Integrating aptasensor with an explosive mass-tag signal amplification strategy for ultrasensitive and multiplexed analysis using a miniature mass spectrometer.

Mass probes attached with aptamers and mass tags offer excellent specificity and sensitivity for multiplexed detection, wherein the dissociation of mass tags from the mass probes is as important as their labeling. Herein, aggregation-induced emission luminogen (AIEgen)-tagged mass probes (AIEMPs) were established to analyze estrogens, which integrated aptasensor with an explosive mass-tag signal amplification strategy via a simple ultrasound-assisted emulsification of nanoliposomes. The AIEMPs were assembled by the hybridization of aptamer-modified Fe3O4 nanoparticles (Fe NPs@Apt) and nanoliposomes loaded with massive AIEgen mass tags and partially complementary DNA strands (AIE NLs@cDNA). The aptamer was preferentially and specifically bound to estrogen, resulting in the detachment of AIE NLs from AIEMPs. Subsequently, the AIEMPs were deposited with electrospray solvents for explosive release of mass tags. Using nanoelectrospray ionization mass spectrometry (nanoESI-MS), the AIEMP-based aptasensor achieved ultrasensitive analysis of estrogens with limits of detection of 0.168-0.543 pg/mL and accuracies in the range of 87.9-114.0%. Compared to direct nanoESI-MS detection, the AIEMP-based aptasensor provides a signal amplification of four orders of magnitude. Furthermore, the utilization of different AIEMPs enables multiplexed detection of three estrogens with a miniature mass spectrometer, showing promising potential for on-site detection. This work expands the diversity of mass-tagging strategy and provides a versatile mass probe-based aptasensor platform for routine MS detection of trace analytes.

CRISPR/Cas12a and G-quadruplex DNAzyme-driven multimodal biosensor for visual detection of Aflatoxin B1.

Aflatoxin B1 (AFB1) contamination severely threatens human and animal health, it is thus critical to construct a strategy for its rapid, accurate, and visual detection. Herein, a multimodal biosensor was proposed based on CRISPR/Cas12a cleaved G-quadruplex (G4) for AFB1 detection. Briefly, specific binding of AFB1 to the aptamer occupied the binding site of the complementary DNA (cDNA), and cDNA then activated Cas12a to cleave G4 into fragments. Meanwhile, the intact G4-DNAzyme could catalyze 3, 3', 5, 5'-tetramethylbenzidine (TMB) to form colourimetric/SERS/fluorescent signal-enhanced TMBox, and the yellow solution produced by TMBox under acidic conditions could be integrated with a smartphone application for visual detection. The colourimetric/SERS/fluorescent biosensor yielded detection limits of 0.85, 0.79, and 1.65 pg·mL-1, respectively, and was applied for detecting AFB1 in peanut, maize, and badam samples. The method is suitable for visual detection in naturally contaminated peanut samples and has prospective applications in the food industry.

Cell-Free Display Techniques for Protein Evolution.

Cell-free protein synthesis (CFPS) with flexibility and controllability can provide a powerful platform for high-throughput screening of biomolecules, especially in the evolution of peptides or proteins. In this chapter, the emerging strategies for enhancing the protein expression level using different source strains, energy systems, and template designs in constructing CFPS systems are summarized and discussed in detail. In addition, we provide an overview of the ribosome display, mRNA display, cDNA display, and CIS display in vitro display technologies, which can couple genotype and phenotype by forming fusion complexes. Moreover, we point out the trend that improving the protein yields of CFPS itself can offer more favorable conditions for maintaining library diversity and display efficiency. It is hoped that the novel CFPS system can accelerate the development of protein evolution in biotechnological and medical applications.

Dual-signal output fluorescent aptasensor based on DNA programmability and gold nanoflowers for multiple mycotoxins detection.

Herein, a dual-signal output fluorescent aptamer sensor was constructed for the simultaneous detection of aflatoxin B1 (AFB1) and ochratoxin A (OTA) using the specific recognition ability of aptamers and the programmability of DNA. A functional capture probe (cDNA) was designed with the black hole quenching motif BHQ1 labeled at the 5' end and biotin (bio) labeled at the 3' end. The fluorescent dye Cy3-labeled aflatoxin B1 aptamer (AFB1-Apt) and the carboxyfluorescein FAM-labeled ochratoxin A aptamer (OTA-Apt) were used as two fluorescent probes. The cDNA is anchored to the quenching material gold nanoflowers (AuNFs) by the action of streptavidin (SA) and biotin. Its ends can be complementarily paired with two fluorescent probe bases to form a double-stranded structure. The fluorescence of Cy3 was quenched by AuNFs, and the fluorescence of FAM was quenched by BHQ1 through the fluorescence energy resonance transfer (FRET) effect, forming a fluorescence quenching system. Due to the high affinity of the target and the aptamer, the structure of the aptamer probe changes and detaches from the sensor when AFB1 and OTA are present, resulting in enhanced fluorescence. Under optimal conditions, the linear range of AFB1 was 0.1-100 ng/mL (R2 = 0.996), the limit of detection (LOD) was as low as 0.014 ng/mL, and the limit of quantification (LOQ) was 0.046 ng/mL. The linear range of OTA was 0.1-100 ng/mL (R2 = 0.995), the limit of detection (LOD) was as low as 0.027 ng/mL, and the limit of quantification (LOQ) was 0.089 ng/mL. The sensor had high accuracy in detecting both AFB1 and OTA in real sample analysis. The results of the t test show that there is no significant difference between the results of this study and the high-performance liquid phase (HPLC) method, indicating that the prepared sensor can be used as a potential platform for multiple mycotoxins detection.

A highly sensitive competitive aptasensor for AFB1 detection based on an exonuclease-assisted target recycling amplification strategy.

Aflatoxin B1 (AFB1) is a typical mycotoxin found in agricultural products, and poses a huge threat to both humans and animals. Accurate and rapid measurement of AFB1 is essential for environmental analysis and food safety. Based on molecular docking simulation design and exonuclease-assisted target recycling amplification, we designed a competitive fluorescence aptasensor to detect AFB1 rapidly and sensitively. According to the molecular docking simulations, a complementary strand (cDNA) was designed by searching for potential binding sites of the aptamer, which had the lowest binding energy. Magnetic beads modified with biotin-Apt were used as the capture probe, while FAM-labeled cDNA acted as the reporter probe. By using EXO I for target recycling amplification, this aptasensor was highly sensitive and selective for AFB1. The detection limit of the suggested aptasensor under optimal conditions was 0.36 ng mL-1 (S/N = 3) in the range of 1-1000 ng mL-1 (R2 = 0.991). The developed aptasensor was successfully used to analyze AFB1 in oil samples.

An electrochemical biosensor for the detection of aflatoxin B1 based on the specific aptamer and HCR biological magnification.

Aflatoxin B1 (AFB1) is a highly toxic mycotoxin, which causes severe acute or cumulative poisoning. Therefore, it is important to develop sensitive and selective detection methods for AFB1 for the safety of food and medicinal herbs. Herein, we have developed a "signal-on" electrochemical aptasensor based on the high specificity of the aptamer and hybridization chain reaction (HCR) biological amplification for AFB1 detection. In this work, thiol-modified complementary DNA (cDNA) immobilized on the surface of a gold electrode (GE) served as an initiator DNA. When AFB1 was present, it competed with the cDNA for binding to the aptamers, which resulted in the detaching of aptamers from the cDNA-aptamer duplexes. Then, the single-stranded cDNA acted as an initiator to trigger the HCR signal amplification. Therefore, long double-stranded DNA (dsDNA) products were produced, which could load large amounts of methylene blue (MB) molecules to generate a distinct electrochemical signal. Under the optimized conditions, the proposed electrochemical aptasensor achieved the ultrasensitive detection of AFB1 with a linear detection range of 0.01-100 pg mL-1, and a limit of detection (LOD) down to 2.84 fg mL-1. Furthermore, the electrochemical aptasensor was successfully applied for detecting AFB1 in corn and two kinds of traditional Chinese medicine samples, indicating the potential value for AFB1 detection in practical samples.

Molecular characterization and expression profiling of FOXL2 gene in goose (Anser cygnoides).

Forkhead box L2 (FOXL2) is a forkhead transcription factor essential for proper reproductive function in females and plays a crucial role in ovarian development in many species of vertebrates. However, little research on goose FOXL2 gene has been conducted. In this study, the cDNA and genomic DNA sequences of goose FOXL2 gene were cloned and characterized. The goose FOXL2 is a single exon gene and contains one open reading frame of 918 bp encoding a protein of 305 amino acids. Bioinformatics analysis displays that the deduced FOXL2 amino acid sequence contains the highly conserved forkhead box domain, which shares greatest similarity to avian species, especially to that of ducks and chicken. RT-qPCR analysis indicates that the FOXL2 mRNA is widely expressed in all examined tissues of fertilized female eggs (28 days), and differentially expressed in female adult (70 days) and laying Zhedong White geese (270 days). Meanwhile, FOXL2 is highly expressed in the hypothalamic-pituitary-ovarian axis, especially in the ovary tissues of the adult and laying geese. Furthermore, one microsatellite (TGTC1415-1418----) and five single nucleotide polymorphisms (A1290G, G1495A, T1554C, T1692A, C1695G and T1697G) were identified in the 3'-untranslated regions. All the information derived from this study could be valuable and facilitate further researches on the function of FOXL2 gene in geese.

Dipeptide nanoparticle and aptamer-based hybrid fluorescence platform for enrofloxacin determination.

A novel fluorescence platform was fabricated for enrofloxacin determination by using cDNA-modified dipeptide fluorescence nanoparticles (FDNP-cDNA) and aptamer-modified magnetic Fe3O4 nanoparticles (Fe3O4-Apt). The FDNP were prepared via tryptophan-phenylalanine self-assembling. When magnetic Fe3O4-Apt incubated with standard solution or sample extracts, the target enrofloxacin was selectively captured by the aptamer on the surface of the Fe3O4 nanoparticles. After removing interference by washing with phosphate-buffered saline, the FDNP-cDNA was added, which can bind to the aptamer on the surface of the Fe3O4 nanoparticles not occupied by the analyte. The higher the concentration of the target enrofloxacin in the standard or sample solution is, the less the FDNP-cDNA can be bound with the Fe3O4 nanoparticles, and the more the FDNP-cDNA can be observed in the supernatant. Fluorescence intensity (Ex/Em = 310/380 nm) increased linearly in the enrofloxacin concentration range 0.70 to 10.0 ng/mL with a detection limit of 0.26 ng/mL (S/N = 3). Good recoveries (88.17-99.30%) were obtained in spiked lake water, chicken, and eel samples with relative standard deviation of 2.7-6.2% (n = 3).

Separable structural requirements for cDNA synthesis, nontemplated extension, and template jumping by a non-LTR retroelement reverse transcriptase.

Broad evolutionary expansion of polymerase families has enabled specialization of their activities for distinct cellular roles. In addition to template-complementary synthesis, many polymerases extend their duplex products by nontemplated nucleotide addition (NTA). This activity is exploited for laboratory strategies of cloning and sequencing nucleic acids and could have important biological function, although the latter has been challenging to test without separation-of-function mutations. Several retroelement and retroviral reverse transcriptases (RTs) support NTA and also template jumping, by which the RT performs continuous complementary DNA (cDNA) synthesis using physically separate templates. Previous studies that aimed to dissect the relationship between NTA and template jumping leave open questions about structural requirements for each activity and their interdependence. Here, we characterize the structural requirements for cDNA synthesis, NTA, template jumping, and the unique terminal transferase activity of Bombyx mori R2 non-long terminal repeat retroelement RT. With sequence alignments and structure modeling to guide mutagenesis, we generated enzyme variants across motifs generally conserved or specific to RT subgroups. Enzyme variants had diverse NTA profiles not correlated with other changes in cDNA synthesis activity or template jumping. Using these enzyme variants and panels of activity assay conditions, we show that template jumping requires NTA. However, template jumping by NTA-deficient enzymes can be rescued using primer duplex with a specific length of 3' overhang. Our findings clarify the relationship between NTA and template jumping as well as additional activities of non-long terminal repeat RTs, with implications for the specialization of RT biological functions and laboratory applications.

Characterization of a single-domain von Willebrand factor type C protein (HaSVC) from the salivary gland of the tick Hyalomma asiaticum.

As a widely distributed arthropod and vector for various pathogens, Hyalomma asiaticum presents great risk and potential losses in animal husbandry. Effective measures, including the use of vaccines, are necessary for controlling ticks and tick-borne diseases. A concise understanding of the tick-host interaction associated molecules and pathways is required for vaccine development. In the present study, a protein containing a single-domain von Willebrand factor type C (HaSVC) was isolated from H. asiaticum and was subjected to functional identification. As a result, the full-length sequence of the HaSVC (506 bp) gene was obtained, which putatively encodes 100 amino acids with a predicted molecular mass of 11 kDa, excluding the 23-amino acid signal peptide. HaSVC contains 8 cysteines to form 4 disulfide bonds. The native HaSVC protein was detected in multiple tick organs. HaSVC neither attenuated the anti-coagulation process nor directly affected the blood feeding of adult ticks. However, the purified recombinant protein HaSVC (rHaSVC/GST) significantly increased the proliferation of mice spleen cells. This might suggest a regulatory function for HaSVC on inflammation, thus providing new information that may explain the "crosstalk" between ticks and hosts.

Identifying proximal RNA interactions from cDNA-encoded crosslinks with ShapeJumper.

SHAPE-JuMP is a concise strategy for identifying close-in-space interactions in RNA molecules. Nucleotides in close three-dimensional proximity are crosslinked with a bi-reactive reagent that covalently links the 2'-hydroxyl groups of the ribose moieties. The identities of crosslinked nucleotides are determined using an engineered reverse transcriptase that jumps across crosslinked sites, resulting in a deletion in the cDNA that is detected using massively parallel sequencing. Here we introduce ShapeJumper, a bioinformatics pipeline to process SHAPE-JuMP sequencing data and to accurately identify through-space interactions, as observed in complex JuMP datasets. ShapeJumper identifies proximal interactions with near-nucleotide resolution using an alignment strategy that is optimized to tolerate the unique non-templated reverse-transcription profile of the engineered crosslink-traversing reverse-transcriptase. JuMP-inspired strategies are now poised to replace adapter-ligation for detecting RNA-RNA interactions in most crosslinking experiments.

Kinetic DNA Self-Assembly: Simultaneously Co-folding Complementary DNA Strands into Identical Nanostructures.

DNA origami is a powerful method for constructing DNA nanostructures. It requires long single-stranded DNAs. The preparation of such long DNA strands is often quite tedious and has a limited production yield. In contrast, duplex DNAs can be easily prepared via enzymatic reactions in large quantities. Thus, we ask a question: can we design DNA nanostructures in such a way that the two complementary strands can simultaneously fold into the designed structures in the same solution instead of hybridizing with each other to form a DNA duplex? By engineering DNA interaction kinetics, herein we are able to provide multiple examples to concretely demonstrate a positive answer to this question. The resulting DNA nanostructures have been thoroughly characterized by electrophoresis and atomic force microscopy imaging. The reported strategy is compatible with the DNA cloning method and thus would provide a convenient method for the large-scale production of the designed DNA nanostructures.

Shear-Thinning and Designable Responsive Supramolecular DNA Hydrogels Based on Chemically Branched DNA.

A novel supramolecular DNA hydrogel system was designed based on a directly synthesized chemically branched DNA. For the hydrogel formation, a self-dimer DNA with two sticky ends was designed as the linker to induce the gelation of B-Y. By programing the linker sequence, thermal and metal-ion responsiveness could be introduced into this hydrogel system. This supramolecular DNA hydrogel shows shear-thinning, designable responsiveness, and good biocompatibility, which will simplify the hydrogel composition and preparation process of the supramolecular DNA hydrogel and accelerate its biomedical applications.

A MOF-Shell-Confined I-Motif-Based pH Probe (MOFC-i) Strategy for Sensitive and Dynamic Imaging of Cell Surface pH.

Dynamic imaging of cell surface pH is extremely challenging due to the slight changes in pH and the fast diffusion of secreted acid to the extracellular environment. In this work, we construct a novel metal-organic framework (MOF)-shell-confined i-motif-based pH probe (MOFC-i) strategy that enables sensitive and dynamic imaging of cell surface pH. The CY3- and CY5-labeled i-motif, which is hybridized via its short complementary chain with two-base mismatches, is optimized for sensing at physiological pH. After efficiently anchoring the optimized pH probes onto the cell membrane with the aid of cholesterol groups, a biocompatible microporous MOF shell is then formed around the cell by cross-linking ZIF-8 nanoparticles via tannic acid. The microporous MOF shell can confine secreted acid without inhibiting the normal physiological activities of cells; thus, the MOFC-i strategy can be used to monitor dynamic changes in the cell surface pH of living cells. Furthermore, this method can not only clearly distinguish the different metabolic behaviors of cancer cells and normal cells but also reveal drug effects on the cell surface pH or metabolism, providing promising prospects in pH-related diagnostics and drug screening.

Expression of a human cDNA in moss results in spliced mRNAs and fragmentary protein isoforms.

Production of biopharmaceuticals relies on the expression of mammalian cDNAs in host organisms. Here we show that the expression of a human cDNA in the moss Physcomitrium patens generates the expected full-length and four additional transcripts due to unexpected splicing. This mRNA splicing results in non-functional protein isoforms, cellular misallocation of the proteins and low product yields. We integrated these results together with the results of our analysis of all 32,926 protein-encoding Physcomitrella genes and their 87,533 annotated transcripts in a web application, physCO, for automatized optimization. A thus optimized cDNA results in about twelve times more protein, which correctly localizes to the ER. An analysis of codon preferences of different production hosts suggests that similar effects occur also in non-plant hosts. We anticipate that the use of our methodology will prevent so far undetected mRNA heterosplicing resulting in maximized functional protein amounts for basic biology and biotechnology.

Oligonucleotide capture sequencing of the SARS-CoV-2 genome and subgenomic fragments from COVID-19 individuals.

The newly emerged and rapidly spreading SARS-CoV-2 causes coronavirus disease 2019 (COVID-19). To facilitate a deeper understanding of the viral biology we developed a capture sequencing methodology to generate SARS-CoV-2 genomic and transcriptome sequences from infected patients. We utilized an oligonucleotide probe-set representing the full-length genome to obtain both genomic and transcriptome (subgenomic open reading frames [ORFs]) sequences from 45 SARS-CoV-2 clinical samples with varying viral titers. For samples with higher viral loads (cycle threshold value under 33, based on the CDC qPCR assay) complete genomes were generated. Analysis of junction reads revealed regions of differential transcriptional activity among samples. Mixed allelic frequencies along the 20kb ORF1ab gene in one sample, suggested the presence of a defective viral RNA species subpopulation maintained in mixture with functional RNA in one sample. The associated workflow is straightforward, and hybridization-based capture offers an effective and scalable approach for sequencing SARS-CoV-2 from patient samples.

First detection and molecular analysis of SARS-CoV-2 from a naturally infected cat from Argentina.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of coronavirus disease 2019 (COVID-19), has rapidly spread worldwide. Studies of transmission of the virus carried out in animals have suggested that certain animals may be susceptible to infection with SARS-CoV-2. The aim of the present study was to investigate the infection of SARS-CoV-2 in pets (18 cats and 20 dogs) from owners previously confirmed as COVID-19-positive. Oropharyngeal and rectal swabs were taken and analyzed by real-time RT-PCR assays, while blood samples were taken for antibody detection. Of the total pets analyzed, one cat was found reactive to SARS-CoV-2 by real-time RT-PCR of an oropharyngeal and a rectal swab. This cat presented only sneezing as a clinical sign. Serological analysis confirmed the presence of antibodies in the serum sample from this cat, as well as in the serum from another cat non-reactive to real-time RT-PCR. Complete sequence and phylogenetic analysis allowed determining that the SARS-CoV-2 genome belonged to the B.1.499 lineage. This lineage has been reported in different provinces of Argentina, mainly in the Metropolitan Area of Buenos Aires. This study notifies the first detection of the natural infection and molecular analysis of SARS-CoV-2 in a cat from Argentina whose owner where COVID-19-positive. Although there is currently no evidence that cats can spread COVID-19, results suggest that health authorities should test pets with COVID-19-positive owners.

Direct Mapping of Higher-Order RNA Interactions by SHAPE-JuMP.

Higher-order structure governs function for many RNAs. However, discerning this structure for large RNA molecules in solution is an unresolved challenge. Here, we present SHAPE-JuMP (selective 2'-hydroxyl acylation analyzed by primer extension and juxtaposed merged pairs) to interrogate through-space RNA tertiary interactions. A bifunctional small molecule is used to chemically link proximal nucleotides in an RNA structure. The RNA cross-link site is then encoded into complementary DNA (cDNA) in a single, direct step using an engineered reverse transcriptase that "jumps" across cross-linked nucleotides. The resulting cDNAs contain a deletion relative to the native RNA sequence, which can be detected by sequencing, that indicates the sites of cross-linked nucleotides. SHAPE-JuMP measures RNA tertiary structure proximity concisely across large RNA molecules at nanometer resolution. SHAPE-JuMP is especially effective at measuring interactions in multihelix junctions and loop-to-helix packing, enables modeling of the global fold for RNAs up to several hundred nucleotides in length, facilitates ranking of structural models by consistency with through-space restraints, and is poised to enable solution-phase structural interrogation and modeling of complex RNAs.

Plant Rho GTPase signaling promotes autophagy.

The roles of Rho family guanosine triphosphatases (GTPases) of plants (ROPs) in modulating plant growth and development have been well characterized. However, little is known about the roles of ROP signaling pathways in regulating plant autophagy and autophagosome formation. In this study, we identify a unique ROP signaling mechanism, which mediates developmental to autophagic transition under stress conditions in the model plant Arabidopsis. Loss-of-function mutants of ROP8 showed stress-induced hypersensitive phenotypes and compromised autophagic flux. Similar to other ROPs in the ROP/RAC family, ROP8 exhibits both plasma membrane and cytosolic punctate localization patterns. Upon autophagic induction, active ROP8 puncta colocalize with autophagosomal markers and are degraded inside the vacuole. In human cells, RalB, an RAS subfamily GTPase, engages its effector Exo84 for autophagosome assembly. However, a RalB counterpart is missing in the plant lineage. Intriguingly, we discovered that plant ROP8 promotes autophagy via its downstream effector Sec5. Live-cell super-resolution imaging showed that ROP8 and Sec5 reside on phagophores for autophagosome formation. Taken together, our findings highlight a previously unappreciated role of an ROP8-Sec5 signaling axis in autophagy promotion, providing new insights into how plants utilize versatile ROP signaling networks to coordinate developmental and autophagic responses depending on environmental changes.