Phylogenetic relationships of the Mangrove Hummingbird, "Amazilia" boucardi (Apodiformes: Trochilidae) of Costa Rica / Relaciones filogenéticas del Colibrí manglero "Amazilia" boucardi (Apodiformes: Trochilidae) de Costa Rica

Abstract Introduction: A recent revision of the generic classification of the Trochilidae based on DNA sequences revealed many inconsistencies with the current generic classification, largely based on plumage characters subject to homoplasy, especially in the Trochilini, the largest tribe. A thorough generic reorganization brought the classification into accord with the phylogeny, but due to lack of genetic data, two species remained unclassified. One of these was the Mangrove Hummingbird, "Amazilia" boucardi, endemic to Costa Rica and included in the IUCN red list of threatened species. Objective: To obtain molecular evidence to clarify the generic relationships of "A." boucardi. Methods: We isolated DNA from tissues of this species and amplified 4 nuclear and 4 mitochondrial fragments and compared these with homologous fragments from 56 species in the Trochilini, constructing phylogenetic trees with maximum likelihood and Bayesian methods. Results: Our phylogenetic analyses confirmed the placement of boucardi in the Trochilini and definitely excluded it from Amazilia but placed it with high confidence in the genus Chrysuronia Bonaparte, 1850, within which its closest relative is C. coeruleogularis, which also inhabits mangroves. Conclusions: Our genetic data based on nuclear and mitochondrial regions clearly indicate the relationship of A. boucardi and L. coeruleogularis. Moreover, it is also supported by their habitat distribution in the mangroves of the Pacific coast of Costa Rica and Western Panama. Therefore, we suggested to exclude A. boucardi as "incertae sedis".

Resumen Introducción: Una revisión reciente de la clasificación de la familia Trochilidae con base en secuencias de ADN demostró muchas incongruencias con la clasificación genérica previa, que había sido hecho con base en caracteres del plumaje muy sujetos a homoplasia, especialmente en la tribu más grande, Trochillini. Una reorganización de los géneros logró llevar su clasificación genérica a la concordancia con la filogenia, pero debido a la ausencia de datos genéticos, dos especies permanecieron sin clasificar. Una de estas fue el colibrí de manglar Amazilia boucardi, una especie endémica de Costa Rica, considerada como amenazada en la lista roja de la UICN. Objetivo: Obtener evidencia molecular para esclarecer las relaciones genéricas de A. boucardi. Métodos: Se aisló ADN de tejidos de esta especie y se amplificaron 4 fragmentos de ADN del núcleo y 5 de la mitocondria, y se compararon con fragmentos homólogos de 56 especies en la tribu Trochillini, generando árboles filogenéticos con métodos de máxima verosimilitud y bayesiano. Resultados: Los análisis filogénticos obtenidos confirmaron la ubicación de boucardi en Trochilini y definitivamente la excluyó del género Amazilia, pero la ubicó con un alto grado de confianza en el género Chrysuronia Bonaparte, 1850, dentro los cuales su pariente más cercano es C. coeruleogularis, que también habita manglares. Conclusiones: Nuestros datos genéticos basados en regiones nucleares y mitocondriales indican claramente la relación entre A. boucardi and L. coeruleogularis. Es más, lo anterior se sustenta por su distribución en los manglares de la costa Pacífica de Costa Rica y oeste de Panamá. Por lo tanto, sugerimos excluir a A. boucardi como "incertae sedis".

Ultrasensitive Electrochemiluminescence Biosensor Based on DNA-Bio-Bar-Code and Hybridization Chain Reaction Dual Signal Amplification for Exosomes Detection.

Exosomes have received considerable attention as potent reference markers for the diagnosis of various neoplasms due to their close and direct relationship with the proliferation, adhesion, and migration of tumor. The ultrasensitive detection of cancer-derived low-abundance exosomes is imperative, but still a great challenge. Herein, we report an electrochemiluminescence (ECL) biosensor based on the DNA-bio-bar-code and hybridization chain reaction (HCR)-mediated dual signal amplification for the ultrasensitive detection of cancer-derived exosomes. In this system, two types of aptamers were modified on the magnetic nanoprobe (MNPs) and gold nanoparticles (AuNPs) with numerous bio-bar-code DNA, respectively, which formed "sandwich" structures in the presence of specific target exosomes. The "sandwich" structures were separated under magnetic field, and the numerous bio-bar-code DNA were released by dissolving AuNPs. The released bio-bar-code DNA triggered the HCR procedure to produce a good deal of long DNA duplex structure for embedding in hemin, which generated strong ECL signal in the presence of coreactors for ultrasensitive detection of exosomes. Under the optimal conditions, it exhibited a good linearly of exosomes ranging from 10 to 104 exosomes particle µL-1 with limit of detection down to 5.01 exosome particle µL-1. Furthermore, the high ratio of ECL signal and minor change of ECL intensity indicated the good specificity, stability, and repeatability of this ECL biosensor. Given the good performance for exosome analysis, this ultrasensitive ECL biosensor has a promising application in the clinical diagnosis of early cancers.

Single Methylation Sensitive Restriction Endonuclease-Based Cascade Exponential Amplification Assay for Visual Detection of DNA Methylation at Single-Molecule Level.

Function as a potential cancer biomarker, DNA methylation shows great significance in cancer diagnosis, prognosis, and treatment monitoring. While the lack of an ultrasensitive, specific, and accurate method at the single-molecule level hinders the analysis of the exceedingly low levels of DNA methylation. Herein, based on the outstanding recognition and digestion ability of methylation-sensitive restriction endonuclease (MSRE), we established a single MSRE-based cascade exponential amplification method, which requires only two ingeniously designed primers and only one recognition site of MSRE for the detection of DNA methylation. Differentiated by MSRE digestion, the cleaved unmethylated DNA is too short to induce any amplification reactions, while methylated DNA remains intact to trigger cascade exponential amplification and the subsequent CRISPR/Cas12a system. By integrating the two exponential amplification reactions, as low as 1 aM methylated DNA can be accurately detected, which corresponds to 6 molecules in a 10 µL system, indicating that our method is more sensitive than single amplification-based methods with the ability to detect DNA methylation at the single-molecule level. In addition, 0.1% methylated DNA can be effectively distinguished from large amounts of unmethylated DNA. Our method is further introduced to exploit the expression difference of DNA methylation among normal cells and cancer cells. Moreover, the visual detection of DNA methylation is also realized by the full hybridization between amplification products and the crRNA of CRISPR/Cas12a. Therefore, the proposed method has great potential to be a promising and robust bisulfite-free method for the detection of DNA methylation at the single-molecule level, which is of great importance for early diagnosis of cancer.

Enhanced Recognition of a Herbal Compound Epiberberine by a DNA Quadruplex-Duplex Structure.

The small molecule epiberberine (EPI) is a natural alkaloid with versatile bioactivities against several diseases including cancer and bacterial infection. EPI can induce the formation of a unique binding pocket at the 5' side of a human telomeric G-quadruplex (HTG) sequence with four telomeric repeats (Q4), resulting in a nanomolar binding affinity (KD approximately 26 nM) with significant fluorescence enhancement upon binding. It is important to understand (1) how EPI binding affects HTG structural stability and (2) how enhanced EPI binding may be achieved through the engineering of the DNA binding pocket. In this work, the EPI-binding-induced HTG structure stabilization effect was probed by a peptide nucleic acid (PNA) invasion assay in combination with a series of biophysical techniques. We show that the PNA invasion-based method may be useful for the characterization of compounds binding to DNA (and RNA) structures under physiological conditions without the need to vary the solution temperature or buffer components, which are typically needed for structural stability characterization. Importantly, the combination of theoretical modeling and experimental quantification allows us to successfully engineer Q4 derivative Q4-ds-A by a simple extension of a duplex structure to Q4 at the 5' end. Q4-ds-A is an excellent EPI binder with a KD of 8 nM, with the binding enhancement achieved through the preformation of a binding pocket and a reduced dissociation rate. The tight binding of Q4 and Q4-ds-A with EPI allows us to develop a novel magnetic bead-based affinity purification system to effectively extract EPI from Rhizoma coptidis (Huang Lian) extracts.

The unusual structural properties and potential biological relevance of switchback DNA.

Synthetic DNA motifs form the basis of nucleic acid nanotechnology. The biochemical and biophysical properties of these motifs determine their applications. Here, we present a detailed characterization of switchback DNA, a globally left-handed structure composed of two parallel DNA strands. Compared to a conventional duplex, switchback DNA shows lower thermodynamic stability and requires higher magnesium concentration for assembly but exhibits enhanced biostability against some nucleases. Strand competition and strand displacement experiments show that component sequences have an absolute preference for duplex complements instead of their switchback partners. Further, we hypothesize a potential role for switchback DNA as an alternate structure in sequences containing short tandem repeats. Together with small molecule binding experiments and cell studies, our results open new avenues for switchback DNA in biology and nanotechnology.

BertSNR: an interpretable deep learning framework for single-nucleotide resolution identification of transcription factor binding sites based on DNA language model.

MOTIVATION: Transcription factors are pivotal in the regulation of gene expression, and accurate identification of transcription factor binding sites (TFBSs) at high resolution is crucial for understanding the mechanisms underlying gene regulation. The task of identifying TFBSs from DNA sequences is a significant challenge in the field of computational biology today. To address this challenge, a variety of computational approaches have been developed. However, these methods face limitations in their ability to achieve high-resolution identification and often lack interpretability. RESULTS: We propose BertSNR, an interpretable deep learning framework for identifying TFBSs at single-nucleotide resolution. BertSNR integrates sequence-level and token-level information by multi-task learning based on pre-trained DNA language models. Benchmarking comparisons show that our BertSNR outperforms the existing state-of-the-art methods in TFBS predictions. Importantly, we enhanced the interpretability of the model through attentional weight visualization and motif analysis, and discovered the subtle relationship between attention weight and motif. Moreover, BertSNR effectively identifies TFBSs in promoter regions, facilitating the study of intricate gene regulation. AVAILABILITY AND IMPLEMENTATION: The BertSNR source code can be found at https://github.com/lhy0322/BertSNR.

Quantitative analysis of dose dependent DNA fragmentation in dry pBR322 plasmid using long read sequencing and Monte Carlo simulations.

Exposure to ionizing radiation can induce genetic aberrations via unrepaired DNA strand breaks. To investigate quantitatively the dose-effect relationship at the molecular level, we irradiated dry pBR322 plasmid DNA with 3 MeV protons and assessed fragmentation yields at different radiation doses using long-read sequencing from Oxford Nanopore Technologies. This technology applied to a reference DNA model revealed dose-dependent fragmentation, as evidenced by read length distributions, showing no discernible radiation sensitivity in specific genetic sequences. In addition, we propose a method for directly measuring the single-strand break (SSB) yield. Furthermore, through a comparative study with a collection of previous works on dry DNA irradiation, we show that the irradiation protocol leads to biases in the definition of ionizing sources. We support this scenario by discussing the size distributions of nanopore sequencing reads in the light of Geant4 and Geant4-DNA simulation toolkit predictions. We show that integrating long-read sequencing technologies with advanced Monte Carlo simulations paves a promising path toward advancing our comprehension and prediction of radiation-induced DNA fragmentation.

Altering traits and fates of wild populations with Mendelian DNA sequence modifying Allele Sails.

Population-scale genome modification can alter the composition or fate of wild populations. Synthetic gene drives provide one set of tools, but their use is complicated by scientific, regulatory, and social issues associated with transgene persistence and flow. Here we propose an alternative approach. An Allele Sail consists of a genome editor (the Wind) that introduces DNA sequence edits, and is inherited in a Mendelian fashion. Meanwhile, the edits (the Sail) experience an arithmetic, Super-Mendelian increase in frequency. We model this system and identify contexts in which a single, low frequency release of an editor brings edits to a very high frequency. We also identify conditions in which manipulation of sex determination can bring about population suppression. In regulatory frameworks that distinguish between transgenics (GMO) and their edited non-transgenic progeny (non-GMO) Allele Sails may prove useful since the spread and persistence of the GM component can be limited.

Rapid enzyme-free detection of miRNA-21 in human ovarian cancerous cells using a fluorescent nanobiosensor designed based on hairpin DNA-templated silver nanoclusters.

BACKGROUND: Cancer is known as one of the main non-communicable diseases and the leading cause of death in the new era. Early diagnosis of cancer requires the identification of special biomarkers. Currently, microRNAs (miRNAs) have attracted the attention of researchers as useful biomarkers for cancer early detection. Hence, various methods have been recently developed for detecting and monitoring miRNAs. Among all miRNAs, detection of miRNA-21 (miR-21) is important because it is abnormally overexpressed in most cancers. Here, a new biosensor based on silver nanoclusters (AgNCs) is introduced for detecting miR-21. RESULTS: As a fluorescent probe, a rationally designed hairpin sequence containing a poly-cytosine motif was used to facilitate the formation of AgNCs. A guanine-rich sequence was also employed to enhance the sensing signal. It was found that in the absence of miR-21, adding a guanine-rich sequence to the detecting probe caused only a slight change in the fluorescence emission intensity of AgNCs. While in the presence of miR-21, the emission signal enhanced. A direct correlation was observed between the increase in the fluorescence of AgNCs and the concentration of miR-21. The performance of the proposed biosensor was characterized thoroughly and confirmed. The biosensor detected miR-21 in an applicable linear range from 9 pM to 1.55 nM (LOD: 2 pM). SIGNIFICANCE: The designed biosensor was successfully applied for detecting miR-21 in human plasma samples and also in human normal and lung and ovarian cancer cells. This biosensing strategy can be used as a model for detecting other miRNAs. The designed nanobiosensor can measure miR-21 without using any enzymes, with fewer experimental steps, and at a low cost compared to the reported biosensors in this field.

[Application of EXODUS system combined with allosteric DNA nanoswitches in the detection of miR-107 among plasma exosomes of Parkinson's disease patients].

This study aimed to achieve rapid detection of Parkinson's disease (PD) plasma exosome miR-107. A case-control design was used to collect ten Parkinson's disease and ten healthy control plasma samples from the Department of Laboratory Medicine, Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology from December 2023 to January 2024. Exosome detection via the ultrafast-isolation system (EXODUS) was used to isolate plasma exosomes. The nanoparticle tracking analysis technology and electron microscopy were used to identify exosome particle size and morphology. The Qiagen miRNeasy Micro Kit was used to extract RNA. The microRNA-activated conditional looping of engineered switches (miRacles) was used to detect miR-107, and the relative expression was analyzed by agarose gel electrophoresis. Thermo Fisher RevertAid RT Reverse Transcription Kit was used to perform reverse transcription of RNA, and real-time PCR was used to detect miR-107. The independent samples t-test was used for comparison between groups. EXODUS system completed the isolation of exosomes from 500 µl plasma within 1.5 hours. The exosome concentration (mean±SD) was (4.82±2.02)×1010 particles/ml in the control group and (5.08±2.34)×1010 particles/ml in the PD group. There was no significant difference in exosome concentration between PD patients and healthy controls (t=-0.168, P=0.872). The morphology of exosomes was confirmed by electron microscopy. The miRacles nanoswitch could detect fM-level miR-107 and also effectively distinguish miR-107 from its family members, including miR-15a and miR-16. Agarose gel electrophoresis showed that the mean±SD of relative grey value content was 1.00±0.26 in the control group and 1.86±0.21 in the PD group. The miR-107 in the PD group was significantly higher than that in the control (t=-8.143, P<0.001), which was consistent with the result of real-time PCR. EXODUS combined with miRacles could achieve rapid, non-enzymatic and cheap detection of plasma exosomal miR-107 in PD patients.

Resilience of DNA chains to molecular fracture after PCR heating cycles and implications on PCR reliability.

Soon after its introduction in 1987, polymerase chain reaction (PCR) has become a technique widely employed in diagnostic medical devices and forensic science with the intention of amplifying genetic information. PCR prescribes that each of its cycles must include a heating subprocess at 95 °C or more (denominated DNA denaturation and provided for allowing a claimed orderly separation of the two complementary nucleotides strands), which can produce significant damage to DNA, caused by high-speed collisions with surrounding molecules. Since such disruption should be prevented in order to reliably employ PCR, a study of the mechanics of such loss of structural integrity is herein presented, preceded by a review of the fundamental literature which has elucidated the effects of molecular agitation on DNA fragmentation. The main conclusion of this retrospective survey is that the body of examined theoretical and experimental evidence consistently and redundantly confirms scarce resilience and significant loss of structural integrity when DNA is heated at temperatures above 90 °C, even for 1 minute. Such conclusion contradicts the claimed paradigm of PCR fidelity and raises the concern that, at least for long sequences, if PCR can amplify some information, such amplified information may be unreliable for diagnostic or forensic applications, since it originates from sequences of nucleotides subjected to random fragmentation and reaggregation. Such a low-reliability scenario should be preventively considered in the various fields where DNA amplification methodologies are employed which provide for high-temperature heating under conditions equal to or similar to those prescribed by the PCR protocols reviewed in this study.

Effect of ethanol on the elasticities of double-stranded RNA and DNA revealed by magnetic tweezers and simulations.

The elasticities of double-stranded (ds) DNA and RNA, which are critical to their biological functions and applications in materials science, can be significantly modulated by solution conditions such as ions and temperature. However, there is still a lack of a comprehensive understanding of the role of solvents in the elasticities of dsRNA and dsDNA in a comparative way. In this work, we explored the effect of ethanol solvent on the elasticities of dsRNA and dsDNA by magnetic tweezers and all-atom molecular dynamics simulations. We found that the bending persistence lengths and contour lengths of dsRNA and dsDNA decrease monotonically with the increase in ethanol concentration. Furthermore, the addition of ethanol weakens the positive twist-stretch coupling of dsRNA, while promotes the negative twist-stretch coupling of dsDNA. Counter-intuitively, the lower dielectric environment of ethanol causes a significant re-distribution of counterions and enhanced ion neutralization, which overwhelms the enhanced repulsion along dsRNA/dsDNA, ultimately leading to the softening in bending for dsRNA and dsDNA. Moreover, for dsRNA, ethanol causes slight ion-clamping across the major groove, which weakens the major groove-mediated twist-stretch coupling, while for dsDNA, ethanol promotes the stretch-radius correlation due to enhanced ion binding and consequently enhances the helical radius-mediated twist-stretch coupling.

A mean-field theory for characterizing the closing rates of DNA origami hinges.

The evolution of dynamic DNA nanostructures has propelled DNA nanotechnology into a robust and versatile field, offering groundbreaking applications in nanoscale communication, drug delivery, and molecular computing. Yet, the full potential of this technology awaits further enhancement through optimization of kinetic properties governing conformational changes. In this work, we introduce a mean-field theory to characterize the kinetic behavior of a dynamic DNA origami hinge where each arm bears complementary single-stranded DNA overhangs of different lengths, which can latch the hinge at a closed conformation. This device is currently being investigated for multiple applications, being of particular interest the development of DNA-based rapid diagnostic tests for coronavirus. Drawing from classical statistical mechanics theories, we derive analytical expressions for the mean binding time of these overhangs within a constant hinge. This analysis is then extended to flexible hinges, where the angle diffuses within a predetermined energy landscape. We validate our model by comparing it with experimental measurements of the closing rates of DNA nanocalipers with different energy landscapes and overhang lengths, demonstrating excellent agreement and suggesting fast angular relaxation relative to binding. These findings offer insights that can guide the optimization of devices for specific state lifetimes. Moreover, the framework introduced here lays the groundwork for further advancements in modeling the kinetics of dynamic DNA nanostructures.

DNA-DISK: Automated end-to-end data storage via enzymatic single-nucleotide DNA synthesis and sequencing on digital microfluidics.

In the age of information explosion, the exponential growth of digital data far exceeds the capacity of current mainstream storage media. DNA is emerging as a promising alternative due to its higher storage density, longer retention time, and lower power consumption. To date, commercially mature DNA synthesis and sequencing technologies allow for writing and reading of information on DNA with customization and convenience at the research level. However, under the disconnected and nonspecialized mode, DNA data storage encounters practical challenges, including susceptibility to errors, long storage latency, resource-intensive requirements, and elevated information security risks. Herein, we introduce a platform named DNA-DISK that seamlessly streamlined DNA synthesis, storage, and sequencing on digital microfluidics coupled with a tabletop device for automated end-to-end information storage. The single-nucleotide enzymatic DNA synthesis with biocapping strategy is utilized, offering an ecofriendly and cost-effective approach for data writing. A DNA encapsulation using thermo-responsive agarose is developed for on-chip solidification, not only eliminating data clutter but also preventing DNA degradation. Pyrosequencing is employed for in situ and accurate data reading. As a proof of concept, DNA-DISK successfully stored and retrieved a musical sheet file (228 bits) with lower write-to-read latency (4.4 min of latency per bit) as well as superior automation compared to other platforms, demonstrating its potential to evolve into a DNA Hard Disk Drive in the future.

Molecular and toxicological mechanisms behind the effects of chromium (VI) on the male reproductive system of Mytilus galloprovincialis: First evidence for poly-ADP-ribosylation of protamine-like II.

Studies on the molecular mechanisms of heavy metal toxicity in invertebrate reproduction are limited. Given that PARP-catalysed ADP-ribosylation is also involved in counteracting heavy metal toxicity and maintaining genomic integrity, and that PARylation is implicated in chromatin remodelling but its role in sperm chromatin remains to be elucidated, we investigated the effects of chromium(VI) at 1, 10 and 100 nM on the reproductive health of Mytilus galloprovincialis. The damage to the gonads was assessed by morphological analyses and the damage indices PARP and É£H2A.X were measured. Changes in the binding of protamine-like (PL) to DNA and the possibility of poly(ADP-ribosyl)ation of PL proteins were also investigated. Gonadal chromium accumulation and morphological damage were found, especially when the mussels were exposed to the highest dose of chromium(VI). In addition, the maximum expression of gonadal É£H2A.X and PARP were obtained at 100 and 10 nM Cr(VI), respectively. Interestingly, for the first time in all exposed conditions, poly(ADP)-ribosylation was detected on PL-II, which, together with PL-III and PL-IV, are the major nuclear basic proteins of Mytilus galloprovincialis sperm chromatin. Since PL-II is involved in the final high level of sperm chromatin compaction, this post-translational modification altered the binding of the PL protein to DNA, favouring the action of micrococcal nuclease on sperm chromatin. This study provides new insights into the effects of chromium(VI) on Mytilus galloprovincialis reproductive system and proposes a molecular mechanism hypothesis describing the toxic effects of this metal on PL-DNA binding, sperm chromatin and gonads.

DSS1 restrains BRCA2's engagement with dsDNA for homologous recombination, replication fork protection, and R-loop homeostasis.

DSS1, essential for BRCA2-RAD51 dependent homologous recombination (HR), associates with the helical domain (HD) and OB fold 1 (OB1) of the BRCA2 DSS1/DNA-binding domain (DBD) which is frequently targeted by cancer-associated pathogenic variants. Herein, we reveal robust ss/dsDNA binding abilities in HD-OB1 subdomains and find that DSS1 shuts down HD-OB1's DNA binding to enable ssDNA targeting of the BRCA2-RAD51 complex. We show that C-terminal helix mutations of DSS1, including the cancer-associated R57Q mutation, disrupt this DSS1 regulation and permit dsDNA binding of HD-OB1/BRCA2-DBD. Importantly, these DSS1 mutations impair BRCA2/RAD51 ssDNA loading and focus formation and cause decreased HR efficiency, destabilization of stalled forks and R-loop accumulation, and hypersensitize cells to DNA-damaging agents. We propose that DSS1 restrains the intrinsic dsDNA binding of BRCA2-DBD to ensure BRCA2/RAD51 targeting to ssDNA, thereby promoting optimal execution of HR, and potentially replication fork protection and R-loop suppression.

Ultrafast DNA detection based on turn-back loop primer-accelerated LAMP (TLAMP).

Rapid DNA detection is a long-pursuing goal in molecular detection, especially in combating infectious diseases. Loop-mediated isothermal amplification (LAMP) is a robust and prevailing DNA detection method in pathogen detection, which has been drawing broad interest in improving its performance. Herein, we reported a new strategy and developed a new LAMP variant named TLAMP with a superior amplification rate. In this strategy, the turn-back loop primers (TLPs) were devised by ingeniously extending the 5' end of the original loop primer, which conferred the new role of being the inner primer for TLPs while retaining its original function as the loop primer. In theory, based on the bifunctional TLPs, a total of eight basic dumbbell-like structures and four cyclic amplification pathways were produced to significantly enhance the amplification efficiency of TLAMP. With the enhancing effect of TLPs, TLAMP exhibited a significantly reduced amplification-to-result time compared to the conventional six-primer LAMP (typically 1 h), enabling rapid DNA detection within 20 min. Furthermore, TLAMP proved to be about 10 min faster than the fast LAMP variants reported so far, while still presenting comparable sensitivity and higher repeatability. Finally, TLAMP successfully achieved an ultrafast diagnosis of Monkeypox virus (MPXV), capable of detecting as few as 10 copies (0.67copies/µL) of pseudovirus within 20 min using real-time fluorescence assay or within 30 min using a colorimetric assay, suggesting that the proposed TLAMP offers a sensitive, specific, reliable, and, most importantly, ultrafast DNA detection method when facing the challenges posed by infectious diseases.

Dual amplification dynamic DNA network system for CRISPR/Cas12a based p53 gene detection.

BACKGROUND: It is estimated that over 50 % of human cancers are caused by mutations in the p53 gene. Early sensitive and accurate detection of the p53 gene is important for diagnosis of cancers in the early stage. However, conventional detection techniques often suffer from strict reaction conditions, or unsatisfied sensitivity, so we need to develop a new strategy for accurate detection of p53 gene with smart designability, multiple signal amplification in mild reaction conditions. RESULTS: In this study, CRISPR/Cas system is exploited in entropy-driven catalysis (EDC) and hybridization chain reaction (CHA) dual signal amplification sensing strategies. The products of both reactions can efficiently and separately activate CRISPR/Cas12a which greatly amplifies the fluorescent signal. The method has good linearity in p53 detection with the concentration ranged from 0.1 fM to 0.5 pM with ultra-low detection limit of 0.096 fM. It also showed good performance in serum, offering potentials for early disease detection. SIGNIFICANCE: The designed dual amplification dynamic DNA network system exhibits an ultra-sensitive fluorescence biosensing for p53 gene identification. The method is simple to operate and requires only one buffer for the experiment, and meanwhile shows smart designability which could be used for a wide range of markers. Thus, we believe the present work will provide a potential tool for the construction and development of sensitive fluorescent biosensors for diseases.

3D DNA origami pincers that multitask on giant unilamellar vesicles.

Proteins self-assemble to function in living cells. They may execute essential tasks in the form of monomers, complexes, or supramolecular cages via oligomerization, achieving a sophisticated balance between structural topology and functional dynamics. The modularity and programmability make DNA origami unique in mimicking these key features. Here, we demonstrate three-dimensional reconfigurable DNA origami pincers (DOPs) that multitask on giant unilamellar vesicles (GUVs). By programmably adjusting their pinching angle, the DOPs can dynamically control the degree of GUV remodeling. When oligomerized on the GUV to form origami cages, the DOP units interact with one another and undergo reorganization, resulting in the capture, compartmentalization, and detachment of lipid fragments. This oligomerization process is accompanied with membrane disruptions, enabling the passage of cargo across the membrane. We envisage that interfacing synthetic cells with engineered, multifunctional DNA nanostructures may help to confer customized cellular properties, unleashing the potential of both fields.