Stem-loop Research Articles

CRISPR/Cas13a-Programmed Cu NCs and Z-Scheme T-COF/Ag2S for Photoelectrochemical Biosensing of circRNA.

Circular RNAs (circRNAs), as a class of noncoding RNA molecules with a circular structure exhibit high stability and spatiotemporal-specific expression, making them ideal cancer biomarkers for liquid biopsy. Herein, a new photoelectrochemical (PEC) biosensor for a highly sensitive circRNA assay in the whole blood of lung cancer patients was designed based on CRISPR/Cas13a-programmed Cu nanoclusters (Cu NCs) and a Z-scheme covalent organic framework/silver sulfide (T-COF/Ag2S) composite. This Z-scheme T-COF/Ag2S composite accelerates electron transfer and produces an excellent initial photocurrent. When CRISPR/Cas13a precisely targets circRNA, it nonspecifically cleaves the triple-helix molecular structure to release DNA fragments (C'/C"). After the C'/C" opens the DNA hairpin probe (HP) modified on the electrode, hybridization chain reactions are performed to produce abundant AT-rich double-stranded DNA with the addition of H1 and H2 probes. Upon the incubation of Cu2+, Cu NCs are in situ formed via the A-Cu2+-T bonds and can effectively quench the photocurrent of the Z-scheme T-COF/Ag2S due to the energy transfer process. This developed PEC biosensor for the circRNA assay shows a low limit of detection of 0.5 fM, and the reusability of DNA-modified magnetic beads (MB-DNA) reduces the detection cost. Moreover, the PEC biosensor can accurately quantify the circRNA level and distinguish the circRNA expression in whole blood from healthy controls and lung cancer patients, offering strong potential in clinical diagnosis.

Identification and Expression Analysis of miR166 Gene Family in Response to Salt Stress in Chrysanthemum

cgr-miR166 was observed to be significantly enhanced in Chrysanthemum under 200 mM NaCl treatment. Here, ten family members were identified by aligning cgr-miR166 with scaffold sequences from the Chrysanthemum nankingense genome database, naming them from cgr-miR166a to cgr-miR166j, and their precursors could form stable stem-loop structures. The mature regions were observed to be highly conserved, with the 3′ end being more conserved than the 5′ end. miR166s promoters have been found to contain cis-acting elements responsive to diverse stimuli like the phytohormones ABA and IAA. qRT-RCR results demonstrated that the transcriptome sequencing results were reliable and miR166 was present at different levels in the roots, stems, leaves and flowers of Chrysanthemum. Furthermore, the HD-ZipIII transcription factor was validated to be the target gene of Chrysanthemum miR166s by degradome sequencing. Taken together, the cgr-miR166 family exhibited both evolutionary conservation and diversification. The expression level of miR166 was upregulated in root under salt stress, while the expression level of the target gene HD-ZipIII was downregulated. These findings established the foundation for further understanding the mechanism of miR166-HD-ZipIII modules in salt response and tolerance.

Mutational Analysis Supports Three-Hairpin Model of Attenuator for Transcription Regulation of ilvBNC Operon in Corynebacterium glutamicum

The ilvBNC operon in Corynebacterium glutamicum encodes key enzymes for the biosynthesis of branched-chain amino acids (L-isoleucine, L-leucine, and L-valine). This operon has been studied for quite a long time, and it is assumed that three hairpin mRNA structures can be formed in its regulatory region; however, their functionality and role in the attenuation mechanism of the ilvBNC operon are not completely clear. In the present work, we performed a mutational analysis of mRNA secondary structures in the regulatory region of the C. glutamicum ilvBNC operon, which allowed us to propose a model of the regulation of its transcription involving three mRNA hairpins that essentially act as a transcription terminator, an antiterminator, and an antiantiterminator. In this work, we proved the existence of a transcription terminator in this operon and experimentally confirmed the effectiveness of its influence on the expression of the ilvBNC operon, AHAS enzyme activity, and valine production. We demonstrated the unique functional features of this attenuator, which, due to the overlapping of the terminator and antiterminator hairpins, is capable of rapid low-energy transitions between them without the complete disruption of the hairpin structures.

Spatioselective Imaging of Noncoding RNAs in Mitochondria via an Organelle-Specific DNA Assembly Strategy.

Precise imaging of noncoding RNAs (ncRNAs) in specific organelles allows decoding of their functions at subcellular level but lacks advanced tools. Here we present a DNA-based nanobiotechnology for spatially selective imaging of ncRNA (e.g., microRNA (miRNA)) in mitochondria via an organelle-specific DNA assembly strategy. The target miRNA-initiated assembly of DNA hairpins is inhibited by the block of toehold-mediated strand displacement reaction but can be exclusively activated by a mitochondria-encoded ribosomal RNA (rRNA) for hybridization chain reaction, enabling spatial control over miRNA imaging. We demonstrate that the conditionally controlled DNA assembly technology allows for minimization of nonspecific activation and thus improves the spatial precision of miRNA detection. In addition, the strategy is adaptable to visualizing other ncRNAs such as long noncoding RNAs in mitochondria, highlighting the universality of the approach. Overall, this work provides a useful tool for spatially selective imaging of ncRNAs and investigating the functions of organelle-located RNA.

Topologically constrained DNA-mediated one-pot CRISPR assay for rapid detection of viral RNA with single nucleotide resolution.

The widespread and evolution of RNA viruses, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), highlights the importance of fast identification of virus subtypes, particularly in non-laboratory settings. Rapid and inexpensive at-home testing of viral nucleic acids with single-base resolution remains a challenge. Topologically constrained DNA ring is engineered as substrates for the trans-cleavage of Cas13a to yield an accelerated post isothermal amplification. The capacity of CRISPR/Cas13a for discriminating single nucleotide variant (SNV) in viral genome is leveraged by designing synthetic mismatches and hairpin structure in CRISPR RNA (crRNA), enabling robust discrimination of different SARS-CoV-2 variants. Via optimisation of CasTDR3pot to be one-pot assay, CasTDR1pot can detect Omicron and its subvariants, with only a few copies in clinical samples in less than 30min without pre-amplification. The detection system boasts high sensitivity (0.1 aM), single-base specificity, and the advantage of a rapid "sample-to-answer" process, which takes only 30min. In the detection of SARS-CoV-2 clinical samples and their variant strains, CasTDR1pot has achieved 100% accuracy. Furthermore, the design of a portable signal-reading device facilitates user-friendly result interpretation. For the detection needs of different RNA viruses, the system can be adapted simply by designing the corresponding crRNA. Our study provides a rapid and accurate molecular diagnostic tool for point-of-care testing, epidemiological screening, and the detection of diseases associated with other RNA biomarkers with excellent single nucleotide differentiation, high sensitivity, and simplicity. National Key Research and Development Program of China (No. 2023YFB3208302), National Natural Science Foundation of China (No. 22377110, 22034004, 82402749, 82073787, 22122409), National Key Research and Development Program of China (No. 2021YFA1200104), Henan Province Fund for Cultivating Advantageous Disciplines (No. 222301420019).

Non-Invasive Detection of Interferon-Gamma in Sweat Using a Wearable DNA Hydrogel-Based Electrochemical Sensor

Monitoring of immune factors, including interferon-gamma (IFN-γ), holds great importance for understanding immune responses and disease diagnosis. Wearable sensors enable continuous and non-invasive detection of immune markers in sweat, drawing significant attention to their potential in real-time health monitoring and personalized medicine. Among these, electrochemical sensors are particularly advantageous, due to their excellent signal responsiveness, cost-effectiveness, miniaturization, and broad applicability, making them ideal for constructing wearable sweat sensors. In this study, we present a flexible and sensitive wearable platform for the detection of IFN-γ, utilizing a DNA hydrogel with favorable loading performance and sample collection capability, and the application of mobile software achieves immediate data analysis and processing. This platform integrates three-dimensional DNA hydrogel functionalized with IFN-γ-specific aptamers for precise target recognition and efficient sweat collection. Signal amplification is achieved through target-triggered catalytic hairpin assembly (CHA), with DNA hairpins remarkably enhancing sensitivity. Ferrocene-labeled reporting strands immobilized on a screen-printed carbon electrode are displayed via CHA-mediated strand displacement, leading to a measurable reduction in electrical signals. These changes are transmitted to a custom-developed mobile application via a portable electrochemical workstation for real-time data analysis and recording. This wearable sensor platform combines the specificity of DNA aptamers, advanced signal amplification, and the convenience of mobile data processing. It offers a high-sensitivity approach to detecting low-abundance targets in sweat, paving the way for new applications in point-of-care diagnostics and wearable health monitoring.

Early events in G-quadruplex folding captured by time-resolved small-angle X-ray scattering.

Time-resolved small-angle X-ray experiments are reported here that capture and quantify a previously unknown rapid collapse of the unfolded oligonucleotide as an early step in the folding of hybrid 1 and hybrid 2 telomeric G-quadruplex structures. The rapid collapse, initiated by a pH jump, is characterized by an exponential decrease in the radius of gyration from 24.3 to 12.6 Å. The collapse is monophasic and is complete in <600 ms. Additional hand-mixing pH-jump kinetic studies show that slower kinetic steps follow the collapse. The folded and unfolded states at equilibrium were further characterized by SAXS studies and other biophysical tools, showing that G4 unfolding was complete at alkaline pH, but not in LiCl solution as is often claimed. The SAXS Ensemble Optimization Method analysis reveals models of the unfolded state as a dynamic ensemble of flexible oligonucleotide chains with a variety of transient hairpin structures. These results suggest a G4 folding pathway in which a rapid collapse, analogous to molten globule formation seen in proteins, is followed by a confined conformational search within the collapsed particle to form the native contacts ultimately found in the stable folded form.

Advancing DNA Structural Analysis: A SERS Approach Free from Citrate Interference Combined with Machine Learning.

Surface-enhanced Raman spectroscopy (SERS) has become an indispensable tool for biomolecular analysis, yet the detection of DNA signals remains hindered by spectral interference from citrate ions, which overlap with key DNA features. This study introduces an innovative, ultrasensitive SERS platform utilizing thiol-modified silver nanoparticles (Ag@SDCNPs) that overcomes this challenge by eliminating citrate interference. This platform enables direct, interference-free detection and structural characterization of a wide range of DNA conformations, including single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), i-motif, hairpin, G-quadruplex, and triple-stranded DNA (tsDNA). Employing calcium ions as aggregating agents and deuterated methanol as an internal standard, the system achieved high spectral quality and reproducibility. Machine learning (ML) techniques, such as linear discriminant analysis (LDA) and t-distributed stochastic neighbor embedding (t-SNE), were utilized for spectral classification, alongside support vector machines (SVM) for predictive modeling, yielding accuracies above 99%. These findings establish a robust and versatile platform for DNA structural analysis, offering transformative potential for applications in clinical diagnostics and biomedical research.

Novel relatives of Mecsek Mountains mammarenavirus (family Arenaviridae) in hedgehogs living in different sampling areas in Hungary

Mammarenaviruses (genus Mammarenavirus, family Arenaviridae) are rodent-borne zoonotic viruses consisting of 52 viral species, including ten that are pathogenic to humans. Currently, only two endemic mammarenavirus species are known in Europe: the human pathogenic Mammarenavirus choriomeningitidis (LCMV) and the recently discovered hedgehog-origin Mammarenavirus mecsekense (MEMV). In this study, 59 faecal specimens from Northern white-breasted hedgehogs (Erinaceus roumanicus) from different geographic regions in Hungary were investigated for mammarenavirus presence and complete genome characterization using newly designed screening primers by RT-semi-nested PCR and sequencing methods. Five (8.5%) of the 59 samples tested positive for mammarenavirus RNA (ER8, ER15, ER27, ER33, and ER39, GenBank accession numbers PQ441959-PQ441968). The L- and S-segments of these strains showed 66–93% and 73–92% nt identity to the closest known mammarenavirus, MEMV, respectively. The NP protein exhibited 86–97% aa sequence identity compared to the corresponding protein of MEMV. Notably, the S-segment intergenic region (S-IGR) of strains ER8, ER15, ER27 and ER33 exceeded the average nt length among known mammarenaviruses and contained two, highly similar stem-loop structures with conserved self-complementary nucleotide motifs. Based on the sequence- and phylogenetic analysis these strains (ER8, ER15, ER27 and ER33) potentially represent a novel mammarenavirus species, tentatively named Pannonia mammarenavirus (PANV).

Complex and Non-sequential Current Signatures of a β-Hairpin Peptide Confined in a Nanopore.

Nanopore sensing is widely used for single-molecule detection, originally applied to nucleic acids and now extended to protein sensing. Our study focuses on the complex conformational changes of peptides in nanopores, which may have implications for peptide fingerprinting and protein identification. Specifically, we investigated the interaction of a β-hairpin peptide (SV28) within an α-hemolysin (αHL) nanopore. Our experiments revealed that SV28 is captured via dielectrophoresis and exhibits long dwell times within the nanopore, leading to multiple current blockade levels. Unlike DNA hairpins, the peptide showed non-sequential transitions among four distinct blockade levels. This complex behavior indicates that the peptide dynamics in nanopores cannot be simply modeled along a single reaction coordinate. Our findings provide insights into peptide-nanopore interactions, which are potentially useful for developing nanopore-based peptide identification technologies.

Ultrasensitive detection of microRNAs based on cascade amplification strategy of RCA-PER and Cas12a.

Since microRNAs (miRNAs) serve as markers for early cancer diagnosis, it is crucial to develop a novel biosensor to detect miRNAs quickly, sensitively and selectively. Hence, we developed a fluorescence biosensor based on target miRNA-initiated rolling circle amplification (RCA) to generate RCA products with multiple tandem catalytic hairpin DNA templates that trigger primer exchange reactions (PER) which extend short single-strand DNA (ssDNA) primers into long ssDNA. Subsequently, the long ssDNA activates the trans-cleavage activity of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system to cleave a fluorescent reporter chain, enabling ultrasensitive detection of miRNAs through the output fluorescence signal. The biosensor could quantify miRNA-141 concentrations from 100 to 105 pM, with a detection limit of 94 fM. Therefore, the biosensing strategy proposed in this study offers a robust technique for the clinical diagnosis of miRNA-141.

An Ultrasensitive Duplex Aptasensor Featuring the Target-Triggered Lanthanide Luminescence Antenna Effect for Wash-Free Detection of Epithelial Cancerous Exosomes.

Fluorescence sensing is widely used in in vitro detection due to its high sensitivity and rapid result delivery. However, detection systems based on nanomaterials involving complex and cumbersome purification steps can lead to sample loss and significantly reduce the accuracy of the results. To address this issue, we proposed a lanthanide-based aptasensor featuring the target-triggered antenna effect to significantly enhance the time-resolved luminescence (TRL) of chelated Tb3+ combined with a wash-free strategy. This sensor enabled highly sensitive detection of the epithelial cell adhesion molecule (EpCAM) on exosomes from epithelial tumors, eliminating the necessity for purification steps and thereby delivering more stable and reliable results. Specifically, using computer-aided design, a sequence capable of self-folding into a stable hairpin structure (Antenna-Hairpin-Tb) was obtained. After hybridization with an EpCAM-specific aptamer (AptEpCAM), a duplex aptasensor (Probe-Tb) was constructed for detection. When the targeted binding occurred between the AptEpCAM and exosomes, the Antenna-Hairpin-Tb dehybridized from the Probe-Tb and underwent self-folding, leading to a significant sensitized TRL signal due to the antenna effect-induced energy transfer from the antenna ligand to the chelated Tb3+. Over a broad range of exosome concentrations (spanning from 6.19 × 102 to 6.19 × 108 particles/mL), the emission intensity of chelated Tb3+ at 543 nm linearly increased with the exosome concentration (linear relationship: Y = 332.62 lg(Nexosomes) + 3690.5, R2 = 0.9956), achieving a theoretical detection limit as low as 17 particles/mL in the buffer. Furthermore, Probe-Tb enabled a swift and effective differentiation of all epithelial cancer serum samples from nonepithelial ones within just 30 min of a wash-free operation, achieving 100% sensitivity, 100% specificity, and 100% accuracy.

SARS-CoV-2 CoCoPUTs: analyzing GISAID and NCBI data to obtain codon statistics, mutations, and free energy over a multiyear period.

A consistent area of interest since the beginning of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has been the sequence composition of the virus and how it has changed over time. Many resources have been developed for the storage and analysis of SARS-CoV-2 data, such as GISAID (Global Initiative on Sharing All Influenza Data), NCBI, Nextstrain, and outbreak.info. However, relatively little has been done to compile codon usage data, codon-level mutation data, and secondary structure data into a single database. Here, we assemble the aforementioned data and many additional virus attributes in a new database entitled SARS-CoV-2 CoCoPUTs. We begin with an overview of the composition and overlap between two of the largest sources of SARS-CoV-2 sequence data: GISAID and NCBI Virus (GenBank). We then evaluate different types of sequence curation strategies to reduce the dataset of millions of sequences to only one sequence per Pango lineage variant. We then performed specific analyses on the coding sequences (CDSs), including calculating codon usage, codon pair usage, dinucleotides, junction dinucleotides, mutations, GC content, effective number of codons (ENCs), and effective number of codon pairs (ENCPs). We have also performed whole-genome secondary RNA structure prediction calculations for each variant, using the LinearPartition software and modified selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) data that are available online. Finally, we compiled all the data into our resource, SARS-CoV-2 CoCoPUTs, and paired many of the resulting statistics with variant proportion data over time in order to derive trends in viral evolution. Although the overall codon usage of SARS-CoV-2 did not change drastically, in line with the previous literature on this subject, we did observe that while overall GC% content decreased, GC% of the third position in the codon was more positive relative to overall GC% content between February 2021 and July 2023. Over the same interval, we noted that both synonymous and nonsynonymous mutations increased in number, with nonsynonymous mutations outpacing synonymous mutations at a rate of 3:1. We noted that the predicted whole-genome secondary structures nearly all contained the previously described virus-activated inhibitor of translation (VAIT) stem loops, validating for the first time their existence in a whole-genome secondary structure prediction for many SARS-CoV-2 variants (as opposed to previous local secondary structure predictions). We also separately produced a synonymous mutation-deprived set of SARS-CoV-2 variant sequences and repeated the secondary structure calculations on this set. This revealed an interesting trend of reduced ensemble free energy compared to the unaltered variant structures, indicating that synonymous mutations play a role in increasing the free energy of viral RNA molecules. These data both validate previous studies describing increases in viral free energy in human viruses over time and indicate a possible role for synonymous mutations in viral biology.

Simultaneous and Ultraspecific Optical Detection of Multiple miRNAs Using a Liquid Flow-Based Microfluidic Assay.

Recent studies have reported that the cause and progression of many diseases are closely related to complex and diverse gene regulation involving multiple microRNAs (miRNAs). However, most existing methods for miRNA detection typically deal with one sample at a time, which limits the achievement of high diagnostic accuracy for diseases associated with multiple gene dysregulations. Herein, we develop a liquid flow-based microfluidic optical assay for the simple and reliable detection of two different target miRNAs simultaneously at room temperature without any enzymatic reactions. This assay utilizes the catalytic hairpin assembly cycling reaction in a mixture containing four types of hairpin DNAs to amplify two different dimeric DNA probes, each of which specifically recognizes one of the two different target miRNAs. The resultant two dimeric DNA probes effectively hybridize with anchor DNA grafted into two outlet channels of a microfluidic device, thus enabling i-motif-driven compact DNA hydrogels to form in the channels under acidic conditions. With this setup, the presence of two target miRNAs can be confirmed by the naked-eye observation of red-colored gold nanoparticles encountering a flow blockage in the two outlet channels. Notably, the developed assay demonstrates sensitive and sequence-specific detection that can precisely distinguish a single base mismatch mutant miRNA within 1.5 h. Our assay thus has the potential to serve as a powerful sensing platform for the simple and simultaneous detection of multiple miRNAs in clinical diagnostics at room temperature without analytic equipment or enzymatic reactions.

Logic Circuits for Intelligent Microcystin Monitoring Based on Aptamer Recognition and Toehold-Mediated Hairpin DNA Self-Assembly.

A sensitive fluorescence biosensor was developed for microcystin-LR (MC-LR) detection using H1, H2, and H3 DNA probes as sensing elements. The aptamer in H1 can recognize the target. H2 was labeled with FAM and BHQ. The MC-LR and H1 binding will activate the H2 and H3 self-assemblies through toehold-mediated strand displacement. In the formed products (MC-LR/H1/nH2/nH3), FAM and BHQ will be separated and a high FAM fluorescence signal can be observed for the MC-LR assay. The biosensor is sensitive with a detection limit of 53 fM. We further constructed several logic circuits (AND-AND cascaded circuit, feedforward circuit, and resource allocation circuit) using MC-LR, MC-LA, and MC-YR as the three inputs. The numbers 0 and 1 are used to code the input and output signals. The AND-AND cascade circuit can produce a high output signal only in the (111) input combination. In the feedforward circuit, MC-LR and MC-LA can activate the logic circuit to produce high signals, and MC-YR will inhibit the self-assembly and execute the negative feedforward operation. Through the rational design of the DNA probe hybridizations on four different magnetic beads (MBs), the resource allocation circuit can achieve an intelligent allocation of input information. Our proposed fluorescence biosensor can not only provide a sensitive platform for microcystin detection but also serve as a smart and intelligent logic system for microcystin sensing.

Identification and functional characterization of a fructose-inducible phosphotransferase system in Azospirillum brasilense Sp7.

Plant growth-promoting rhizobacterium Azospirillum brasilense Sp7 utilizes fructose efficiently via a fructose phosphotransferase system (Fru-PTS). Its genome encodes two putative Fru-PTS, each consisting of FruB (EIIA), FruK (Pfk), and FruA (EIIBC) proteins. We compared the proteomes of A. brasilense Sp7 grown with malate or fructose as sole carbon source, and noticed upregulation of the constituent proteins of Fru-PTS1 only on fructose. Inactivation of fruA gene of both the Fru-PTS showed that Fru-PTS1 is the main PTS involved in fructose utilization. Overexpression of fruA1 in A. brasilense Sp7 enhanced its growth on fructose showing improved consumption of fructose. This suggested that fructose utilization in A. brasilense Sp7 is limited due to the limitation of EIIBC component. A FruR-type regulator, encoded divergently to the Fru-PTS1 operon, was required for chemotaxis toward fructose. Although not an absolute necessity for the growth of fructose, FruR was required for the optimal growth of fructose. The fruB1 promoter was activated by fructose and repressed by malate, but FruR does not seem to regulate its expression. A 27-nucleotide stem-loop structure located between the -125 and -99 promoter proximal region of fruB1 was involved in fructose inducibility and malate repression. Fructose also upregulated several proteins involved in the biogenesis of a Type 6 secretion system. Here, we have shown that A. brasilense Sp7 was able to inhibit the growth of Escherichia coli and Agrobacterium tumefaciens in the presence of fructose, and that an intact T6SS was required for contact-dependent growth inhibition of the two Gram-negative bacteria.IMPORTANCEAzospirillum brasilense, a plant growth-promoting rhizobacterium, has limited ability to utilize carbohydrates and sugars. Although it is known to utilize fructose via a fructose phosphotransferase system (fructose-PTS), the genes involved in fructose utilization and the role of fructose in its biology were not well characterized. This study has shown that out of the two units of fructose-PTS encoded in its genome, fructose-PTS1 plays the major role in fructose utilization. Overexpression of the membrane component (EIIBC) improved the growth of A. brasilense on fructose. The ability of fructose to induce proteins of the Type 6 Secretion System (T6SS) enables A. brasilense to cause contact-dependent inhibition of the growth of Escherichia coli as well as A. tumefaciens. This is the first report on the fructose inducibility of T6SS in A. brasilense, which may provide a handle to control the growth of undesirable bacteria using T6SS of A. brasilense in a mixed culture.

A High-Efficiency Autocatalysis-Oriented Cascade Circuit via Reciprocal Hug-Amplification for Assay-to-Treat Application.

Developing a DNA autocatalysis-oriented cascade circuit (AOCC) via reciprocal navigation of two enzyme-free hug-amplifiers might be desirable for constructing a rapid, efficient, and sensitive assay-to-treat platform. In response to a specific trigger (T), seven functional DNA hairpins were designed to execute three-branched assembly (TBA) and three isotropic hybridization chain reaction (3HCR) events for operating the AOCC. This was because three new inducers were reconstructed in TBA arms to initiate 3HCR (TBA-to-3HCR) and periodic T repeats were resultantly reassembled in the tandem nicks of polymeric nanowires to rapidly activate TBA in the opposite direction (3HCR-to-TBA) without steric hindrance, thereby cooperatively manipulating sustainable AOCC progress for exponential hug-amplification (1:3Nn). Our experimental verifications manifested that the T-dependent AOCC amplifier achieved fast input transduction and efficient fluorescence readout. As predicted, the flexible programming of reactive hairpin species endowed the repeating nicks in productive 3HCR nanowires with great possibilities and accessibilities to graft tailored modular elements, such as G-rich AS1411 aptamers capable of adopting G-quadruplex conformations (G4) that readily facilitated the embedding of zinc(II) protoporphyrin IX (ZnPPIX), a kind of heme oxygenase-1 enzyme inhibitor. Thus, the cascading ZnPPIX/G4 entities acted as fluorescent signal reporters, photosensitizers and anticancer drugs, thereby creating an updated AOCC-based assay-to-treat platform for ultrasensitive biosensing, discernible cell imaging and efficient photodynamic therapy of cancer cells. This would offer a new paradigm to advance the rational integration of dynamic DNA assembly and amplifiable recycling circuits for applicable bioassay and theranostics.

The complete mitochondrial genome of Gyrodactylus pseudorasborae (Platyhelminthes: Monogenea) with a phylogeny of Gyrodactylidae parasites

BackgroundGyrodactylus von Nordmann, 1832, a genus of viviparous parasites within the family Gyrodactylidae, contains one of the largest nominal species in the world. Gyrodactylus pseudorasborae Ondračková, Seifertová & Tkachenko, 2023 widely distributed in Europe and China, although its mitochondrial genome remains unclear. This study aims to sequence the mitogenome of G.pseudorasborae and clarify its phylogenetic relationship within the Gyrodactylidea. The mitochondrial genome of G. pseudorasborae was amplified in six parts from a single parasite, sequenced using primer walking, annotated and analyzed using bioinformatic tools.ResultsThe mitochondrial genome of G. pseudorasborae is 14,189 bp in length, containing 12 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs), and two major non-coding regions (NCR: NC1 and NC2). The overall A + T content of the mitogenome is 73.1%, a medium content compared with all reported mitochondrial genomes of monogeneans. The mitogenome of G. pseudorasborae presents a clear bias in nucleotide composition with a negative AT skew and a positive GC skew except for NCR. All tRNAs have the typical cloverleaf secondary structure except for tRNACys, tRNASer1, and tRNASer2, which lack the dihydrouridine (DHU) arm. Furthermore, one repetitive non-coding region of 32 bp repeats occurred in the NC1 region with poly-T stretch, stem-loop structure, and TAn motif. The gene order is identical to the mitochondrial genomes reported from other Gyrodactylus species except Gyrodactylus nyanzae Paperna, 1973 and Gyrodactylus sp. FZ-2021. Phylogenetic analyses show that G. pseudorasborae and Gyrodactylus parvae You, Easy & Cone, 2008 cluster together with high nodal support based on 12 PCGs sequences and amino acid sequences, Gyrodactylidae forms independent monophyletic clade within Gyrodactylidea.ConclusionBoth the mitochondrial genome and phylogenetic analyses support G. pseudorasborae is a member of the genus Gyrodactylus and Gyrodactylidae forms an independent monophyletic clade within Gyrodactylidea. Furthermore, the mitochondrial genome of G. pseudorasborae is the shortest in the Gyrodactylidea species compared with size differences in NCR.

APE1-Activated and NIR-II Photothermal-Enhanced Chemodynamic Therapy Guided by Amplified Fluorescence Imaging.

The development of intelligent nanotheranostic technology that integrates diagnostic and therapeutic functions holds great promise for personalized nanomedicine. However, most of the nanotheranostic agents exhibit "always-on" properties and do not involve an amplification step, which may largely limit imaging contrast and restrict therapeutic efficacy. Herein, we construct a novel nanotheranostic platform (Hemin/DHPs/PDA@CuS nanocomposite) by assembling DNA hairpin probes (DHPs) and hemin on the surface of PDA@CuS nanosheets that enables amplified fluorescence imaging and activatable chemodynamic therapy (CDT) of tumors. The cancer-relevant APE1 triggers nucleic acid amplification with DHPs to generate activatable and amplified fluorescence signals for discriminating cancer cells from normal cells. Meanwhile, excessive G-quadruplex/hemin-based DNAzyme are also activated, and they function as Fenton-like catalysts to catalyze the production of highly toxic hydroxyl radicals (•OH) for CDT. Moreover, owing to the excellent photothermal conversion efficiency in the near-infrared-II (NIR-II) window, the PDA@CuS not only improves the catalytic performance of CDT but also furnishes PTT. A remarkable antitumor therapeutic effect is demonstrated both in vitro and in vivo. Therefore, the Hemin/DHPs/PDA@CuS nanocomposite is expected to provide a promising avenue for precise imaging-guided antitumor therapy.

Fluorescent aptasensor based on target-induced hairpin conformation switch coupled with nicking enzyme-assisted signal amplification for detection of beta-amyloid oligomers in cerebrospinal fluid.

Afluorescent aptasensor was developedbased on target-induced hairpin conformation switch coupled with nicking enzyme-assisted signal amplification (NESA) to detect the oligomeric form of ß-amyolidpeptide (AβO) in cerebrospinal fluid. The hairpin DNA probe (HP) was specifically designed to recognize AβO. When AβO is present in the sensing system, it induces an HP conformational switch and triggers the NESA reaction. After evaluating the parameters of the aptasensor system, we selected Hairpin10, which has a 10-nucleotide extended sequence, as the hairpin sequence that interacts with AβO. The quantitative linear range of the proposed aptasensor is from11.3 to 113 ng mL-1 in artificial cerebrospinal fluid (aCSF), and the detection limit was 7.29 ng mL-1. The present work realized the assay of AβO in aCSF with satisfactory quantitative results.