Z-DNA is a left-handed duplex structure that exists in all living organisms and plays important biological functions through specific binding to some proteins in vivo. However, quantitative analysis of binding dynamics has been challenging due to the difficulties in obtaining unmodified Z-DNA that can be immobilized on sensors. In this study, a bait (Bait-cc) for biofilm interference without chemical modification at the Z-DNA region was constructed by using a topological constraint. The corresponding binding dynamics were measured by immobilizing Bait-cc on the sensor surface of bio-layer interferometry (BLI) via biotin-streptavidin interactions. This enabled quantitative evaluation of interactions between Z-DNA and the Z-DNA-binding proteins (in solution) across varying ionic strengths. Interestingly, our study revealed that the Z22 antibody likely exhibits a binding preference at or near the B-Z junction region, with an affinity for the initial binding site over the subsequent binding site by more than two orders of magnitude. Unexpectedly, Z22 binding demonstrated no sequence preference once the B-Z junction and Z-DNA were formed. Additionally, the binding dynamics between ZBP1 and unmodified Z-DNA were also quantitatively assessed. The CD spectrum analysis demonstrated that ZBP1 underwent structural changes upon binding to Z-DNA. This study not only enhances our understanding of Z-DNA's biological functions but also provides theoretical support for developing therapeutics targeting Z-DNA-related diseases.

Nucleic acids are essential dietary components with diverse physiological functions. Numerous studies have focused on the biological functions of nucleotides, nucleosides, and functional RNAs such as microRNAs. However, the nutritional value of ribosomal RNA (rRNA)-derived oligonucleotides, which are likely the predominant nucleic acid-derived components in foods, remains largely unexplored. Here, yeast was used as a food-associated eukaryotic model organism to investigate the uptake and utilization of rRNA-derived oligonucleotides. Yeast efficiently utilized short RNA oligonucleotides (approximately 5-30 nt) as nutrient sources, supporting robust cell growth. Confocal microscopy confirmed rapid uptake of Cy5-labeled RNA oligonucleotides by yeast cells. Proteomic analysis further revealed marked upregulation of proteins involved in endocytosis and autophagy in yeast cultured with RNA oligonucleotides. Collectively, these findings demonstrate that yeast can internalize and metabolize rRNA-derived oligonucleotides as efficient nutrient sources, likely through coordinated endocytic and autophagic pathways. This study highlights the nutritional potential of rRNA-derived oligonucleotides and provides a foundation for their future application in functional foods and fermentation systems.

Efficient and specific adapter ligation is essential for high-quality library construction in high-throughput sequencing. Conventional blunt-end adapters offer superior ligation efficiency compared to 3 '-T overhang adapters but suffer from severe self-ligation, resulting in unwanted adapter dimers and reduced library purity. Here, we report a new class of Non-Self-Ligating Blunt-End (NSL-BE) adapters enabled by the intrinsically asymmetric DNA-binding requirements of T4 DNA ligase, which imposes distinct minimal duplex-length constraints at the 3 '-hydroxyl and 5 '-phosphate sides of a ligation junction. By rationally designing blunt-end adapters consisting of a 5-bp duplex (serving as the blunt-end) and adjacent mismatches, self-ligation is effectively suppressed, while efficient ligation to target fragments is retained. Under standard T4 DNA ligase buffer conditions, NSL-BE adapters achieve >85% paired-end ligation efficiency, substantially outperforming T-adapters. Importantly, NSL-BE adapters incorporating only a minimal ligation module were successfully applied to high-throughput sequencing (Illumina) of genomic DNA, demonstrating their practical sequencability and compatibility with real library construction workflows. Modular extension of the adapter structure allows the incorporation of sequencing elements, functional domains, and nanostructure interfaces, supporting adaptation across diverse library formats and sequencing platforms. Beyond blunt-end ligation, this strategy has been successfully extended to sticky-end adapters with palindromic overhangs, demonstrating its broader utility. Altogether, this work presents a facile and powerful solution for eliminating adapter self-ligation with broad applications in high-throughput sequencing, biotechnology, and enzymology.

Vibrio anguillarum poses a serious threat to food safety, human health and aquaculture. At present, sensitive visual detection methods for V. anguillarum remain scarce. In this study, a novel dual-signal sensing system was constructed for the highly specific and visual detection of V. anguillarum. The system leverages loop-mediated isothermal amplification (LAMP) reaction to generate abundant AT-rich double-strand DNA products in the presence of the target pathogen. These products serve as templates for in-situ synthesis of copper nanoclusters (CuNCs), which exhibit both strong fluorescence and peroxidase-mimicking activity. The fluorescence intensity of CuNCs was positively correlated with the concentration of V. anguillarum, enabling quantitative fluorescence detection. Simultaneously, the CuNCs with peroxidase-like activity can catalyze the oxidation of colorless 3,3’,5,5’-tetramethylbenzidine (TMB) into blue oxTMB, allowing straightforward colorimetric readout. This dual-mode approach achieved outstanding sensitivity, with detection limits as low as 18 CFU/mL for fluorescence and 108 CFU/mL for colorimetry. The system also demonstrated high specificity against related pathogenic bacteria and was successfully applied to detect V. anguillarum in real turbot samples, showing excellent recovery and reliability. Notably, a closed-vial configuration effectively minimized aerosol-derived carryover contamination, and a portable colorimetric test paper further enabled visual point-of-care analysis. This work provides a robust and versatile strategy for rapid, visual, and accurate detection of V. anguillarum, and serves as a reference for developing detection platforms for other pathogens.

The sarcopenia burden is escalating in super-aged societies. The “gut-muscle axis” hypothesis suggests gut dysbiosis contributes to muscle wasting via inflammation and metabolic dysregulation, yet identifying consistent markers is complicated by heterogeneity. This study determined prevalence, risk factors, and microbiota signatures in a homogeneous cohort of community-dwelling men aged ≥ 75 receiving chronic disease management in Shanghai. We employed a cross-sectional design, enrolling 172 older men (mean age 82 ± 3 years). Sarcopenia was diagnosed using AWGS 2019 criteria. Comprehensive clinical data, including serological markers and medication history, were collected. Fecal samples from a subgroup (n = 45) underwent 16 S rRNA sequencing. Clinical risk factors were identified using backward stepwise multivariate logistic regression with Bootstrap internal validation. Multivariate logistic regression and Linear Discriminant Analysis Effect Size (LEfSe) were used to identify independent factors. To mitigate potential overfitting and small-sample bias in the microbiota analysis, complementary differential abundance testing (e.g., ANCOM-BC) and exploratory penalized multivariable modeling (LASSO feature selection and Firth’s penalized regression) were strictly applied. Sarcopenia prevalence was 25.6

Rational design of RNA-cleaving DNAzymes is an effective approach to enhance catalytic activity and broaden their applications in analytical chemistry. However, existing strategies often rely on peripheral stem-loop modifications and multi-molecular assembly, both of which compromise the universality of applications and structural controllability. In this study, we report an allosteric double-hairpin DNAzyme (hp-Dz)-based analytical strategy that directly employs two consecutive hairpins inserted into the catalytic core, providing new regulatory sites for activity modulation and aptamer integration without relying on pre-existing hairpin structures. The resulting hp-Dz exhibits up to a 1.8-fold enhancement in catalytic activity when configured with 6-bp stems and 3-nt loops. Furthermore, by replacing one of the hairpins with an Hg2+ specific aptamer, an allosteric aptazyme is engineered to enable Hg2+-triggered signal generation. This system allows facile Hg2+ detection with a limit of detection of 24.0 nM within 40 min. Overall, this work establishes a versatile allosteric DNAzyme platform for simplified and tunable analytical systems.

Given the resilience of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on frozen food, there is a risk that contaminated products could serve as vectors for viral transmission. Yet, methods capable of inactivating the virus at sub-zero temperatures without compromising the food's taste and quality are scarce. The high-pressure environment that arises spontaneously during isochoric freezing has demonstrated efficacy in suppressing or inactivating harmful microorganisms, such as bacteria; however, its effectiveness against coronavirus and the mechanisms involved remain unclear. In this study, we employed Porcine epidemic diarrhea virus (PEDV) as a proxy for SARS-CoV-2 to examine the effects of isochoric freezing on PEDV infectivity and to evaluate post-treatment alterations in the integrity of viral nucleic acids and envelopes, as well as changes in antigenic properties. Our experimental findings indicate that after a 6-h isochoric freezing treatment at -20 °C and 201 MPa, the titer decreased by 1.18 log10(TCID50/mL). While the viral nucleic acid remained intact post-treatment, the envelope's integrity was significantly impaired, accounting for the loss of infectivity. Moreover, the antigenic properties of the virus showed a slight increase following isochoric freezing treatment. This study pioneers the exploration of isochoric freezing technology's inactivation effects on coronaviruses, including preliminary mechanisms, offering novel perspectives for managing coronavirus contamination in frozen food and highlighting the potential of isochoric freezing in vaccine inactivation processes.

The objective of the current study was to investigate and compare the impact of enzymatic hydrolysis and pre-fermentation with probiotics on the biological activity and metabolic profile of melon juice. We found that fermentation with Lactiplantibacillus plantarum N13, not only decreased the juice pH, total soluble solids and reducing sugar, but also promoted the release of phenolic compounds, increased antioxidant activity and improved juice's aroma profile. Although pectinase and cellulase aided in the release of reducing sugar and total phenols in the juice, the heating process involved in the enzymatic treatment decreased total soluble solids and phenolics in the juice. Altogether, both enzymatic treatment and fermentation with probiotics could aid in the release of bioactive compounds, while fermentation with probiotics demonstrated greater impact on the biological activity and metabolic profile of melon juice. The findings of the current study suggested the use of probiotic cultivars in the pre-alcohol-fermentation process.

Rolling circle amplification (RCA) at ambient temperature is prone to false positive signals during nucleic acid detection, which makes it challenging to establish an efficient RCA detection method. The false positive signals are primarily caused by binding of non-target nucleic acids to the circular single-stranded template, leading to non-specific amplification. Here, we present an RCA method for miRNA detection at 37 °C using two circular ssDNAs, each of which is formed by ligating the intramolecularly formed nick (without any splint) in a secondary structure. The specific target recognition is realized by utilizing low concentrations (0.1 nM) of circular ssDNA1 (C1). A phosphorothioate modification is present at G*AATTC on C1 to generate a nick for primer extension during the primer self-generated rolling circle amplification (PG-RCA). The fragmented amplification products are used as primers for the following RCA that serves as signal amplification using circular ssDNA2 (C2). Notably, the absence of splints and the low concentration of C1 significantly inhibits non-target binding, thus minimizing false positive signals. A high concentration (10 nM) of C2 is used to carry out linear rolling circle amplification (LRCA), which is highly specific. This strategy demonstrates a good linear response to 0.01–100 pM of miRNA with a detection limit of 7.76 fM (miR-155). Moreover, it can distinguish single-nucleotide mismatch in the target miRNA, enabling the rapid one-pot detection of miRNA at 37 °C. Accordingly, this method performs with high specificity and sensitivity. This approach is suitable for clinical serum sample analysis and offers a strategy for developing specific biosensors and diagnostic tools.

Z-DNA, a left-handed DNA conformation, plays critical roles in transcriptional regulation, genetic recombination, genomic instability, immunity, and human diseases. In 2019, a stable LR-chimera containing Z-DNA (Lk = 0) under physiological ionic conditions was prepared by hybridizing two complementary circular ssDNAs. However, the difficulty in preparing circular ssDNA precursors and the excessively long Z-DNA segment in the chimera limit its applications. In this study, using a splint-free circularization method, we prepared two circular ssDNAs (each with a hairpin structure). Hybridization of these two circles whose loops are complementary (but not the two hairpins) yielded a Stem-LR chimera containing short Z-DNA and B-DNA and two hairpins that could not hybridize with each other. Stability analysis revealed that the 18-34 bp Z-DNA segment with only unmodified nucleotides in the Stem-LR chimera remained stable under physiological conditions (10 mM Mg2+, 37 °C). When hairpins were far apart (180°), multiple Stem-LR chimera isomers (varying in B-Z junction numbers and Z-DNA lengths) formed. Intriguingly, higher hybridization temperatures (60 °C) favored continuous B-DNA and Z-DNA segments (minimal B-Z junctions). When hairpins were adjacent (0° orientation), exclusively continuous B-DNA/Z-DNA was obtained, even for hybridization at 10 °C. As expected, Stem-LR chimeras exhibited enhanced resistance to topoisomerase I compared to chimeras without hairpins. This approach holds promise for delivery into cells or organisms to investigate the impact of Z-DNA and its biological functions under physiological conditions.

Nucleic acids are significant components of daily diet and have attracted attention regarding their metabolic and nutritional roles. Numerous studies have explored the biological functions of nucleotides, nucleosides, and functional nucleic acids like microRNAs. However, the nutritional value and metabolic mechanisms of RNA oligonucleotides derived from ribosomal RNA (rRNA)—a major form of nucleic acids in nature remain underexplored. Here, yeast was utilized as a model organism to investigate the absorption and metabolism of oligonucleotides obtained from rRNA. We cultured yeast directly using RNA oligonucleotides as one nutrient, demonstrating that yeast can efficiently utilize RNA oligonucleotides (length < 30 nt) as a nitrogen source. Through proteomic analysis to assess the expression levels of key proteins associated with transport and metabolic processes, we found that the key proteins involved in endocytosis, autophagy, and RNA degradation were upregulated. These results clearly demonstrate that yeast directly uptakes RNA oligonucleotides via endocytosis, which are subsequently degraded into nucleosides, ammonia and β-Alanine through autophagy and RNA degradation, thus providing substrates for synthesizing nucleic acid and other organic nitrogenous metabolites. Our findings and proposed mechanisms for RNA absorption and metabolism in eukaryotic cells can promote future research in both nutrition and nucleic acid metabolism.

The larynx is vital for swallowing, breathing, coughing, and voice production, supported by its unique microbial and immunological environment. We hypothesized the existence of a gut-larynx axis, where resident gut and laryngeal microbiota influence immune modulation in the larynx. To test this, conventionally raised, wild-type C57BL/6 J mice were treated with an oral antibiotic regimen to disrupt gut microbiota and compared with untreated controls. Antibiotic treatment significantly disrupted gut microbiota but left laryngeal microbiota largely unaffected. However, antibiotic-treated mice showed notable changes in laryngeal epithelial and immune cells, as well as fibroblasts. Differential gene expression analysis revealed alterations in pathways related to epithelial barrier integrity, immune signaling, and bacterial response. Gene regulatory network analysis identified significant changes in regulons Etv4(+), Irf3(+), Hltf(+), Mga(+), and Nfil3(+). Additionally, cell-cell communication, particularly immune-epithelial interactions, was altered, with integrin-mediated signaling emerging as a key pathway. These findings suggest that gut and laryngeal microbiota may synergistically modulate immune responses, highlighting the importance of gut-larynx interactions in respiratory immunity. IMPORTANCE:This study investigates the gut-larynx axis, revealing how gut dysbiosis impacts immune responses in the larynx. Although laryngeal microbiota remained stable, significant immunological and cellular changes occurred following gut microbiota disruption. Transcriptomic alterations in epithelial integrity, immune signaling, and cell communication underscore the systemic impact of gut dysbiosis. The identification of integrin-mediated signaling as a key pathway in immune-epithelial interactions emphasizes the complexity of host-microbe dynamics. These findings suggest that gut health plays a critical role in shaping respiratory immunity, providing a foundation for future research into microbiota-driven immune modulation in the upper airway.

Short DNA catenanes [circular double-stranded DNA (dsDNA)] have attracted considerable interest for constructing nanostructures and nanomachines, as well as understanding DNA topology. The study of topoisomers of a circular dsDNA with a definite linking number (Lk) is essential but very difficult for simplifying the complex problems about DNA topology. The topoisomers are difficult to prepare, especially in the case that two strands are completely complementary. In this study, using a model system, we prepared all eight topoisomers (Lk0-Lk7) of a 79-bp-long circular dsDNA (8-14 nm in size) by utilizing aid-DNA to prevent undesired hybridization. By rapid ligation before strand displacement, high selectivity (>75%) for most topoisomers (31% for Lk1) was achieved under the strict topological control. All eight topoisomers with high purity were obtained after purification. Using a gel shift assay with Z-DNA-specific binding proteins, as well as by circular dichroism chromatography and enzymatic digestion, it was found that Z-DNA forms for topoisomers Lk0-Lk6, and Lk0-Lk5 can be converted to Lk6 by topoisomerase I. The approach developed in this study can significantly contribute to DNA or RNA topology, particularly the effect of topological constraints on DNA structures and functions.

Consumption of fructo- (FOS) and galacto-oligosaccharides (GOS) has health benefits which have been linked in part to short-chain fatty acids (SCFA) production by the gut microbiota. However, detailed knowledge of this process in the human intestine is lacking. We aimed to determine the acute fermentation kinetics of a FOS:GOS mixture in healthy males using a naso-intestinal catheter for sampling directly in the ileum or colon. We studied the fate of SCFA as substrates for glucose and lipid metabolism by the host after infusion of 13C-SCFA. In the human distal ileum, no fermentation of FOS:GOS, nor SCFA production, or bacterial cross-feeding was observed. The relative composition of intestinal microbiota changed rapidly during the test day, which demonstrates the relevance of postprandial intestinal sampling to track acute responses of the microbial community toward interventions. SCFA were vividly taken up and metabolized by the host as shown by incorporation of 13C in various host metabolites.

The efficient preparation of single-stranded DNA (ssDNA) rings, as a macromolecular construction approach with topological features, has aroused much interest due to the ssDNA rings' numerous applications in biotechnology and DNA nanotechnology. However, an extra splint is essential for enzymatic circularization, and by-products of multimers are usually present at high concentrations. Here, we proposed a simple and robust strategy using permuted precursor (linear ssDNA) for circularization by forming an intramolecular dynamic nick using a part of the linear ssDNA substrate itself as the template. After the simulation of the secondary structure for desired circular ssDNA, the linear ssDNA substrate is designed to have its ends on the duplex part (≥5 bp). By using this permuted substrate with 5'-phosphate, the splint-free circularization is simply carried out by T4 DNA ligase. Very interestingly, formation of only several base pairs (2-4) flanking the nick is enough for ligation, although they form only instantaneously under ligation conditions. More significantly, the 5-bp intramolecular duplex part commonly exists in genomes or functional DNA, demonstrating the high generality of our approach. Our findings are also helpful for understanding the mechanism of enzymatic DNA ligation from the viewpoint of substrate binding.

By forming a nick instantaneously, nucleic acids are efficiently adenylated by T4 DNA ligase without further ligation.

Microbiota play a critical role in the development and training of host innate and adaptive immunity. We present the cellular landscape of the upper airway, specifically the larynx, by establishing a reference single-cell atlas, while dissecting the role of microbiota in cell development and function at single-cell resolution. We highlight the larynx's cellular heterogeneity with the identification of 16 cell types and 34 distinct subclusters. Our data demonstrate that commensal microbiota have extensive impact on the laryngeal immune system by regulating cell differentiation, increasing the expression of genes associated with host defense, and altering gene regulatory networks. We uncover macrophages, innate lymphoid cells, and multiple secretory epithelial cells, whose cell proportions and expressions vary with microbial exposure. These cell types play pivotal roles in maintaining laryngeal and upper airway health and provide specific guidance into understanding the mechanism of immune system regulation by microbiota in laryngeal health and disease.

Although fecal microbiota composition is considered to preserve relevant and representative information for distal colonic content, it is evident that it does not represent microbial communities inhabiting the small intestine. Nevertheless, studies investigating the human small intestinal microbiome and its response to dietary intervention are still scarce. The current study investigated the spatio-temporal dynamics of the small intestinal microbiome within a day and over 20 days, as well as its responses to a 14-day synbiotic or placebo control supplementation in 20 healthy subjects. Microbial composition and metabolome of luminal content of duodenum, jejunum, proximal ileum and feces differed significantly from each other. Additionally, differences in microbiota composition along the small intestine were most pronounced in the morning after overnight fasting, whereas differences in composition were not always measurable around noon or in the afternoon. Although overall small intestinal microbiota composition did not change significantly within 1 day and during 20 days, remarkable, individual-specific temporal dynamics were observed in individual subjects. In response to the synbiotic supplementation, only the microbial diversity in jejunum changed significantly. Increased metabolic activity of probiotic strains during intestinal passage, as assessed by metatranscriptome analysis, was not observed. Nevertheless, synbiotic supplementation led to a short-term spike in the relative abundance of genera included in the product in the small intestine approximately 2 hours post-ingestion. Collectively, small intestinal microbiota are highly dynamic. Ingested probiotic bacteria could lead to a transient spike in the relative abundance of corresponding genera and ASVs, suggesting their passage through the entire gastrointestinal tract. This study was registered to http://www.clinicaltrials.gov, NCT02018900.

DNase II, identified in 1947 and named in 1953, is an acidic DNA endonuclease prevalent across organisms and crucial for normal growth. Despite its expression in nearly all human tissues, as well as its biological significance, DNase II’s detailed functions and corresponding mechanisms remain unclear. Although many groups are trying to figure this out, progress is very limited. It is very hard to connect its indispensability with its DNA cleavage activity. In this study, we find that DNase II secreted to saliva can digest RNA in mildly acidic conditions, prompting us to hypothesize that salivary DNase II might digest RNA in the stomach. This finding is consistent with the interesting discovery reported in 1964 that RNA could inhibit DNase II’s activity, which has been largely overlooked. This RNA digestion activity is further confirmed by using purified DNase II, showing activity to digest both DNA and RNA effectively. Here, we suggest redesignating DNase II as DNase II (RNase). The biological functions of DNase II are suggested to recycle intracellular RNA or digest external nucleic acids (both RNA and DNA) as nutrients. This discovery may untangle the mystery of DNase II and its significant biofunctions.