RNA Repair: Hiding in Plain Sight.

Enzymes that phosphorylate, dephosphorylate, and ligate RNA 5' and 3' ends were discovered more than half a century ago and were eventually shown to repair purposeful site-specific endonucleolytic breaks in the RNA phosphodiester backbone. The pace of discovery and characterization of new candidate RNA repair activities in taxa from all phylogenetic domains greatly exceeds our understanding of the biological pathways in which they act. The key questions anent RNA break repair in vivo are (a) identifying the triggers, agents, and targets of RNA cleavage and (b) determining whether RNA repair results in restoration of the original RNA, modification of the RNA (by loss or gain at the ends), or rearrangements of the broken RNA segments (i.e., RNA recombination). This review provides a perspective on the discovery, mechanisms, and physiology of purposeful RNA break repair, highlighting exemplary repair pathways (e.g., tRNA restriction-repair and tRNA splicing) for which genetics has figured prominently in their elucidation.

A cooperative PNPase-Hfq-RNA carrier complex facilitates bacterial riboregulation.

Polynucleotide phosphorylase (PNPase) is an ancient exoribonuclease conserved in the course of evolution and is found in species as diverse as bacteria and humans. Paradoxically, Escherichia coli PNPase can act not only as an RNA degrading enzyme but also by an unknown mechanism as a chaperone for small regulatory RNAs (sRNAs), with pleiotropic consequences for gene regulation. We present structures of the ternary assembly formed by PNPase, the RNA chaperone Hfq, and sRNA and show that this complex boosts sRNA stability in vitro. Comparison of structures for PNPase in RNA carrier and degradation modes reveals how the RNA is rerouted away from the active site through interactions with Hfq and the KH and S1 domains. Together, these data explain how PNPase is repurposed to protect sRNAs from cellular ribonucleases such as RNase E and could aid RNA presentation to facilitate regulatory actions on target genes.

PRC2 direct transfer from G-quadruplex RNA to dsDNA has implications for RNA-binding chromatin modifiers.

The chromatin-modifying enzyme, Polycomb Repressive Complex 2 (PRC2), deposits the H3K27me3 epigenetic mark to negatively regulate expression at numerous target genes, and this activity has been implicated in embryonic development, cell differentiation, and various cancers. A biological role for RNA binding in regulating PRC2 histone methyltransferase activity is generally accepted, but the nature and mechanism of this relationship remains an area of active investigation. Notably, many in vitro studies demonstrate that RNA inhibits PRC2 activity on nucleosomes through mutually antagonistic binding, while some in vivo studies indicate that PRC2's RNA-binding activity is critical for facilitating its biological function(s). Here we use biochemical, biophysical, and computational approaches to interrogate PRC2's RNA and DNA-binding kinetics. Our findings demonstrate that PRC2-polynucleotide dissociation rates are dependent on the concentration of free ligand, indicating the potential for direct transfer between nucleic acid ligands without a free-enzyme intermediate. Direct transfer explains the variation in previously reported dissociation kinetics, allows reconciliation of prior in vitro and in vivo studies, and expands the potential mechanisms of RNA-mediated PRC2 regulation. Moreover, simulations indicate that such a direct transfer mechanism could be obligatory for RNA to recruit proteins to chromatin.

The Bacterial Ro60 Protein and Its Noncoding Y RNA Regulators.

Ro60 ribonucleoproteins (RNPs), composed of the ring-shaped Ro 60-kDa (Ro60) protein and noncoding RNAs called Y RNAs, are present in all three domains of life. Ro60 was first described as an autoantigen in patients with rheumatic disease, and Ro60 orthologs have been identified in 3% to 5% of bacterial genomes, spanning the majority of phyla. Their functions have been characterized primarily in Deinococcus radiodurans, the first sequenced bacterium with a recognizable ortholog. In D. radiodurans, the Ro60 ortholog enhances the ability of 3'-to-5' exoribonucleases to degrade structured RNA during several forms of environmental stress. Y RNAs are regulators that inhibit or allow the interactions of Ro60 with other proteins and RNAs. Studies of Ro60 RNPs in other bacteria hint at additional functions, since the most conserved Y RNA contains a domain that is a close tRNA mimic and Ro60 RNPs are often encoded adjacent to components of RNA repair systems.

Target recognition by RNase E RNA-binding domain AR2 drives sRNA decay in the absence of PNPase.

The C-terminal domain (CTD) of the major endoribonuclease RNase E not only serves as a scaffold for the central RNA decay machinery in gram-negative bacteria but also mediates coupled degradation of small regulatory RNAs (sRNAs) and their cognate target transcripts following RNA chaperone Hfq-facilitated sRNA-mRNA base pairing. Despite the crucial role of RNase E CTD in sRNA-dependent gene regulation, the contribution of particular residues within this domain in recruiting sRNAs and mRNAs upon base pairing remains unknown. We have previously shown that in Escherichia coli, the highly conserved 3'-5'-exoribonuclease polynucleotide phosphorylase (PNPase) paradoxically stabilizes sRNAs by limiting access of RNase E to Hfq-bound sRNAs and by degrading target mRNA fragments that would otherwise promote sRNA decay. Here, we report that in the absence of PNPase, the RNA-binding region AR2 in the CTD is required for RNase E to initiate degradation of the Hfq-dependent sRNAs CyaR and RyhB. Additionally, we show that introducing mutations in either hfq that disrupts target mRNA binding to Hfq or the AR2 coding region of rne impairs RNase E binding to sRNAs. Altogether, our data support a model where sRNAs are recruited via bound mRNA targets to RNase E by its AR2 domain after Hfq catalyzes sRNA-mRNA pairing. These results also support our conclusion that in a PNPase-deficient strain, more rapid decay of sRNAs occurs due to accelerated pairing with mRNA targets as a consequence of their accumulation. Our findings provide insights into the mechanisms by which sRNAs and mRNAs are regulated by RNase E.

Cryo-EM structures of Mycobacterium tuberculosis polynucleotide phosphorylase suggest a potential mechanism for its RNA substrate degradation.

As one of the oldest infectious diseases in the world, tuberculosis (TB) is the second most deadly infectious disease after COVID-19. Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), which can attack various organs of the human body. Up to now, drug-resistant TB continues to be a public health threat. Pyrazinamide (PZA) is regarded as a sterilizing drug in the treatment of TB due to its distinct ability to target Mtb persisters. Previously we demonstrated that a D67N mutation in Mycobacterium tuberculosis polynucleotide phosphorylase (MtbPNPase, Rv2783c) confers resistance to PZA and Rv2783c is a potential target for PZA, but the mechanism leading to PZA resistance remains unclear. To gain further insight into the MtbPNPase, we determined the cryo-EM structures of apo Rv2783c, its mutant form and its complex with RNA. Our studies revealed the Rv2783c structure at atomic resolution and identified its enzymatic functional groups essential for its phosphorylase activities. We also investigated the molecular mechanisms underlying the resistance to PZA conferred by the mutation. Our research findings provide structural and functional insights enabling the development of new anti-tuberculosis drugs.

High-resolution 3-D scanning electron microscopy (SEM) images of DOTTM polynucleotides (PN): Unique scaffold characteristics and potential applications in biomedicine.

INTRODUCTION: Polynucleotides (PN) are becoming more prominent in aesthetic medicine. However, the structural characteristics of PN have not been published and PN from different companies may have different structural characteristics. This study aimed to elucidate the structural attributes of DOT™ PN and distinguish differences with polydeoxyribonucleotides (PDRN) using high-resolution scanning electron microscopy (SEM) imaging. MATERIALS AND METHODS: DOT™ PN was examined using a Quanta 3-D field emission gun (FEG) Scanning Electron Microscope (SEM). Sample preparation involved cryogenic cooling, cleavage, etching, and metal coating to facilitate high-resolution imaging. Cryo-FIB/SEM techniques were employed for in-depth structural analysis. RESULTS: PDRN exhibited an amorphous structure without distinct features. In contrast, DOT™ PN displayed well-defined polyhedral shapes with smooth, uniformly thick walls. These cells were empty, with diameters ranging from 3 to 8 micrometers, forming a seamless tessellation pattern. DISCUSSION: DOT™ PN's distinct geometric tessellation design conforms to the principles of biotensegrity, providing both structural reinforcement and integrity. The presence of delicate partitions and vacant compartments hints at possible uses in the field of pharmaceutical delivery systems. Within the realms of beauty enhancement and regenerative medicine, DOT™ PN's capacity to bolster cell growth and tissue mending could potentially transform approaches to rejuvenation treatments. Its adaptability becomes apparent when considering its contributions to drug administration and surgical procedures. CONCLUSION: This study unveils the intricate structural scaffold features of DOT™ PN for the first time, setting it apart from PDRN and inspiring innovation in biomedicine and materials science. DOT™ PN's unique attributes open doors to potential applications across healthcare and beyond.

The Effect of Polynucleotide-Hyaluronic Acid Hydrogel in the Recovery After Mechanical Skin Barrier Disruption.

BACKGROUND: The epidermal barrier acts as a defense against external agents as well as helps to maintain body homeostasis. Polynucleotides (PN), exogenous DNA fragments, promote wound repair through their stimulatory and anti-inflammatory effects. Recent findings indicate a synergistic effect of PN and hyaluronic acid (HA) combinations in regulating inflammation and promoting cell proliferation. This study aims to elucidate the effects of PN and HA on repairing the epidermal barrier following its disruption by tape stripping (TS) in a mouse model. MATERIALS AND METHODS: After disrupting the epidermal barrier using TS, a formulation containing PN (14 mg/mL) and HA (6 mg/mL) was applied. Trans-epidermal water loss (TEWL) was measured at 0, 3, 6, 24, 48, and 72 h. Mice were euthanized after the final application at 72 h, and tissue samples were analyzed for epidermal/dermal thickness, neutrophil infiltration, and filaggrin expression. RESULTS: We observed a significant reduction in TEWL in the PN+HA group compared to that in the control group (20.8 ± 0.5 vs. 43.7 ± 0.5 g/m2h at 72 h, p < 0.05), indicating an improvement in barrier function. Histological evaluation showed decreased epidermal and dermal thickening in the PN+HA group compared to that in the control group (epidermal: 29.4 ± 2.2 vs. 57.9 ± 3.5 µm; dermal: 464.8 ± 25.9 vs. 825.9 ± 44.8 µm, both p < 0.05). Additionally, neutrophil infiltration in the dermis was significantly reduced, and filaggrin protein levels were significantly higher in the PN+HA group compared to those in the control group (4.8 ± 0.4 vs. 21.1 ± 3.3 for neutrophils; 0.84 ± 0.04 vs. 0.42 ± 0.03 for filaggrin, both p < 0.05). CONCLUSION: These results suggest that PN+HA may be an effective therapeutic strategy for repairing skin barrier damage.

Effects of a Combination of Polynucleotide and Hyaluronic Acid for Treating Osteoarthritis.

Knee osteoarthritis (OA), an age-related degenerative disease characterized by severe pain and disability, is treated using polynucleotides (PNs) and hyaluronic acid (HA). The intra-articular (IA) injection of HA has been studied extensively in both animal models and in humans; however, the efficacy and mechanisms of action remain unclear. In addition, there has been a paucity of research regarding the use of PN alone or in combination with HA in OA. To investigate the effect of the combined injection of PN and HA in vivo, pathological and behavioral changes were assessed in an OA model. Anterior cruciate ligament transection and medial meniscectomy were performed in Sprague-Dawley rats to create the OA animal model. The locomotor activity improved following PNHA injection, while the OARSI grade improved in the medial tibia and femur. In mild OA, TNFα levels decreased histologically in the PN, HA, and PNHA groups but only the PNHA group showed behavioral improvement in terms of distance. In conclusion, PNHA exhibited anti-inflammatory effects during OA progression and improved locomotor activity regardless of the OARSI grade.

Maturation of the E. coli Glu2, Ile1, and Ala1B tRNAs utilizes a complex processing pathway.

Despite significant progress in understanding the diversity of tRNA processing pathways in Escherichia coli, the mechanism for the maturation of tRNAs encoded within the rRNA operons has not received much attention. Here, we show that the Glu2, Ile1, and Ala1B tRNAs, encoded by 10 genes located between the 16S and 23S rRNAs in the seven rRNA operons, are matured via a RNase E-independent processing pathway that utilizes at least six different enzymes. It has been shown that the Glu2 and Ile1-Ala1B pre-tRNAs released by initial RNase III cleavages of the 30S primary rRNA transcripts retain extended 5'-leader (35-139 nt) and 3'-trailer (166-185 nt) sequences. However, the 5' maturation of the tRNAs by RNase P is inhibited until the trailer sequences are shortened to 1-4 nucleotides, initially by a second RNase III cleavage at 31-42 nucleotides downstream of the CCA determinant followed by exonucleolytic trimming. The RNase III cleaved Glu2 and Ile1-Ala1B trailer fragments are degraded via PAP I- dependent exonucleolytic decay. Compared to the six previously characterized tRNA processing pathways, maturation of the Glu2, Ile1, and Ala1B tRNAs is considerably more complex and appears to be distinct from what occurs in Gram-positive bacteria.

Systematic Analysis of Diverse Polynucleotide Kinase Clp1 Family Proteins in Eukaryotes: Three Unique Clp1 Proteins of Trypanosoma brucei.

The Clp1 family proteins, consisting of the Clp1 and Nol9/Grc3 groups, have polynucleotide kinase (PNK) activity at the 5' end of RNA strands and are important enzymes in the processing of some precursor RNAs. However, it remains unclear how this enzyme family diversified in the eukaryotes. We performed a large-scale molecular evolutionary analysis of the full-length genomes of 358 eukaryotic species to classify the diverse Clp1 family proteins. The average number of Clp1 family proteins in eukaryotes was 2.3 ± 1.0, and most representative species had both Clp1 and Nol9/Grc3 proteins, suggesting that the Clp1 and Nol9/Grc3 groups were already formed in the eukaryotic ancestor by gene duplication. We also detected an average of 4.1 ± 0.4 Clp1 family proteins in members of the protist phylum Euglenozoa. For example, in Trypanosoma brucei, there are three genes of the Clp1 group and one gene of the Nol9/Grc3 group. In the Clp1 group proteins encoded by these three genes, the C-terminal domains have been replaced by unique characteristics domains, so we designated these proteins Tb-Clp1-t1, Tb-Clp1-t2, and Tb-Clp1-t3. Experimental validation showed that only Tb-Clp1-t2 has PNK activity against RNA strands. As in this example, N-terminal and C-terminal domain replacement also contributed to the diversification of the Clp1 family proteins in other eukaryotic species. Our analysis also revealed that the Clp1 family proteins in humans and plants diversified through isoforms created by alternative splicing.

Zika Virus Induces Mitotic Catastrophe in Human Neural Progenitors by Triggering Unscheduled Mitotic Entry in the Presence of DNA Damage While Functionally Depleting Nuclear PNKP.

Vertical transmission of Zika virus (ZIKV) leads with high frequency to congenital ZIKV syndrome (CZS), whose worst outcome is microcephaly. However, the mechanisms of congenital ZIKV neurodevelopmental pathologies, including direct cytotoxicity to neural progenitor cells (NPC), placental insufficiency, and immune responses, remain incompletely understood. At the cellular level, microcephaly typically results from death or insufficient proliferation of NPC or cortical neurons. NPC replicate fast, requiring efficient DNA damage responses to ensure genome stability. Like congenital ZIKV infection, mutations in the polynucleotide 5'-kinase 3'-phosphatase (PNKP) gene, which encodes a critical DNA damage repair enzyme, result in recessive syndromes often characterized by congenital microcephaly with seizures (MCSZ). We thus tested whether there were any links between ZIKV and PNKP. Here, we show that two PNKP phosphatase inhibitors or PNKP knockout inhibited ZIKV replication. PNKP relocalized from the nucleus to the cytoplasm in infected cells, colocalizing with the marker of ZIKV replication factories (RF) NS1 and resulting in functional nuclear PNKP depletion. Although infected NPC accumulated DNA damage, they failed to activate the DNA damage checkpoint kinases Chk1 and Chk2. ZIKV also induced activation of cytoplasmic CycA/CDK1 complexes, which trigger unscheduled mitotic entry. Inhibition of CDK1 activity inhibited ZIKV replication and the formation of RF, supporting a role of cytoplasmic CycA/CDK1 in RF morphogenesis. In brief, ZIKV infection induces mitotic catastrophe resulting from unscheduled mitotic entry in the presence of DNA damage. PNKP and CycA/CDK1 are thus host factors participating in ZIKV replication in NPC, and pathogenesis to neural progenitor cells. IMPORTANCE The 2015-2017 Zika virus (ZIKV) outbreak in Brazil and subsequent international epidemic revealed the strong association between ZIKV infection and congenital malformations, mostly neurodevelopmental defects up to microcephaly. The scale and global expansion of the epidemic, the new ZIKV outbreaks (Kerala state, India, 2021), and the potential burden of future ones pose a serious ongoing risk. However, the cellular and molecular mechanisms resulting in microcephaly remain incompletely understood. Here, we show that ZIKV infection of neuronal progenitor cells results in cytoplasmic sequestration of an essential DNA repair protein itself associated with microcephaly, with the consequent accumulation of DNA damage, together with an unscheduled activation of cytoplasmic CDK1/Cyclin A complexes in the presence of DNA damage. These alterations result in mitotic catastrophe of neuronal progenitors, which would lead to a depletion of cortical neurons during development.

Comparison of Polynucleotide, Sodium Hyaluronate, and Crosslinked Sodium Hyaluronate for the Management of Painful Knee Osteoarthritis: A Multi-Center, Randomized, Double-Blind, Parallel-Group Study.

OBJECTIVE: To compare the clinical effectiveness of sodium polynucleotide, classic hyaluronic acid, and crosslinked hyaluronic acid for the management of painful knee osteoarthritis. DESIGN: Randomized, double-blind, parallel-group clinical trial. SETTING: Multicenter study. SUBJECTS: Patients with chronic painful knee osteoarthritis. METHODS: Ninety patients were selected and randomized into polynucleotide, classic hyaluronic acid, and crosslinked hyaluronic acid groups (30 per group). Intra-articular injections of the viscosupplement for each group were administered to the patients three times at one-week intervals. The primary outcome was differences in changes of weight-bearing pain scores at 16 weeks between the groups. The secondary outcomes were changes in the intensity of knee pain during weight-bearing, walking, and rest, and functional disability, quality of life, and adverse events during the 16-week follow-up period. RESULTS: At 16 weeks, the polynucleotide group showed a higher reduction in pain score using a Visual Analog Scale score (0-100) than the classic hyaluronic acid (-17.6 [95% CI = -35.1 to -0.1]; P = .048) and crosslinked hyaluronic acid (-22.4 [95% CI = -41.5 to -3.3]; P = .016) groups. The polynucleotide and crosslinked hyaluronic acid groups showed an early-onset reduction in knee pain during weight-bearing, walking, and rest. All three groups showed reductions in functional disability and improved quality of life at 16 weeks without inter-group differences. No severe adverse events were reported throughout the study period. CONCLUSION: Polynucleotide significantly relieves pain more and relieves pain faster in patients with knee osteoarthritis than classic and crosslinked hyaluronic acid, with improved health-related quality of life.

An electrochemical biosensor for T4 polynucleotide kinase activity identification according to host-guest recognition among phosphate pillar[5]arene@palladium nanoparticles@reduced graphene oxide nanocomposite and toluidine blue.

T4 polynucleotide kinase (T4 PNK) helps with DNA recombination and repair. In this work, a phosphate pillar[5]arene@palladium nanoparticles@reduced graphene oxide nanocomposite (PP5@PdNPs@rGO)-based electrochemical biosensor was created to identify T4 PNK activities. The PP5 used to complex toluidine blue (TB) guest molecules is water-soluble. With T4 PNK and ATP, the substrate DNA, which included a 5'-hydroxyl group, initially self-assembled over the gold electrode surface by chemical adsorption of the thiol units. Strong phosphate-Zr4+-phosphate chemistry allowed Zr4+ to act as a bridge between phosphorylated DNA and PP5@PdNPs@rGO. Through a supramolecular host-guest recognition connection, TB molecules were able to penetrate the PP5 cavity, where they produced a stronger electrochemical response. With a 5 × 10-7 U mL-1 detection limit, the electrochemical signal is linear in the 10-6 to 1 U mL-1 T4 PNK concentration range. It was also effective in measuring HeLa cell lysate-related PNK activities and screening PNK inhibitors. Nucleotide kinase-target drug development, clinical diagnostics, and screening for inhibitors all stand to benefit greatly from the suggested technology, which offers a unique sensing mechanism for kinase activity measurement.

Polynucleotides Suppress Inflammation and Stimulate Matrix Synthesis in an In Vitro Cell-Based Osteoarthritis Model.

Osteoarthritis (OA) is characterized by degeneration of the joint cartilage, inflammation, and a change in the chondrocyte phenotype. Inflammation also promotes cell hypertrophy in human articular chondrocytes (HC-a) by activating the NF-κB pathway. Chondrocyte hypertrophy and inflammation promote extracellular matrix degradation (ECM). Chondrocytes depend on Smad signaling to control and regulate cell hypertrophy as well as to maintain the ECM. The involvement of these two pathways is crucial for preserving the homeostasis of articular cartilage. In recent years, Polynucleotides Highly Purified Technology (PN-HPT) has emerged as a promising area of research for the treatment of OA. PN-HPT involves the use of polynucleotide-based agents with controlled natural origins and high purification levels. In this study, we focused on evaluating the efficacy of a specific polynucleotide sodium agent, known as CONJURAN, which is derived from fish sperm. Polynucleotides (PN), which are physiologically present in the matrix and function as water-soluble nucleic acids with a gel-like property, have been used to treat patients with OA. However, the specific mechanisms underlying the effect remain unclear. Therefore, we investigated the effect of PN in an OA cell model in which HC-a cells were stimulated with interleukin-1ß (IL-1ß) with or without PN treatment. The CCK-8 assay was used to assess the cytotoxic effects of PN. Furthermore, the enzyme-linked immunosorbent assay was utilized to detect MMP13 levels, and the nitric oxide assay was utilized to determine the effect of PN on inflammation. The anti-inflammatory effects of PN and related mechanisms were investigated using quantitative PCR, Western blot analysis, and immunofluorescence to examine and analyze relative markers. PN inhibited IL-1ß induced destruction of genes and proteins by downregulating the expression of MMP3, MMP13, iNOS, and COX-2 while increasing the expression of aggrecan (ACAN) and collagen II (COL2A1). This study demonstrates, for the first time, that PN exerted anti-inflammatory effects by partially inhibiting the NF-κB pathway and increasing the Smad2/3 pathway. Based on our findings, PN can potentially serve as a treatment for OA.

Phenanthridine-pyrene conjugates as fluorescent probes for DNA/RNA and an inactive mutant of dipeptidyl peptidase enzyme.

Two novel conjugate molecules were designed: pyrene and phenanthridine-amino acid units with a different linker length between the aromatic fragments. Molecular modelling combined with spectrophotometric experiments revealed that in neutral and acidic buffered water solutions conjugates predominantly exist in intramolecularly stacked conformations because of the π-π stacking interaction between pyrene and phenanthridine moieties. The investigated systems exhibited a pH-dependent excimer formation that is significantly red-shifted relative to the pyrene and phenanthridine fluorescence. While the conjugate with a short linker showed negligible spectrophotometric changes due to the polynucleotide addition, the conjugate with a longer and more flexible linker exhibited a micromolar and submicromolar binding affinity for ds-polynucleotides and inactivated a mutant of dipeptidyl peptidase enzyme E451A. Confocal microscopy revealed that the conjugate with the longer linker entered the HeLa cell membranes and blue fluorescence was visualized as the dye accumulated in the cell membrane.

Prolonged severe acute respiratory syndrome coronavirus 2 persistence, attenuated immunologic response, and viral evolution in a solid organ transplant patient.

Unlike immunocompetent hosts, the duration of viral persistence after infection with severe acute respiratory syndrome coronavirus 2 can be prolonged in immunosuppressed patients. Here, we present a case of viral persistence for over 19 weeks in a patient with a history of solid organ transplant and explore the clinical, virologic, and immunologic course. Our patient still demonstrated viral persistence at 138 days with low polymerase chain reaction cycle threshold values and evidence of continuing viral sequence evolution indicative of ongoing virus replication. These findings have important implications for infection prevention and control recommendations in immunosuppressed patients. Immune response, including neutralizing antibody titers, T cell activity, and cytokine levels, peaked around days 44-72 after diagnosis. Anti-S trimer antibodies were low at all time points, and T cell response was attenuated by day 119. As immune response waned and viral load increased, increased genetic diversity emerged, suggesting a mechanism for the development of viral variants.

A label-free T4 polynucleotide kinase fluorescence sensor based on split dimeric G-quadruplex and ligation-induced dimeric G-quadruplex/thioflavin T conformation.

The phosphorylation process of DNA by T4 polynucleotide kinase (T4 PNK) plays a crucial role in DNA recombination, DNA replication, and DNA repair. Traditional monomeric G-quadruplex (G4) systems are always activated by single cation such as K+ or Na+. The conformation transformation caused by the coexistence of multiple cations may interfere with the signal readout and limit their applications in physiological system. In view of the stability of dimeric G4 in multiple cation solution, we reported a label-free T4 PNK fluorescence sensor based on split dimeric G4 and ligation-induced dimeric G4/thioflavin T (ThT) conformation. The dimeric G4 was divided into two independent pieces of one normal monomeric G4 and the other monomeric G4 fragment phosphorylated by T4 PNK in order to decrease the background signal. With the introduction of template DNA, DNA ligase, and invasive DNA, the dimeric G4 could be generated and liberated to combine with ThT to show obvious fluorescence signal. Using our strategy, the linear range from 0.005 to 0.5 U mL-1, and the detection limit of 0.0021 U mL-1 could be achieved without the consideration of interference caused by the coexistence of multiple cations. Additionally, research in real sample determination and inhibition effect investigations indicated its further potential application value in biochemical process research and clinic diagnostics.

Nano-hybrid luminophores of Ti3C2TX quantum dots-gold nanoparticles based on in situ generation for sensitive electrochemiluminescence biosensing.

Nanomaterial-derived quantum dots (QDs) are excellent electrochemiluminescence (ECL) luminophores and play an important role in optical sensing due to their excellent water solubility, good biocompatibility and tunable molecular size. In this work, a novel strategy was designed to form nano-hybrid Ti3C2 QDs-AuNPs in situ as a luminophore based on the unique reducibility of Ti3C2 QDs, which showed remarkable and stable ECL performance. Here, AuNPs were formed in situ without the addition of reducing agents and stabilizers, leading to threefold enhancement of the ECL signal of Ti3C2 QDs due to their excellent charge transfer capability. Meanwhile, Ti3C2 QDs-AuNPs with abundant Ti atoms also acted as recognition units. Through skillful combination with hybridization chain reaction (HCR) to expose more phosphate, an ECL platform was constructed to detect polynucleotide kinase (PNK) with good specificity and sensitivity. A lower limit of detection limit of 2.7×10-5 U mL-1 was achieved, with a wide linear relationship ranging from 0.0001 to 10 U mL-1. This novel strategy provides a guide for the application of nano-hybrid Ti3C2 QDs-AuNPs as a luminophore in the field of ECL bioanalysis. Novel in situ-formed nano-hybrid Ti3C2 QDs-AuNPs were prepared as a luminophore, with threefold enhancement of the ECL signal of Ti3C2 QDs.

CRISPR-Cas13a cascade-based viral RNA assay for detecting SARS-CoV-2 and its mutations in clinical samples.

SARS-CoV-2 is one of the greatest threats to global human health. Point-of-care diagnostic tools for SARS-CoV-2 could facilitate rapid therapeutic intervention and mitigate transmission. In this work, we report CRISPR-Cas13a cascade-based viral RNA (Cas13C) assay for label-free and isothermal determination of SARS-CoV-2 and its mutations in clinical samples. Cas13a/crRNA was utilized to directly recognize the target of SARS-CoV-2 RNA, and the recognition events sequentially initiate the transcription amplification to produce light-up RNA aptamers for output fluorescence signal. The recognition of viral RNA via Cas13a-guide RNA ensures a high specificity to distinguish SARS-CoV-2 from MERS-CoV and SARS-CoV, as well as viral mutations. A post transcription amplification strategy was triggered after CRISPR-Cas13a recognition contributes to an amplification cascade that achieves high sensitivity for detecting SARS-CoV-2 RNA, with a limit of detection of 0.216 fM. In addition, the Cas13C assay could be able to discriminate single-nucleotide mutation, which was proven with N501Y in SARS-Cov-2 variant. This method was validated by a 100% agreement with RT-qPCR results from 12 clinical throat swab specimens. The Cas13C assay has the potential to be used as a routine nucleic acid test of SARS-CoV-2 virus in resource-limited regions.