Neurophysiological Characteristics in Type II and Type III 5q Spinal Muscular Atrophy Patients: Impact of Nusinersen Treatment.

Objective: This study aimed to observe the neurophysiological characteristics of type II and type III 5q spinal muscular atrophy (SMA) patients and the changes in peripheral motor nerve electrophysiology after Nusinersen treatment, as well as the influencing factors. Methods: This single-center retrospective case-control study collected clinical data and peripheral motor nerve CMAP parameters from 42 5qSMA patients and 42 healthy controls at the Second Affiliated Hospital of Xi'an Jiaotong University (January 2021 to December 2022). It evaluated changes in motor function and CMAP amplitude before and after Nusinersen treatment. Results: Our investigation encompassed all symptomatic and genetically confirmed SMA patients, consisting of 32 type II and 10 type III cases, with a median age of 57 months (29.5 to 96 months). Comparative analysis with healthy controls revealed substantial reductions in CMAP amplitudes across various nerves in both type II and type III patients. Despite the administration of Nusinersen treatment for 6 or 14 months to the entire cohort, discernible alterations in motor nerve amplitudes were not observed, except for a significant improvement in younger patients (≤36 months) at the 14-month mark. Further scrutiny within the type II subgroup unveiled that individuals with a disease duration ≤12 months experienced a noteworthy upswing in femoral nerve amplitude, a statistically significant difference when compared to those with >12 months of disease duration. Conclusion: Motor nerve amplitudes were significantly decreased in type II and type III 5q SMA patients compared to healthy controls. Nusinersen treatment showed better improvement in motor nerve amplitudes in younger age groups and those with shorter disease duration, indicating a treatment-time dependence.

Cross-Reactive Polyclonal Antibodies Raised Against GalNAc-Conjugated siRNA Recognize Mostly the GalNAc Moiety.

Small interfering RNA (siRNA) is gaining momentum as a therapeutic modality with six approved products. Since siRNA has the potential to elicit undesired immune responses in patients, immunogenicity assessment is required during clinical development by regulatory authorities. In this study, anti-siRNA polyclonal antibodies were generated through animal immunization. These cross-reactive polyclonal antibodies recognized mostly the N-acetylgalactosamine (GalNAc) moiety with a small fraction against sequence-independent epitopes. We demonstrate that the polyclonal antibodies can be utilized as immunogenicity assay positive controls for the same class of GalNAc-conjugated siRNAs. In addition, anti-GalNAc mAbs showed desired sensitivity and drug tolerance, supporting their use as alternative surrogate positive controls. These findings can guide positive control selection and immunogenicity assay development for GalNAc-conjugated siRNAs and other oligonucleotide therapeutics.

Photoelectrochemical sensing of isoniazid and streptomycin based on metal Bi-doped BiOI microspheres grown on book-shape layers of Ti3C2 heterostructures.

Isoniazid and streptomycin are vital drugs for treating tuberculosis, which are utilized as efficient anti-tuberculosis agents. This paper presents a novel visible-light-driven composite photocatalyst Ti3C2/Bi/BiOI, which was built from Ti3C2 nanosheets and Bi/BiOI microspheres. Photoelectrochemical (PEC) sensors based on Ti3C2/Bi/BiOI were synthesized for isoniazid identification, which showed a linear concentration range of 0.1-125 µM with a detection limit of 0.05 µM (S/N = 3). Moreover, we designed a PEC aptasensors based on aptamer/Ti3C2/Bi/BiOI to detect streptomycin in 0.1 M PBS covering the electron donor isoniazid, because the isoniazid consumes photogenerated holes thus increasing the photocurrent effectively and preventing photogenerated electron-hole pairs from being recombined. Furthermore, PEC aptasensors based on aptamer/Ti3C2/Bi/BiOI were synthesized for streptomycin identification, which exhibited a linear concentration range of 0.01-1000 nM with a detection limit of 2.3 × 10-3 nM (S/N = 3), and are well stable in streptomycin sensing.

Recent advances in the synthesis of single-stranded DNA in vitro.

Single-stranded DNA (ssDNA) is the foundation of modern biology, with wide applications in gene editing, sequencing, DNA information storage, and materials science. However, synthesizing ssDNA with high efficiency, high throughput, and low error rate in vitro remains a major challenge. Various methods have been developed for ssDNA synthesis, and some significant results have been achieved. In this review, six main methods were introduced, including solid-phase oligonucleotide synthesis, terminal deoxynucleotidyl transferase-based ssDNA synthesis, reverse transcription, primer exchange reaction, asymmetric polymerase chain reaction, and rolling circle amplification. The advantages and limitations of each method were compared, as well as illustrate their representative achievements and applications. Especially, rolling circle amplification has received significant attention, including ssDNA synthesis, assembly, and application based on recent work. Finally, the future challenges and opportunities of ssDNA synthesis were summarized and discussed. Envisioning the development of new methods and significant progress will be made in the near future with the efforts of scientists around the world.

IMPATIENT-qPCR: monitoring SELEX success during in vitro aptamer evolution.

SELEX (Systematic Evolution of Ligands by Exponential enrichment) processes aim on the evolution of high-affinity aptamers as binding entities in diagnostics and biosensing. Aptamers can represent game-changers as constituents of diagnostic assays for the management of instantly occurring infectious diseases or other health threats. Without in-process quality control measures SELEX suffers from low overall success rates. We present a quantitative PCR method for fast and easy quantification of aptamers bound to their targets. Simultaneous determination of melting temperatures (Tm) of each SELEX round delivers information on the evolutionary success via the correlation of increasing GC content and Tm alone with a round-wise increase of aptamer affinity to the respective target. Based on nine successful and published previous SELEX processes, in which the evolution/selection of aptamer affinity/specificity was demonstrated, we here show the functionality of the IMPATIENT-qPCR for polyclonal aptamer libraries and resulting individual aptamers. Based on the ease of this new evolution quality control, we hope to introduce it as a valuable tool to accelerate SELEX processes in general. IMPATIENT-qPCR SELEX success monitoring. Selection and evolution of high-affinity aptamers using SELEX technology with direct aptamer evolution monitoring using melting curve shifting analyses to higher Tm by quantitative PCR with fluorescence dye SYBR Green I. KEY POINTS: • Fast and easy analysis. • Universal applicability shown for a series of real successful projects.

Real-life experience with inotersen at CEPARM, Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro.

BACKGROUND: Hereditary transthyretin amyloidosis (ATTRv) is an inherited, progressive, and fatal disease still largely underdiagnosed. Mutations in the transthyretin (TTR) gene cause the TTR protein to destabilize, misfold, aggregate, and deposit in body tissues, which makes ATTRv a disease with heterogeneous clinical phenotype. OBJECTIVE: To describe the long-term efficacy and safety of inotersen therapy in patients with ATTRv peripheral neuropathy (ATTRv-PN). METHODS: Patients who completed the NEURO-TTR pivotal study and the NEURO-TTR OLE open-label extension study migrated to the present study and were followed-up for at least 18 more months to an average of 67 months and up to 76 months since day 1 of the inotersen therapy (D1-first dose of inotersen). Disease progression was evaluated by standard measures. RESULTS: Ten ATTRv-PN patients with Val30Met mutation were included. The mean disease duration on D1 was of 3 years, and the mean age of the patients was of 46.8 years. During an additional 18-month follow up, neurological function, based on the Neuropathy Impairment Score and the Polyneuropathy Disability Score, functionality aspects (Karnofsky Performance Status), and nutritional and cardiac aspects were maintained. No new safety signs have been noted. CONCLUSION: The treatment with inotersen was effective and well tolerated for the average of 67 months and up to 76 months. Our results are consistent with those of larger phase-III trials.

ANTECEDENTES: Amiloidose hereditária por transtirretina (ATTRv) é uma doença hereditária, progressiva e fatal ainda largamente subdiagnosticada. Mutações no gene transtirretina (TTR) promovem desestabilização, desdobramento, agregação e depósito da proteína TTR em tecidos do corpo, o que faz da ATTRv uma doença de fenótipo clínico heterogêneo. OBJETIVO: Descrever a eficácia e segurança da terapia com inotersena no longo prazo em pacientes com neuropatia periférica ATTRv (ATTRv-PN). MéTODOS: Pacientes que completaram o estudo pivotal NEURO-TTR e o estudo de extensão aberta NEURO-TTR OLE migraram para este estudo e foram acompanhados por no mínimo 18 meses adicionais, em média por 67 meses, e por até 76 meses, desde o dia 1 da terapia com inotersena (D1­primeira dose de inotersena). A progressão da doença foi avaliada por medidas padronizadas. RESULTADOS: Dez pacientes com ATTRv-PN com mutação Val30Met foram incluídos. A duração média da doença no D1 era de 3 anos, e a média de idade dos pacientes era de 46,8 anos. Durante o período de acompanhamento adicional de 18 meses, a função neurológica, baseada no Neuropathy Impairment Score e no Polyneuropathy Disability Score, os aspectos de funcionalidade (Karnofsky Performance Status), nutricional e cardíacos estavam mantidos. Não se observou nenhum novo sinal de segurança. CONCLUSãO: O tratamento com inotersena foi eficaz e bem tolerado por 67 meses em média, e por até 76 meses. Nossos resultados são consistentes com os de estudos maiores de fase III.

Mixed insect pest populations of Diaspididae species under control of oligonucleotide insecticides: 3'-end nucleotide matters.

Diaspididae are one of the most serious small herbivorous insects with piercing-sucking mouth parts and are major economic pests as they attack and destroy perennial ornamentals and food crops. Chemical control is the primary management approach for armored scale infestation. However, chemical insecticides do not possess selectivity in action and not always effective enough for the control of armored scale insects. Our previous work showed that green oligonucleotide insecticides (olinscides) are highly effective against armored and soft scale insects. Moreover, olinscides possess affordability, selectivity in action, fast biodegradability, and a low carbon footprint. Insect pest populations undergo microevolution and olinscides should take into account the problem of insecticide resistance. Using sequencing results, it was found that in the mixed populations of insect pests Dynaspidiotus britannicus Newstead and Aonidia lauri Bouche, predominates the population of A. lauri. Individuals of A. lauri comprised for 80% of individuals with the sequence 3'-ATC-GTT-GGC-AT-5' in the 28S rRNA site, and 20% of the population comprised D. britannicus individuals with the sequence 3'-ATC-GTC-GGT-AT-5'. We created olinscides Diasp80-11 (5'-ATG-CCA-ACG-AT-3') and Diasp20-11 (5'-ATA-CCG-ACG-AT-3') with perfect complementarity to each of the sequences. Mortality of insects on the 14th day comprised 98.19 ± 3.12% in Diasp80-11 group, 64.66 ± 0.67% in Diasp20-11 group (p < 0.05), and 3.77 ± 0.94% in the control group. Results indicate that for maximum insecticidal effect it is necessary to use an oligonucleotide insecticide that corresponds to the dominant species. Mortality in Diasp80-11 group was accompanied with significant decrease in target 28S rRNA concentration and was 8.44 ± 0.14 and 1.72 ± 0.36 times lower in comparison with control (p < 0.05) on the 10th and 14th days, respectively. We decided to make single nucleotide substitutions in Diasp20-11 olinscide to understand which nucleotide will play the most important role in insecticidal effect. We created three sequences with single nucleotide transversion substitutions at the 5'-end - Diasp20(5')-11 (A to T), 3'-end - Diasp20(3')-11 (T to A), and in the middle of the sequence - Diasp20(6)-11 (6th nitrogenous base of the sequence; G to C), respectively. As a result, mortality of mixed population of the field experiment decreased and comprised 53.89 ± 7.25% in Diasp20(5')-11 group, 40.68 ± 4.33% in Diasp20(6)-11 group, 35.74 ± 5.51% in Diasp20(3')-11 group, and 3.77 ± 0.94% in the control group on the 14th day. Thus, complementarity of the 3'-end nucleotide to target 28S rRNA was the most important for pronounced insecticidal effect (significance of complementarity of nucleotides for insecticidal effect: 5' nt < 6 nt < 3' nt). As was found in our previous research works, the most important rule to obtain maximum insecticidal effect is complete complementarity to the target rRNA sequence and maximum coverage of target sequence in insect pest populations. However, in this article we also show that the complementarity of 3'-end is a second important factor for insecticidal potential of olinscides. Also in this article we propose 2-step DNA containment mechanism of action of olinscides, recruiting RNase H. The data obtained indicate the selectivity of olinscides and at the same time provide a simple and flexible platform for the creation of effective plant protection products, based on antisense DNA oligonucleotides.

Human Rad51 Protein Requires Higher Concentrations of Calcium Ions for D-Loop Formation than for Oligonucleotide Strand Exchange.

Human Rad51 protein (HsRad51)-promoted DNA strand exchange, a crucial step in homologous recombination, is regulated by proteins and calcium ions. Both the activator protein Swi5/Sfr1 and Ca2+ ions stimulate different reaction steps and induce perpendicular DNA base alignment in the presynaptic complex. To investigate the role of base orientation in the strand exchange reaction, we examined the Ca2+ concentration dependence of strand exchange activities and structural changes in the presynaptic complex. Our results show that optimal D-loop formation (strand exchange with closed circular DNA) required Ca2+ concentrations greater than 5 mM, whereas 1 mM Ca2+ was sufficient for strand exchange between two oligonucleotides. Structural changes indicated by increased fluorescence intensity of poly(dεA) (a poly(dA) analog) reached a plateau at 1 mM Ca2+. Ca2+ > 2 mM was required for saturation of linear dichroism signal intensity at 260 nm, associated with rigid perpendicular DNA base orientation, suggesting a correlation with the stimulation of D-loop formation. Therefore, Ca2+ exerts two different effects. Thermal stability measurements suggest that HsRad51 binds two Ca2+ ions with KD values of 0.2 and 2.5 mM, implying that one step is stimulated by one Ca2+ bond and the other by two Ca2+ bonds. Our results indicate parallels between the Mg2+ activation of RecA and the Ca2+ activation of HsRad51.

Identification of Dhx15 as a Major Regulator of Liver Development, Regeneration, and Tumor Growth in Zebrafish and Mice.

RNA helicase DHX15 plays a significant role in vasculature development and lung metastasis in vertebrates. In addition, several studies have demonstrated the overexpression of DHX15 in the context of hepatocellular carcinoma. Therefore, we hypothesized that this helicase may play a significant role in liver regeneration, physiology, and pathology. Dhx15 gene deficiency was generated by CRISPR/Cas9 in zebrafish and by TALEN-RNA in mice. AUM Antisense-Oligonucleotides were used to silence Dhx15 in wild-type mice. The hepatocellular carcinoma tumor induction model was generated by subcutaneous injection of Hepa 1-6 cells. Homozygous Dhx15 gene deficiency was lethal in zebrafish and mouse embryos. Dhx15 gene deficiency impaired liver organogenesis in zebrafish embryos and liver regeneration after partial hepatectomy in mice. Also, heterozygous mice presented decreased number and size of liver metastasis after Hepa 1-6 cells injection compared to wild-type mice. Dhx15 gene silencing with AUM Antisense-Oligonucleotides in wild-type mice resulted in 80% reduced expression in the liver and a significant reduction in other major organs. In addition, Dhx15 gene silencing significantly hindered primary tumor growth in the hepatocellular carcinoma experimental model. Regarding the potential use of DHX15 as a diagnostic marker for liver disease, patients with hepatocellular carcinoma showed increased levels of DHX15 in blood samples compared with subjects without hepatic affectation. In conclusion, Dhx15 is a key regulator of liver physiology and organogenesis, is increased in the blood of cirrhotic and hepatocellular carcinoma patients, and plays a key role in controlling hepatocellular carcinoma tumor growth and expansion in experimental models.

MicroRNA Expression Profiles in Human Samples and Cell Lines Revealed Nine miRNAs Associated with Cisplatin Resistance in High-Grade Serous Ovarian Cancer.

Metastasis and drug resistance are major contributors to cancer-related fatalities worldwide. In ovarian cancer (OC), a staggering 70% develop resistance to the front-line therapy, cisplatin. Despite proposed mechanisms, the molecular events driving cisplatin resistance remain unclear. Dysregulated microRNAs (miRNAs) play a role in OC initiation, progression, and chemoresistance, yet few studies have compared miRNA expression in OC samples and cell lines. This study aimed to identify key miRNAs involved in the cisplatin resistance of high-grade-serous-ovarian-cancer (HGSOC), the most common gynecological malignancy. MiRNA expression profiles were conducted on RNA isolated from formalin-fixed-paraffin-embedded human ovarian tumor samples and HGSOC cell lines. Nine miRNAs were identified in both sample types. Targeting these with oligonucleotide miRNA inhibitors (OMIs) reduced proliferation by more than 50% for miR-203a, miR-96-5p, miR-10a-5p, miR-141-3p, miR-200c-3p, miR-182-5p, miR-183-5p, and miR-1206. OMIs significantly reduced migration for miR-183-5p, miR-203a, miR-296-5p, and miR-1206. Molecular pathway analysis revealed that the nine miRNAs regulate pathways associated with proliferation, invasion, and chemoresistance through PTEN, ZEB1, FOXO1, and SNAI2. High expression of miR-1206, miR-10a-5p, miR-141-3p, and miR-96-5p correlated with poor prognosis in OC patients according to the KM plotter database. These nine miRNAs could be used as targets for therapy and as markers of cisplatin response.

Neurodegeneration Biomarkers in Adult Spinal Muscular Atrophy (SMA) Patients Treated with Nusinersen.

The objective of this study is to evaluate biomarkers for neurodegenerative disorders in adult SMA patients and their potential for monitoring the response to nusinersen. Biomarkers for neurodegenerative disorders were assessed in plasma and CSF samples obtained from a total of 30 healthy older adult controls and 31 patients with adult SMA type 2 and 3. The samples were collected before and during nusinersen treatment at various time points, approximately at 2, 6, 10, and 22 months. Using ELISA technology, the levels of total tau, pNF-H, NF-L, sAPPß, Aß40, Aß42, and YKL-40 were evaluated in CSF samples. Additionally, plasma samples were used to measure NF-L and total tau levels using SIMOA technology. SMA patients showed improvements in clinical outcomes after nusinersen treatment, which were statistically significant only in walkers, in RULM (p = 0.04) and HFMSE (p = 0.05) at 24 months. A reduction in sAPPß levels was found after nusinersen treatment, but these levels did not correlate with clinical outcomes. Other neurodegeneration biomarkers (NF-L, pNF-H, total tau, YKL-40, Aß40, and Aß42) were not found consistently changed with nusinersen treatment. The slow progression rate and mild treatment response of adult SMA types 2 and 3 may not lead to detectable changes in common markers of axonal degradation, inflammation, or neurodegeneration, since it does not involve large pools of damaged neurons as observed in pediatric forms. However, changes in biomarkers associated with the APP processing pathway might be linked to treatment administration. Further studies are warranted to better understand these findings.

Comprehensive fluorescence profiles of contamination-prone foods applied to the design of microcontact-printed in situ functional oligonucleotide sensors.

With both foodborne illness and food spoilage detrimentally impacting human health and the economy, there is growing interest in the development of in situ sensors that offer real-time monitoring of food quality within enclosed food packages. While oligonucleotide-based fluorescent sensors have illustrated significant promise, the development of such on-food sensors requires consideration towards sensing-relevant fluorescence properties of target food products-information that has not yet been reported. To address this need, comprehensive fluorescence profiles for various contamination-prone food products are established in this study across several wavelengths and timepoints. The intensity of these food backgrounds is further contextualized to biomolecule-mediated sensing using overlaid fluorescent oligonucleotide arrays, which offer perspective towards the viability of distinct wavelengths and fluorophores for in situ food monitoring. Results show that biosensing in the Cyanine3 range is optimal for all tested foods, with the Cyanine5 range offering comparable performance with meat products specifically. Moreover, recognizing that mass fabrication of on-food sensors requires rapid and simple deposition of sensing agents onto packaging substrates, RNA-cleaving fluorescent nucleic acid probes are successfully deposited via microcontact printing for the first time. Direct incorporation onto food packaging yields cost-effective sensors with performance comparable to ones produced using conventional deposition strategies.

Modern approaches to therapeutic oligonucleotide manufacturing.

Therapeutic oligonucleotides are a powerful drug modality with the potential to treat many diseases. The rapidly growing number of therapies that have been approved and that are in advanced clinical trials will place unprecedented demands on our capacity to manufacture oligonucleotides at scale. Existing methods based on solid-phase phosphoramidite chemistry are limited by their scalability and sustainability, and new approaches are urgently needed to deliver the multiton quantities of oligonucleotides that are required for therapeutic applications. The chemistry community has risen to the challenge by rethinking strategies for oligonucleotide production. Advances in chemical synthesis, biocatalysis, and process engineering technologies are leading to increasingly efficient and selective routes to oligonucleotide sequences. We review these developments, along with remaining challenges and opportunities for innovations that will allow the sustainable manufacture of diverse oligonucleotide products.

Monitoring Real-Time Temperature Dynamics of a Short RNA Hairpin Using Förster Resonance Energy Transfer and Circular Dichroism.

RNA molecules play crucial roles in gene expression regulation and cellular signaling, and these functions are governed by the formation of RNA secondary and tertiary structures. These structures are highly dynamic and subject to rapid changes in response to environmental cues, temperature in particular. Thermosensitive RNA secondary structures have been harnessed by multiple organisms to survey their temperature environment and to adjust gene expression accordingly. It is thus highly desirable to observe RNA structural changes in real time over a range of temperatures. Multiple approaches have been developed to study structural dynamics, but many of these require extensive processing of the RNA, large amounts of RNA input, and/or cannot be applied under physiological conditions. Here, we describe the use of a dually fluorescently labeled RNA oligonucleotide (containing a predicted hairpin structure) to monitor subtle RNA structural dynamics in vitro by Förster resonance energy transfer (FRET) and circular dichroism (CD) spectroscopy. These approaches can be employed under physiologically relevant conditions over a range of temperatures and with RNA concentrations as low as 200 nM; they enable us to observe RNA structural dynamics in real time and to correlate these dynamics with changes in biological processes such as translation.

An RNA origami robot that traps and releases a fluorescent aptamer.

RNA nanotechnology aims to use RNA as a programmable material to create self-assembling nanodevices for application in medicine and synthetic biology. The main challenge is to develop advanced RNA robotic devices that both sense, compute, and actuate to obtain enhanced control over molecular processes. Here, we use the RNA origami method to prototype an RNA robotic device, named the "Traptamer," that mechanically traps the fluorescent aptamer, iSpinach. The Traptamer is shown to sense two RNA key strands, acts as a Boolean AND gate, and reversibly controls the fluorescence of the iSpinach aptamer. Cryo-electron microscopy of the closed Traptamer structure at 5.45-angstrom resolution reveals the mechanical mode of distortion of the iSpinach motif. Our study suggests a general approach to distorting RNA motifs and a path forward to build sophisticated RNA machines that through sensing, computing, and actuation modules can be used to precisely control RNA functionalities in cellular systems.

Sensing Characteristics of SARS-CoV-2 Spike Protein Using Aptamer-Functionalized Si-Based Electrolyte-Gated Field-Effect Transistor (EGT).

The sensing responses of SARS-CoV-2 spike protein using top-down-fabricated Si-based electrolyte-gated transistors (EGTs) have been investigated. An aptamer was employed as a receptor for the SARS-CoV-2 spike protein. The EGT demonstrated excellent intrinsic characteristics and higher sensitivity in the subthreshold regime compared to the linear regime. The limit of detection (LOD) was achieved as low as 0.94 pg/mL and 20 pg/mL for the current and voltage sensitivity, respectively. To analyze the sensing responses of EGT in detecting the aptamer-SARS-CoV-2 spike protein conjugate, a lumped-capacitive model with the presence of an effective dipole potential and an effective capacitance of the functionalized layer component was employed. The aptamer-functionalized EGT showed high sensitivity even in 10 mM phosphate-buffered saline (PBS) solution. These results suggest that Si-based EGTs are a highly promising method for detecting SARS-CoV-2 spike proteins.

G4-Ligand-Conjugated Oligonucleotides Mediate Selective Binding and Stabilization of Individual G4 DNA Structures.

G-quadruplex (G4) DNA structures are prevalent secondary DNA structures implicated in fundamental cellular functions, such as replication and transcription. Furthermore, G4 structures are directly correlated to human diseases such as cancer and have been highlighted as promising therapeutic targets for their ability to regulate disease-causing genes, e.g., oncogenes. Small molecules that bind and stabilize these structures are thus valuable from a therapeutic perspective and helpful in studying the biological functions of the G4 structures. However, there are hundreds of thousands of G4 DNA motifs in the human genome, and a long-standing problem in the field is how to achieve specificity among these different G4 structures. Here, we developed a strategy to selectively target an individual G4 DNA structure. The strategy is based on a ligand that binds and stabilizes G4s without selectivity, conjugated to a guide oligonucleotide, that specifically directs the G4-Ligand-conjugated oligo (GL-O) to the single target G4 structure. By employing various biophysical and biochemical techniques, we show that the developed method enables the targeting of a unique, specific G4 structure without impacting other off-target G4 formations. Considering the vast amount of G4s in the human genome, this represents a promising strategy to study the presence and functions of individual G4s but may also hold potential as a future therapeutic modality.

PsAF5 functions as an essential adapter for PsPHB2-mediated mitophagy under ROS stress in Phytophthora sojae.

Host-derived reactive oxygen species (ROS) are an important defense means to protect against pathogens. Although mitochondria are the main intracellular targets of ROS, how pathogens regulate mitochondrial physiology in response to oxidative stress remains elusive. Prohibitin 2 (PHB2) is an inner mitochondrial membrane (IMM) protein, recognized as a mitophagy receptor in animals and fungi. Here, we find that an ANK and FYVE domain-containing protein PsAF5, is an adapter of PsPHB2, interacting with PsATG8 under ROS stress. Unlike animal PHB2 that can recruit ATG8 directly to mitochondria, PsPHB2 in Phytophthora sojae cannot recruit PsATG8 to stressed mitochondria without PsAF5. PsAF5 deletion impairs mitophagy under ROS stress and increases the pathogen's sensitivity to H2O2, resulting in the attenuation of P. sojae virulence. This discovery of a PsPHB2-PsATG8 adapter (PsAF5) in plant-pathogenic oomycetes reveals that mitophagy induction by IMM proteins is conserved in eukaryotes, but with differences in the details of ATG8 recruitment.

Mercury (II) sensing using a simple turn-on fluorescent graphene oxide based aptasensor in serum and water samples.

A simple, highly sensitive, and selective fluorometric aptasensing platform based on aptamer and graphene oxide (GO) is proposed for the determination of mercury (II) ion (Hg2+). In the designed assay, two aptamer probes, a carboxy-fluorescein (FAM) labeled aptamer (aptamer A) and its complementary (aptamer B) with partial complement containing several mismatches and GO as the quencher were used. In the absence of Hg2+, both A and B aptamers were adsorbed on the surface of GO by π-π-stacking, leading to fluorescence quenching of FAM due to fluorescence resonance energy transfer (FRET). Upon exposure to Hg2+, the A and B aptamer strands bind Hg2+ and form T-Hg2+-T complexes, leading to the formation of a stable double-stranded aptamer. The double-stranded aptamer is detached from the GO surface, resulting in the recovery of FAM fluorescence. The fluorescence intensity (FI) of the developed sensor was correlated with the Hg2+ concentration under optimized experimental conditions in two wide linear ranges, even in the presence of 10 divalent cations as interferences. The linear ranges were obtained from 200.0 to 900.0 fM and 5.0 to 33.0 pM, a limit of detection (LOD) of 106.0 fM, and a limit of quantification (LOQ) of 321.3 fM. The concentration of Hg2+ was determined in five real samples containing three water and two serum samples, using spiking and standard addition methods and the results were compared with the spiked amounts and atomic absorption (AAS) as standard method respectively, with acceptable recoveries. Furthermore, in the standard addition method, to overcome the effects of matrix influence of real samples in quantitative predictions, the excitation-emission matrix (EEM) data for samples was simultaneously analyzed by multivariate curve resolution with alternating least squares (MCR-ALS) as a second-order standard addition method (SOSAM).