XPO5, SRSF3, and hnRNPA3 knockdown increases, while their overexpression decreases, N protein-induced pneumonia’s severity. Older mice show lower expression of Dicer, XPO5, SRSF3, and hnRNPA3 in their lung tissues and exhibit more severe N protein-induced pneumonia than younger mice. PJ34, a poly(ADP-ribose) polymerase inhibitor, or anastrozole, an aromatase inhibitor, ameliorates N protein- or SARS-CoV-2-induced pneumonia by restoring Dicer, XPO5, SRSF3, and hnRNPA3 expression. These findings will aid in developing improved treatments for SARS-CoV-2-associated pneumonia.

Unveiling the role of TAGLN2 in glioblastoma: From proneural-mesenchymal transition to Temozolomide resistance

Glioblastoma (GBM) presents a daunting challenge due to its resistance to temozolomide (TMZ), a hurdle exacerbated by the proneural-to-mesenchymal transition (PMT) from a proneural (PN) to a mesenchymal (MES) phenotype. TAGLN2 is prominently expressed in GBM, particularly in the MES subtype compared to low-grade glioma (LGG) and the PN subtype. Our research reveals TAGLN2's involvement in PMT and TMZ resistance through a series of in vitro and in vivo experiments. TAGLN2 knockdown can restrain proliferation and invasion, trigger DNA damage and apoptosis, and heighten TMZ sensitivity in GBM cells. Conversely, elevating TAGLN2 levels amplifies resistance to TMZ in cellular and intracranial xenograft mouse models. We demonstrate the interaction relationship between TAGLN2 and ERK1/2 through co-immunoprecipitation (Co-IP) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) spectrometry analysis. Knockdown of TAGLN2 results in a decrease in the expression of p-ERK1/2, whereas overexpression of TAGLN2 leads to an increase in p-ERK1/2 expression within the nucleus. Subsequently, the regulatory role of TAGLN2 in the expression and control of MGMT has been demonstrated. Finally, the regulation of TAGLN2 by NF-κB has been validated through chromatin immunoprecipitation and ChIP-PCR assays. In conclusion, our results confirm that TAGLN2 exerts its biological functions by interacting with the ERK/MGMT axis and being regulated by NF-κB, thereby facilitating the acquisition of promoting PMT and increased resistance to TMZ therapy in glioblastoma. These results provide valuable insights for the advancement of targeted therapeutic approaches to overcome TMZ resistance in clinical treatments.

Radiotherapy and immunology.

The majority of cancer patients receive radiotherapy during the course of treatment, delivered with curative intent for local tumor control or as part of a multimodality regimen aimed at eliminating distant metastasis. A major focus of research has been DNA damage; however, in the past two decades, emphasis has shifted to the important role the immune system plays in radiotherapy-induced anti-tumor effects. Radiotherapy reprograms the tumor microenvironment, triggering DNA and RNA sensing cascades that activate innate immunity and ultimately enhance adaptive immunity. In opposition, radiotherapy also induces suppression of anti-tumor immunity, including recruitment of regulatory T cells, myeloid-derived suppressor cells, and suppressive macrophages. The balance of pro- and anti-tumor immunity is regulated in part by radiotherapy-induced chemokines and cytokines. Microbiota can also influence radiotherapy outcomes and is under clinical investigation. Blockade of the PD-1/PD-L1 axis and CTLA-4 has been extensively investigated in combination with radiotherapy; we include a review of clinical trials involving inhibition of these immune checkpoints and radiotherapy.

Mebendazole preferentially inhibits cilia formation and exerts anticancer activity by synergistically augmenting DNA damage

The cilium is a microtubule-based organelle that plays a pivotal role in embryonic development and maintenance of physiological functions in the human body. In addition to their function as sensors that transduce diverse extracellular signals, including growth factors, fluid flow, and physical forces, cilia are intricately involved in cell cycle regulation and preservation of DNA integrity, as their formation and resorption dynamics are tightly linked to cell cycle progression. Recently, several studies have linked defects in specific ciliary proteins to the DNA damage response. However, it remains unclear whether and how primary cilia contribute to cancer development. Mebendazole (MBZ) is an anthelmintic drug with anticancer properties in some cancer cells. MBZ is continuously being tested for clinical studies, but the precise mechanism of its anticancer activities remains unknown. Here, using Xenopus laevis embryos as a model system, we discovered that MBZ significantly hinders cilia formation and induces DNA damage. Remarkably, primary cilium-bearing cancer cells exhibited heightened vulnerability to combined treatment with MBZ and conventional anticancer drugs. Our findings shed light on the specific influence of MBZ on cilia, rather than cytosolic microtubules, in triggering DNA damage, elucidating a previously unidentified mechanism underlying potential MBZ-mediated cancer therapy.

Trisomy 21-driven metabolite alterations are linked to cellular injuries in Down syndrome

Down syndrome (DS) arises from a genetic anomaly characterized by an extra copy of chromosome 21 (exCh21). Despite high incidence of congenital diseases among DS patients, direct impacts of exCh21 remain elusive. Here, we established a robust DS model harnessing human-induced pluripotent stem cells (hiPSCs) from mosaic DS patient. These hiPSC lines encompassed both those with standard karyotype and those carrying an extra copy of exCh21, allowing to generate isogenic cell lines with a consistent genetic background. We unraveled that exCh21 inflicted disruption upon the cellular transcriptome, ushering in alterations in metabolic processes and triggering DNA damage. The impact of exCh21 was also manifested in profound modifications in chromatin accessibility patterns. Moreover, we identified two signature metabolites, 5-oxo-ETE and Calcitriol, whose biosynthesis is affected by exCh21. Notably, supplementation with 5-oxo-ETE promoted DNA damage, in stark contrast to the protective effect elicited by Calcitriol against such damage. We also found that exCh21 disrupted cardiogenesis, and that this impairment could be mitigated through supplementation with Calcitriol. Specifically, the deleterious effects of 5-oxo-ETE unfolded in the form of DNA damage induction and the repression of cardiogenesis. On the other hand, Calcitriol emerged as a potent activator of its nuclear receptor VDR, fostering amplified binding to chromatin and subsequent facilitation of gene transcription. Our findings provide a comprehensive understanding of exCh21’s metabolic implications within the context of Down syndrome, offering potential avenues for therapeutic interventions for Down syndrome treatment.

Exponential Amplification-Induced Activation of CRISPR/Cas9 for Sensitive Detection of Exosomal miRNA.

As an important component of highly heterogeneous exosomes, exosomal microRNAs (miRNAs) have great potential as noninvasive biomarkers for cancer diagnosis. Therefore, a sensitive and simple sensor is the key for its clinical application. Herein, we designed an exponential amplification reaction (EXPAR) to induce the reactivation of the CRISPR-associated protein 9/small guide RNA (Cas9/sgRNA) complex, thus achieving sensitive and visual exosomal miRNAs-21 (miR-21) fluorescence sensing. In this design, we inactivated the sgRNA by hybridizing sgRNA and blocker DNA. Then, we used a trigger DNA to hybridize with miR-21 and produced a lot of activated DNA by EXPAR. Those activated DNA further hybridized with blocker DNA and released the free sgRNA to form the activated Cas9/sgRNA complex. Based on the quick cleavage of activated Cas9/sgRNA complex, the reporter DNA labeled by SYBR Green I was released from the surface of the magnetic nanoparticles (MNPs) into the supernatant, and thus was used to sensitively quantify the miRNAs concentration with a limit of detection of 3 × 103 particles/mL. In addition, this fluorescence sensor has also been successfully employed to distinguish healthy people and cancer patients by naked-eye observation of the fluorescence, thus demonstrating its great potential for accurate and point-of-care cancer diagnosis.

A triggered DNA nanomachine with enzyme-free for the rapid detection of telomerase activity in a one-step method

BackgroundTelomerase is considered a biomarker for the early diagnosis and clinical treatment of cancer. The rapid and sensitive detection of telomerase activity is crucial to biological research, clinical diagnosis, and drug development. However, the main obstacles facing the current telomerase activity assay are the cumbersome and time-consuming procedure, the easy degradation of the telomerase RNA template and the need for additional proteases. Therefore, it is necessary to construct a new method for the detection of telomerase activity with easy steps, efficient reaction and strong anti-interference ability. ResultsHerein, an efficient, enzyme-free, economical, sensitive, fluorometric detection method for telomerase activity in one-step, named triggered-DNA (T-DNA) nanomachine, was created based on target-triggered DNAzyme-cleavage activity and catalytic molecular beacon (CMB). Telomerase served as a switch and extended few numbers of (TTAGGG)n repeat sequences to initiate the signal amplification in the T-DNA nanomachine, resulting in a strong fluorescent signal. The reaction was a one-step method with a shortened time of 1 h and a constant temperature of 37 °C, without the addition of any protease. It also sensitively distinguished telomerase activity in various cell lines. The T-DNA nanomachine offered a detection limit of 12 HeLa cells μL−1, 9 SK-Hep-1 cells μL−1 and 3 HuH-7 cells μL−1 with a linear correlation detection range of 0.39 × 102–6.25 × 102 HeLa cells μL−1 for telomerase activity. SignificanceIn conclusion, our study demonstrated that the triggered-DNA nanomachine fulfills the requirements for rapid detection of telomerase activity in one-step under isothermal and enzyme-free conditions with excellent specificity, and its simple and stable structure makes it ideal for complex systems. These findings indicated the application prospect of DNA nanomachines in clinical diagnostics and provided new insights into the field of DNA nanomachine-based bioanalysis.

Heavy Metal-Induced Assembly of DNA Network Biosensor from Double-Loop Hairpin Probes for Ultrasensitive Detection of UO22+ in Water and Soil Samples.

The uranyl ion (UO22+) is the most stable form of uranium, which exhibits high toxicity and bioavailability posing a severe risk to human health. The construction of ultrasensitive, reliable, and robust sensing techniques for UO22+ detection in water and soil samples remains a challenge. Herein, a DNA network biosensor was fabricated for UO22+ detection using DNAzyme as the heavy metal recognition element and double-loop hairpin probes as DNA assembly materials. UO22+-activated specific cleavage of the DNAzyme will liberate the triggered DNA fragment, which can be utilized to launch a double-loop hairpin probe assembly among Hab, Hbc, and Hca. Through multiple cyclic cross-hybridization reactions, hexagonal DNA duplex nanostructures (n[Hab•Hbc•Hca]) were formed. This DNA network sensing system generates a high fluorescence response for UO22+ monitoring. The biosensor is ultrasensitive, with a detection limit of 2 pM. This sensing system also displays an excellent selectivity and robustness, enabling the DNA network biosensor to work even in complex water and soil samples with excellent accuracy and reliability. With the advantages of enzyme-free operation, outstanding specificity, and high sensitivity, our proposed DNA network biosensor provides a reliable, simple, and robust method for trace levels of UO22+ detection in environmental samples.

Intelligent monitoring of the available lead (Pb) and cadmium (Cd) in soil samples based on half adder and half subtractor molecular logic gates

The available heavy metals in soil samples can cause the direct toxicity on ecosystems, plants, and human health. Traditional chemical extraction and recombinant bacterial methods for the available heavy metals assay often suffer from inaccuracy and poor specificity. In this work, we construct half adder and half subtractor molecular logic gates with molecular-level biocomputation capabilities for the intelligent sensing of the available lead (Pb) and cadmium (Cd). The available Pb and Cd can cleave DNAzyme sequences to release the trigger DNA, which can activate the hairpin probe assembly in the logic system. This multifunctional logic system can not only achieve the intelligent recognition of the available Pb and Cd according to the truth tables, but also can realize the simultaneous quantification with high sensitivity, with the detection limits of 2.8 pM and 25.6 pM, respectively. The logic biosensor is robust and has been applied to determination of the available Pb and Cd in soil samples with good accuracy and reliability. The relative error (Re) between the logic biosensor and the DTPA + ICP-MS method was from −8.1 % to 7.9 %. With the advantages of programmability, scalability, and multicomputing capacity, the molecular logic system can provide a simple, rapid, and smart method for intelligent monitoring of the available Pb and Cd in environmental samples.

Abstract B033: Direct and sensitive in-situ detection of DNA breaks by STRIDE and its potential as a new biomarker

Abstract DNA damage is usually detected using indirect methods which require active signaling and triggered DNA repair mechanisms. These methods, although relatively sensitive, are prone to false (positive or negative) results. Methods for direct DNA damage detection, on the other hand, suffer from low sensitivity or require DNA isolation, what can result in misleading artefactual readouts of the analysis. Here we present a method called STRIDE (SensiTive Recognition of Individual DNA Ends), which allows for specific, very sensitive in-situ detection of DNA breaks. This fluorescence-based method, in its two variants (dSTRIDE and sSTRIDE), allows selective labelling of double-or single-strand breaks, respectively. STRIDE enables highly sensitive and specific detection and quantification of single and double-strand DNA breaks in cultured cells and FFPE human tissues, both PBMCs as well as solid tumors. STRIDE is superior to classic methods used to measure DNA damage, as it combines a direct detection that is independent of DNA repair mechanisms with an unprecedented sensitivity. With its versatility it can be used in any type of biological material, including liquid biopsies as well as different types of tumors. STRIDE has the potential to become a very useful biomarker reporting about the efficiency of therapies based on DNA damage induction, such as DNA repair inhibitors or radiotherapy. Citation Format: Kamil Solarczyk, Brian Patterson. Direct and sensitive in-situ detection of DNA breaks by STRIDE and its potential as a new biomarker [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B033.

Interactions of insecticides with DNA–RNA bases and environmental conditions

The toxicological effects of dichlorodiphenyltrichloroethane (DDT) and the stability of methoprene under environmental conditions were investigated in this study by Density Functional Theory. DDT is known to be very persistent in the environment and accumulate in fatty tissues concluding with serious health issues such as birth defects, metabolic disorders, cancers, impaired immune and reproductive systems. Therefore results of the interactions between DDT and DNA–RNA bases were presented at the first part of this study. While there is no deterioration in the structure of DDT or DNA–RNA bases, DDT binds to DNA–RNA bases with high binding energy values which may cause aggregation or trigger DNA methylation. It is important in terms of explaining the interaction of DDT on the basis of DNA–RNA bases for the first time. In the second part, methoprene which is widely used as larvicide in the aquatic mosquito habitats was investigated. Because the chemical stability of the methoprene is an important phenomenon which might present a valuable information in mosquito control efforts, the effects of temperature and atom/molecules which are commonly exist in aquatic nature (Ca, Cl, Mg, Na, CO, CO2, CO3, H2, H2O, H2S, HCO3, N2, NH3, O2, SO4) on methoprene were presented. These atoms/molecules bind to methoprene with low binding energies however, the binding of the molecules to methoprene was disrupted by 313 K (40[Formula: see text]C).

Hybridization chain reaction-enhanced electrochemically mediated ATRP coupling high-efficient magnetic separation for electrochemical aptasensing of cardiac troponin I

The sensitive and accurate detection of cardiac troponin I (cTnI) as a gold biomarker for cardiovascular diseases at an early stage is crucial but has long been a challenge. In this study, we presented such an electrochemical (EC) aptasensor by combining hybridization chain reaction (HCR)-enhanced electrochemically mediated atom transfer radical polymerization (eATRP) amplification with high-efficient separation of magnetic beads (MBs). Aptamer-modified MBs empowered effective recognition and separation of cTnI from complex samples with high specificity. The specific binding of cTnI and aptamer could release triggered DNA (T-DNA) into solution to drive an HCR process, which produced plentiful active sites for eATRP initiators labeling followed by initiating eATRP process. With the development of eATRP, a great many of electroactive polymer probes were continually in situ formed to generate amplified current output for signal enhancement. Compared to no amplification, HCR-enhanced eATRP promoted the signals by ∼10-fold, greatly improving detection sensitivity for low-abundant cTnI analysis. Integrating MBs as capture carriers with HCR-enhanced eATRP as amplification strategy, this EC aptasensor achieved a low detection limit of 10.9 fg/mL for cTnI detection. Furthermore, the reliable detectability and anti-interference were confirmed in serum samples, indicating its promising application toward early diagnosis of cardiovascular diseases.

B-291 Proximity Labeling at Sites of Protein-Protein Interactions Using a Switchable DNA Catalyst

Abstract Background Extracellular protein-protein interactions (PPIs) play a crucial role in cell-cell adhesion, communication, and signaling. The dysregulation of PPIs in cancer caused by differential protein expression can drive oncogenic factors. Traditional methods to study PPIs disfavor weak or transient interacting protein partners, leaving them undiscovered. Proximity labeling (PL) is an emerging tool in which a catalyst is attached to a protein of interest (POI) and creates reactive species that tag nearby endogenous proteins. Tagged proteins are subsequently isolated and identified as potential interacting partners with the POI. If PL could be activated at a more specific subcellular location, such as the site of a PPI, more detailed information could be unveiled concerning the interactome of cancer-relevant PPIs. Recent advances in PL involve the use of a synthetic catalyst that can be localized to a POI via an antibody or ligand and avoids the need for PL enzyme-POI fusion proteins to be constructed, allowing for analysis of primary tissue samples. Methods To create a PL catalyst with pseudo-allosteric regulation, we created a switchable DNA catalyst. The DNA catalyst is composed of synthetic photocatalyst and quencher moieties covalently linked to DNA oligomers such that when hybridized together the photocatalyst is quenched and inactive. The DNA scaffold affords conformational control that is utilized to change the distance between photocatalyst and quencher, thus regulating photocatalyst activity. Split triggers composed of DNA oligos that bind to catalyst-DNA and simultaneously disrupt quencher-DNA hybridization were used to create an AND logic gate where both split triggers need to be in proximity to activate the PL catalyst. The split triggers were localized to POIs via connection to aptamers, short pieces of synthetic DNA that bind targets. Using the c-Met homodimer as a model system, we first labeled c-Met expressing HEK293T cells with aptamer-split trigger DNA oligomers as well as the quenched photocatalyst. After the catalyst was activated at the site of the PPI, we performed proximity labeling using a biotin-phenol substrate in the presence of an oxidizing agent and 450 nm light. Fluorescent streptavidin was used to visualize sites of biotinylation and detected using fluorescence microscopy. Results We observed extracellular biotin signal only of c-Met transfected cells as evidenced by the expression of a co-transfection plasmid encoding a fluorescent nuclear protein. When one of the split triggers had the aptamer motif removed, meaning it couldn’t be localized to the cell surface, no labeling was observed which shows that both split probes must be localized to the cell surface to activate labeling. Additionally, controls including omission of light, photocatalyst, or biotin phenol showed no labeling. Using a free-floating version of the photocatalyst to label cells, we confirmed that lysates contained biotinylated proteins via western blot. Conclusion We localized two split triggers to interacting proteins to activate a PL catalyst and performed proximity labeling at the site of the PPI. This technique demonstrates non-genetic split proximity labeling to enable downstream analysis of cancer cell proteomes in an ultra-specific manner and expands the toolkit for spatiotemporally controlled proximity labeling.