Search

Nucleic acids research / Special publication 48(16), 9387 - 9405 (2020). doi:10.1093/nar/gkaa647, Template-independent terminal ribonucleotide transferases (TENTs) catalyze the addition of nucleotide monophosphates to the 3′-end of RNA molecules regulating their fate. TENTs include poly(U) polymerases (PUPs) with a subgroup of 3′ CUCU-tagging enzymes, such as CutA in Aspergillus nidulans. CutA preferentially incorporates cytosines, processively polymerizes only adenosines and does not incorporate or extend guanosines. The basis of this peculiar specificity remains to be established. Here, we describe crystal structures of the catalytic core of CutA in complex with an incoming non-hydrolyzable CTP analog and an RNA with three adenosines, along with biochemical characterization of the enzyme. The binding of GTP or a primer with terminal guanosine is predicted to induce clashes between 2-NH$_2$ of the guanine and protein, which would explain why CutA is unable to use these ligands as substrates. Processive adenosine polymerization likely results from the preferential binding of a primer ending with at least two adenosines. Intriguingly, we found that the affinities of CutA for the CTP and UTP are very similar and the structures did not reveal any apparent elements for specific NTP binding. Thus, the properties of CutA likely result from an interplay between several factors, which may include a conformational dynamic process of NTP recognition., Published by Information Retrieval Ltd.21517, London

The stability and processing of cellular RNA transcripts are efficiently controlled via non-templated addition of single or multiple nucleotides, which is catalyzed by various nucleotidyltransferases including poly(A) polymerases (PAPs). Germline development defective 2 (GLD-2) is among the first reported cytoplasmic non-canonical PAPs that promotes the translation of germline-specific mRNAs by extending their short poly(A) tails in metazoan, such as Caenorhabditis elegans and Xenopus. On the other hand, the function of mammalian GLD-2 seems more diverse, which includes monoadenylation of certain microRNAs. To understand the structural basis that underlies the difference between mammalian and non-mammalian GLD-2 proteins, we determine crystal structures of two rodent GLD-2s. Different from C. elegans GLD-2, mammalian GLD-2 is an intrinsically robust PAP with an extensively positively charged surface. Rodent and C. elegans GLD-2s have a topological difference in the β-sheet region of the central domain. Whereas C. elegans GLD-2 prefers adenosine-rich RNA substrates, mammalian GLD-2 can work on RNA oligos with various sequences. Coincident with its activity on microRNAs, mammalian GLD-2 structurally resembles the mRNA and miRNA processor terminal uridylyltransferase 7 (TUT7). Our study reveals how GLD-2 structurally evolves to a more versatile nucleotidyltransferase, and provides important clues in understanding its biological function in mammals.

Neurofibrillary tangles composed of aberrantly aggregating tau protein are a hallmark of Alzheimer’s disease and related dementia disorders. Recent work has shown that mammalian suppressor of tauopathy 2 (MSUT2), also named ZC3H14 (Zinc Finger CCCH-Type Containing 14), controls accumulation of pathological tau in cultured human cells and mice. Knocking out MSUT2 protects neurons from neurodegenerative tauopathy and preserves learning and memory. MSUT2 protein functions to bind polyadenosine [poly(A)] tails of mRNA through its C-terminal CCCH type zinc finger domains, and loss of CCCH domain function suppresses tauopathy in Caenorhabditis elegans and mice. Thus, we hypothesized that inhibiting the poly(A):MSUT2 RNA–protein interaction would ameliorate pathological tau accumulation. Here we present a high-throughput screening method for the identification of small molecules inhibiting the poly(A):MSUT2 RNA–protein interaction. We employed a fluorescent polarization assay for initial small molecule discovery with the intention to repurpose hits identified from the NIH Clinical Collection (NIHCC). Our drug repurposing development workflow included validation of hits by dose–response analysis, specificity testing, orthogonal assays of activity, and cytotoxicity. Validated compounds passing through this screening funnel will be evaluated for translational effectiveness in future studies. This preclinical drug development pipeline identified diverse FDA approved drugs duloxetine, saquinavir, and clofazimine as potential repurposing candidates for reducing pathological tau accumulation.

Homopolymeric adenosine RNA plays numerous roles in both cells and noncellular genetic material. We report herein an unusual poly(A) signature in chemical mapping data generated by the Eterna Massive Open Laboratory. Poly(A) sequences of length seven or more show unexpected results in the selective 2'-hydroxyl acylation read out by primer extension (SHAPE) and dimethyl sulfate (DMS) chemical probing. This unusual signature first appears in poly(A) sequences of length seven and grows to its maximum strength at length ∼10. In a long poly(A) sequence, the substitution of a single A by any other nucleotide disrupts the signature, but only for the 6 or so nucleotides on the 5' side of the substitution.

Alternative polyadenylation (APA) produces isoforms with distinct 3′-ends, yet their functional differences remain largely unknown. Here, we introduce the APA-seq method to detect the expression levels of APA isoforms from 3′-end RNA-Seq data by exploiting both paired-end reads for gene isoform identification and quantification. We detected the expression levels of APA isoforms in individual Caenorhabditis elegans embryos at different stages throughout embryogenesis. Examining the correlation between the temporal profiles of isoforms led us to distinguish two classes of genes: those with highly correlated isoforms (HCI) and those with lowly correlated isoforms (LCI) across time. We hypothesized that variants with similar expression profiles may be the product of biological noise, while the LCI variants may be under tighter selection and consequently their distinct 3′ UTR isoforms are more likely to have functional consequences. Supporting this notion, we found that LCI genes have significantly more miRNA binding sites, more correlated expression profiles with those of their targeting miRNAs and a relative lack of correspondence between their transcription and protein abundances. Collectively, our results suggest that a lack of coherence among the regulation of 3′ UTR isoforms is a proxy for selective pressures acting upon APA usage and consequently for their functional relevance.

Aberrant KRAS signaling is a driver of many cancers and yet remains an elusive target for drug therapy. The nuclease hypersensitive element of the KRAS promoter has been reported to form secondary DNA structures called G-quadruplexes (G4s) which may play important roles in regulating KRAS expression, and has spurred interest in structural elucidation studies of the KRAS G-quadruplexes. Here, we report the first high-resolution crystal structure (1.6 Å) of a KRAS G-quadruplex as a 5′-head-to-head dimer with extensive poly-A π-stacking interactions observed across the dimer. Molecular dynamics simulations confirmed that the poly-A π-stacking interactions are also maintained in the G4 monomers. Docking and molecular dynamics simulations with two G4 ligands that display high stabilization of the KRAS G4 indicated the poly-A loop was a binding site for these ligands in addition to the 5′-G-tetrad. Given sequence and structural variability in the loop regions provide the opportunity for small-molecule targeting of specific G4s, we envisage this high-resolution crystal structure for the KRAS G-quadruplex will aid in the rational design of ligands to selectively target KRAS.

3′-end poly(A)+ sequencing is an efficient and economical method for global measurement of mRNA levels and alternative poly(A) site usage. A common method involves oligo(dT)19V reverse-transcription (RT)-based library preparation and high-throughput sequencing with a custom primer ending in (dT)19. While the majority of library products have the first sequenced nucleotide reflect the bona fide poly(A) site (pA), a substantial fraction of sequencing reads arise from various mis-priming events. These can result in incorrect pA site calls anywhere from several nucleotides downstream to several kilobases upstream from the bona fide pA site. While these mis-priming events can be mitigated by increasing annealing stringency (e.g. increasing temperature from 37 °C to 42 °C), they still persist at an appreciable level (∼10%) and computational methods must be used to prevent artifactual calls. Here we present a bioinformatics workflow for precise mapping of poly(A)+ 3′ ends and handling of artifacts due to oligo(dT) mis-priming and sample polymorphisms. We test pA site calling with three different read mapping programs (STAR, BWA, and BBMap), and show that the way in which each handles terminal mismatches and soft clipping has a substantial impact on identifying correct pA sites, with BWA requiring the least post-processing to correct artifacts. We demonstrate the use of this pipeline for mapping pA sites in the model eukaryote S. cerevisiae, and further apply this technology to non-polyadenylated transcripts by employing in vitro polyadenylation prior to library prep (IVP-seq). As proof of principle, we show that a fraction of tRNAs harbor CCU 3′ tails instead of the canonical CCA tail, and globally identify 3′ ends of splicing intermediates arising from inefficiently spliced transcripts.

Grifola frondosa is an edible and medicinal mushroom with great nutritional values and bioactivities. In the present study, a soluble homogeneous β-glucan, GFPS, with high molecular mass of 5.42 × 106 Da was purified from the fruit bodies of Grifola frondosa using 5% cold NaOH. The structure of GFPS was determined with FT-IR, NMR, and monosaccharide composition analysis, and was identified to be a β-D-(1-3)-linked glucan backbone with a single β-D-(1-6)-linked glucopyranosyl residue branched at C-6 on every third residue. Our results indicated that GFPS had a triple helical structure and could form complex with polydeoxyadenylic acid (poly[A]). Further studies demonstrated that GFPS could interact with poly[A] moiety of a designed antisense oligonucleotide (ASO) targeting the primary transcript of proinflammatory cytokine TNFα (TNFα-A60). This GFPS-based complex could incorporate TNFα-A60 into the macrophage cells via dectin-1 receptor and attenuate lipopolysaccharide-induced secretion of TNFα. Our results suggested that GFPS could be applied to deliver therapeutic oligonucleotides for the treatment of diseases such as inflammation and cancers.

Introns in different genes, or even different introns within the same gene, often have different splice sites and differ in splicing efficiency (SE). One expects mass-transcribed genes to have introns with higher SE than weakly transcribed genes. However, such a simple expectation cannot be tested directly because variable SE for these genes is often not measured. Mechanistically, SE should depend on signal strength at key splice sites (SS) such as 5′SS, 3′SS and branchpoint site (BPS), i.e., SE = F(5′SS, 3′SS, BPS). However, without SE, we again cannot model how these splice sites contribute to SE. Here I present an RNA-Seq approach to quantify SE for each of the 304 introns in yeast (Saccharomyces cerevisiae) genes, including 24 in the 5′UTR, by measuring 1) number of reads mapped to exon-exon junctions (NEE) as a proxy for the abundance of spliced form, and 2) number of reads mapped to exon-intron junction (NEI5 and NEI3 at 5′ and 3′ ends of intron) as a proxy for the abundance of unspliced form. The total mRNA is NTotal = NEE + p * NEI5 + (1−p) * NEI3, with the simplest p = 0.5 but statistical methods were presented to estimate p from data. An estimated p is needed because NEI5 is expected to be smaller than NEI3 due to 1) step 1 splicing occurs before step 2 so EI5 is broken before EI3, 2) enrichment of poly(A) mRNA by oligo-dT, and 3) 5′ degradation. SE is defined as the proportion (NEE/NTotal). Application of the method shows that ribosomal protein messages are efficiently and mostly cotranscriptionally spliced. Yeast genes with long introns are also spliced efficiently. HAC1/YFL031W is poorly spliced partly because its splicing involves a nonspliceosome mechanism and partly because Ire1p, which participate in splicing HAC1, is hardly expressed. Many putative yeast genes have low SE, and some splice sites are incorrectly annotated.

The near-field effects in the vicinity of metallic nanoparticle surfaces, as induced by electromagnetic radiation with specific wavelength, give rise to a variety of novel optical properties and attractive applications because of surface plasmons, which are the coherent oscillations of conduction electrons on a metal surface. The interdisciplinary field of plasmonics has witnessed vigorous growth, promoting research on the modulation of plasmon resonance by constructing advanced plasmonic nanoarchitectures with controllable size, morphology, or interparticle coupling. Among diversified tools, deoxyribonucleic nucleic acid (DNA) possesses prominent superiority as a result of its designability, programmability, addressability, and ease of nanomaterial modification. In this review, we focus on the methods and optical applications of plasmon resonance modulation accomplished by DNA nanotechnology. Recent developments in the construction of DNA-mediated plasmonic nanoarchitecture and key ongoing research directions utilizing unique optical features are highlighted. Obstacles and challenges in this field are pointed out, followed by preliminary suggestions on some areas of opportunity that deserve attention.

Messenger RNA (mRNA) vaccines have attracted great interest in recent years due to their high potency, safety profile, and potential of rapid development. Although a number of mRNA vaccines have entered clinical trials, there remain several challenges. Inefficient in vivo delivery of mRNA is the foremost one. Here we synthesized a conjugate composed of β-cyclodextrin (β-CD) and branched polyethyleneimine (molecular weight 2 kDa, bPEI2k) to deliver an mRNA vaccine. The CD-PEI (CP) conjugate helped the encapsulated mRNA molecules pass through the plasma membranes and escape from the endosomes, which consequently ensured high transfection efficiency. On this basis, we optimized several structural elements of mRNA molecules via synthesizing an advanced cap structure and incorporating untranslated regions (UTRs) and an extended poly(A) tail into the sequence. These modifications led to a higher expression level of encoded proteins, which was expected to induce potent immune responses with a relatively low dosage. We also investigated the relevance of the administration route to the immune responses induced by CP-assisted mRNA vaccines with in vivo evidence, providing a basis for the selection of optimum administration route in specific cases. This CP-based mRNA vaccine platform, with an optimized mRNA structure and administrated in a most appropriate route, holds a promise to be applied to specific antigens in the future. Graphical abstract.

Ribonuclease P (RNase P) is essential for the 5′-end maturation of tRNAs in all kingdoms of life. In Escherichia coli, temperature sensitive mutations in either its protein (rnpA49) and or RNA (rnpB709) subunits lead to inviability at nonpermissive temperatures. Using the rnpA49 temperature sensitive allele, which encodes a partially defective RNase P at the permissive temperature, we show here for the first time that the processing of RNase P-dependent polycistronic tRNA operons to release pre-tRNAs is the essential function of the enzyme, since the majority of 5′-immature tRNAs can be aminoacylated unless their 5′-extensions ≥8 nt. Surprisingly, the failure of 5′-end maturation elicits increased polyadenylation of some pre-tRNAs by poly(A) polymerase I (PAP I), which exacerbates inviability. The absence of PAP I led to improved aminoacylation of 5′-immature tRNAs. Our data suggest a more dynamic role for PAP I in maintaining functional tRNA levels in the cell.

Motivation Polyadenylation plays a regulatory role in transcription. The recognition of polyadenylation signal (PAS) motif sequence is an important step in polyadenylation. In the past few years, some statistical machine learning-based and deep learning-based methods have been proposed for PAS identification. Although these methods predict PAS with success, there is room for their improvement on PAS identification. Results In this study, we proposed a deep neural network-based computational method, called SANPolyA, for identifying PAS in human and mouse genomes. SANPolyA requires no manually crafted sequence features. We compared our method SANPolyA with several previous PAS identification methods on several PAS benchmark datasets. Our results showed that SANPolyA outperforms the state-of-art methods. SANPolyA also showed good performance on leave-one-motif-out evaluation. Availability and implementation https://github.com/yuht4/SANPolyA. Supplementary information Supplementary data are available at Bioinformatics online.

The nucleosome is the basic structural element of genomic DNA packaging and plays a role in transcription, replication, and recombination. Poly(dA) tracts are considered major sequence determinants of nucleosome positioning, although their role is not well understood. Here, we show that the homopolymeric character and the low GC content of poly(dA)s play different roles in nucleosome formation. We found that the inherent low GC content of poly(dA) alone can account for the deep and anisotropic nucleosome depletion at structurally and functionally important regions of promoters and origins of replication. We also show that the level of nucleosome occupancy at poly(dA) is strongly related to the local nucleotide background and its high frequency of occurrence in Saccharomyces cerevisiae does not appear merely to be associated with its intrinsic nucleosome-excluding properties. In addition, we show that the GC content alone can predict more than 60% of the in vitro nucleosome map, providing further evidence that the intrinsic nucleosome positioning is more greatly determined by GC content than poly(dA) stretches. Our results are consistent with a model in which poly(dA) stretches act at two distinct levels: first, by its low GC content, which intrinsically contributes to hinder nucleosome formation, and second, by its contiguous runs of dA that selectively drive the recruitment of non-histone proteins with structural and functional roles.

We studied the directed self-assembly of two types of complementary single-stranded DNA (ssDNA) strands [i.e., poly(dA) and poly(dT)] into more complex, organized, and percolating networks in dilute solutions and at surfaces. Understanding ssDNA self-assembly into 2D networks on surfaces is important for the use of such networks in the fabrication of well-defined nanotechnological devices, as, for instance, required in nanoelectronics or for biosensing. To control the formation of 2D networks on surfaces, it is important to know whether DNA assemblies are formed already in dilute solutions or only during the drying/immobilization process at the surface, where the concentration automatically increases. Fluorescence cross-correlation spectroscopy clearly shows the presence of larger DNA complexes in mixed poly(dA) and poly(dT) solutions already at very low DNA concentrations (

Two Ru(II) complexes, [Ru(phen)

Two Ru(II) complexes containing different substituents, [Ru(phen)

Small interfering RNA (siRNA)-based therapeutics represent a novel and compelling drug modality, provided that safe and competent vectors are available for their delivery. Here, we report a biodegradable amphiphilic poly(aminoester) dendrimer for effective siRNA delivery. This dendrimer is readily biodegradable upon enzyme action, and harnesses the delivery features of both lipid and polymer vectors thanks to its lipid/dendrimer hybrid structure. This study opens new perspectives for developing biodegradable and biocompatible vectors for siRNA therapeutics.

Alternative splicing (AS) is necessary for viral proliferation in host cells and a critical regulatory component of viral gene expression. Conventional RNA-seq approaches provide incomplete coverage of AS due to their short read lengths and are susceptible to biases and artifacts introduced in prevailing library preparation methodologies. Moreover, viral splicing studies are often conducted separately from host cell transcriptome analysis, precluding an assessment of the viral manipulation of host splicing machinery. To address current limitations, we developed a quantitative full-length direct cDNA sequencing strategy to simultaneously profile viral and host cell transcripts. This nanopore-based approach couples processive reverse transcriptases with a novel one-step chemical ablation of 3′ RNA ends (termed CASPR), which decreases ribosomal RNA reads and enriches polyadenylated coding sequences. We extensively validate our approach using synthetic reference transcripts and show that CASPR doubles the breadth of coverage per transcript and increases detection of long transcripts (>4 kb), while being functionally equivalent to PolyA+ selection for transcript quantification. We used our approach to interrogate host cell and HIV-1 transcript dynamics during viral reactivation and identified novel putative HIV-1 host factors containing exon skipping or novel intron retentions and delineated the HIV-1 transcriptional state associated with these differentially regulated host factors.

MotivationPoly-alanine (polyA) regions are protein stretches mostly composed of alanines. Despite their abundance in eukaryotic proteomes and their association to nine inherited human diseases, the structural and functional roles exerted by polyA stretches remain poorly understood. In this work we study how the amino acid context in which polyA regions are settled in proteins influences their structure and function.ResultsWe identified glycine and proline as the most abundant amino acids within polyA and in the flanking regions of polyA tracts, in human proteins as well as in 17 additional eukaryotic species. Our analyses indicate that the non-structuring nature of these two amino acids influences the α-helical conformations predicted for polyA, suggesting a relevant role in reducing the inherent aggregation propensity of long polyA. Then, we show how polyA position in protein N-termini relates with their function as transit peptides. PolyA placed just after the initial methionine is often predicted as part of mitochondrial transit peptides, whereas when placed in downstream positions, polyA are part of signal peptides. A few examples from known structures suggest that short polyA can emerge by alanine substitutions in α-helices; but evolution by insertion is observed for longer polyA. Our results showcase the importance of studying the sequence context of homorepeats as a mechanism to shape their structure–function relationships.Availability and implementationThe datasets used and/or analyzed during the current study are available from the corresponding author onreasonable request.Supplementary informationSupplementary data are available at Bioinformatics online.

Herein, we report rapid electrochemical detection of miRNA let-7a based on a DNA probe consisting of a polyA and Fc-co-labeled harpin structure (the polyA-H probe). The polyA-H probe could be facilely immobilized on Au surfaces through the interactions between polyA and Au, followed by its pre-hybridization with a single strand (S1). The probe's surface density could be optimized for minimizing steric hindrance via changing the polyA block length. The target let-7a could be rapidly amplified via loop-mediated isothermal amplification (LAMP) with four simplified primers, followed by inducing the formation of dimeric i-motif (DIM) structure via H+-induced rapid folding of two C-rich sequences of motif strand 1 and strand 2. It was found that, after introducing the as-formed DIM to hybridize the S1, the immobilized polyA20-H probe could rapidly revert to its hairpin structure, sending out a turn-on electrochemical signal of the Fc. The total time for detecting the let-7a was around 80 min, obviously less than that of most of electrochemical DNA sensors reported previously. The biosensor showed a linear relationship of the current response to the let-7a in the range of 10 fM to 50 nM with a limit of detection (LOD) of 5.1 fM. Our biosensors were further tested using human serum spiked with the let-7a and the extracts of the breast adenocarcinoma cells spiked with and without the let-7a, respectively. Satisfied results were obtained. This study shows a potential promising future of development of electrochemical biosensors for rapid detection of miRNAs in the application of clinical practice.

© The Author(s), under exclusive licence to Springer Nature Limited 2022, RNA modifications are important regulators of gene expression1. In Trypanosoma brucei, transcription is polycistronic and thus most regulation happens post-transcriptionally2. N6-methyladenosine (m6A) has been detected in this parasite, but its function remains unknown3. Here we found that m6A is enriched in 342 transcripts using RNA immunoprecipitation, with an enrichment in transcripts encoding variant surface glycoproteins (VSGs). Approximately 50% of the m6A is located in the poly(A) tail of the actively expressed VSG transcripts. m6A residues are removed from the VSG poly(A) tail before deadenylation and mRNA degradation. Computational analysis revealed an association between m6A in the poly(A) tail and a 16-mer motif in the 3' untranslated region of VSG genes. Using genetic tools, we show that the 16-mer motif acts as a cis-acting motif that is required for inclusion of m6A in the poly(A) tail. Removal of this motif from the 3' untranslated region of VSG genes results in poly(A) tails lacking m6A, rapid deadenylation and mRNA degradation. To our knowledge, this is the first identification of an RNA modification in the poly(A) tail of any eukaryote, uncovering a post-transcriptional mechanism of gene regulation., We are grateful to support from the Howard Hughes Medical Institute International Early Career Scientist Program (55007419), a European Molecular Biology Organization Installation grant (2151) and La Caixa Foundation (HR20-00361). This work was also partially supported by the ONEIDA project (LISBOA-01-0145-FEDER-016417) co-funded by Fundos Europeus Estruturais e de Investimento (FEEI) from ‘Programa Operacional Regional Lisboa 2020’ and by national funds from Fundação para a Ciência e a Tecnologia (FCT). S.R.J. was supported by NIH (R35 NS111631). Researchers were funded by individual fellowships from FCT (PD/BD/105838/2014 to I.J.V., 2020.06827.BD to L.S., SFRH/BD/80718/2011 to F.A.-B., PD/BD/138891/2018 to A.T. and CEECIND/03322/2018 to L.M.F.); a Novartis Foundation for Biomedical-Biological research to J.P.d.M.; a Human Frontier Science Programme long-term postdoctoral fellowship to M.D.N. (LT000047/2019); a Marie Skłodowska-Curie Individual Standard European Fellowship to S.S.P. (grant no. 839960); the GlycoPar Marie Curie Initial Training Network (GA 608295) to J.A.R. We thank J. Thomas-Oates (University of York, Centre of Excellence in Mass Spectrometry, Department of Chemistry) for the mass spectrometry analysis. The York Centre of Excellence in Mass Spectrometry was created thanks to a major capital investment through Science City York, supported by Yorkshire Forward with funds from the Northern Way Initiative, and subsequent support from EPSRC (EP/K039660/1 and EP/M028127/1).

Adsorbents with high adsorption capacity, sustainability, and reusability are desired in wastewater treatments. Herein, covalently crosslinked microporous cryogels with efficient removal of cationic dyes are fabricated by freezing radical copolymerization using methacrylated alginate (AlgMA) and sodium p-styrenesulfonate (NaSS). The chemical structure, morphology, and thermal stability of the AlgMA/PNaSS cryogels are characterized. Compared with the AlgMA/PNaSS hydrogels with the same chemical composition fabricated by thermal-initiated polymerization, the cryogels show higher adsorption of methylene blue. The AlgMA/PNaSS (with a mass ratio of 1:2) possesses the maximum adsorption capacity for MB with 2300 mg/g under alkaline condition. In addition, the adsorption process of AlgMA/PNaSS cryogels fits the pseudo-second order model and Freundlich model, respectively. The AlgMA/PNaSS cryogels also reveal selective adsorption capacity and reusability after regeneration, which own unchanged corrected adsorption capacity during five adsorption-desorption cycles. The AlgMA/PNaSS cryogels show great potential for use in wastewater treatments and intelligent separation.

Adsorbents with highly efficient and selective recovery performance towards uranium are significantly demanded for the sustainable nuclear power production. Herein, poly(amidoxime)-graft-magnetic chitosan (P(AO)-g-MC) was synthesized through functionalizing magnetic chitosan with polyacrylonitrile followed by amidoximation process. Under magnetic field, P(AO)-g-MC can be separated from the solution in 10 s. Owing to the strong affinity of high-density amidoxime groups towards uranium, P(AO)-g-MC showed remarkable adsorption capacity, rapid kinetics and good regeneration performance in uranium spiked aqueous solution. Notably, the 7-day uranium adsorption capacity of P(AO)-g-MC from natural seawater in column mode was up to 5.14 mg/g, 12 times that of vanadium. The excellent uranium uptake performance over vanadium originated from the strong coordination by N and O in amidoxime groups according to theoretical simulation. The advantages of easy separating and high selectivity make P(AO)-g-MC a very potential uranium adsorbent in natural seawater.

Poly(A) tails are added to the 3' ends of most mRNAs in a non-templated manner and play essential roles in post-transcriptional regulation, including mRNA export, stability and translation. Measuring poly(A) tails is critical for understanding their regulatory roles in almost every aspect of biological and medical studies. Previous methods for analyzing poly(A) tails require large amounts of input RNA (microgram-level total RNA), which limits their application. We recently developed a poly(A) inclusive full-length RNA isoform-sequencing method (PAIso-seq) at single-oocyte-level sensitivity (a single mammalian oocyte contains ~0.5 ng of total RNA) based on PacBio sequencing that enabled accurate measurement of the poly(A) tail length and non-A residues within the body of poly(A) tails along with the full-length cDNA, providing the opportunity to study precious in vivo samples with very limited input material. Here, we describe a detailed protocol for PAIso-seq library preparation from single mouse oocytes or bulk oocyte samples. In addition, we provide a complete bioinformatic pipeline to perform the analysis from the raw data to downstream analysis. The minimum time required is ~14.5 h for PAIso-seq double-stranded cDNA preparation, 2 d for PacBio sequencing in HiFi mode and 8 h for the initial data analysis.

Kanamycin fluorescence aptasensors were created using a series of di-block oligonucleotide modified gold nanoparticles with various lengths of poly-adenine. In the presence of kanamycin, the double strand structure of the aptamer-reporter strand complex is disrupted, and the dye-labelled reporter strand detaches from the surface of gold nanoparticles, resulting in fluorescence recovery (Ex/Em = 485/520 nm). By adjusting the number of consecutive adenines, the programable aptamer density can be implemented on the gold nanoparticle surface, and the conformation of nucleic acid changed from lying-down to up-right. The apparent binding constant, binding kinetics, and limit of detection of the prepared aptasensors were carefully examined to explore the influence of surface density. Under the optimum condition, the aptasensor had a tenfold lower limit of detection than the thiolated aptamer modified one, as low as 23.6 nM, when a di-block oligonucleotide with twenty consecutive adenines tailed. In addition, satisfactory recoveries ranging from 96.33 to 99.47% were achieved in spiked milk samples with relative standard deviation of 1.2-6.9% (n = 3). This surface density regulation strategy holds great promise in other aptamer-based interfacial recognition and sensing. Schematic presentation of di-block oligonucleotide modified gold nanoparticle with different surface densities and its kanamycin sensing application.

Summary Mutations causing amyotrophic lateral sclerosis (ALS) often affect the condensation properties of RNA-binding proteins (RBPs). However, the role of RBP condensation in the specificity and function of protein-RNA complexes remains unclear. We created a series of TDP-43 C-terminal domain (CTD) variants that exhibited a gradient of low to high condensation propensity, as observed in vitro and by nuclear mobility and foci formation. Notably, a capacity for condensation was required for efficient TDP-43 assembly on subsets of RNA-binding regions, which contain unusually long clusters of motifs of characteristic types and density. These “binding-region condensates” are promoted by homomeric CTD-driven interactions and required for efficient regulation of a subset of bound transcripts, including autoregulation of TDP-43 mRNA. We establish that RBP condensation can occur in a binding-region-specific manner to selectively modulate transcriptome-wide RNA regulation, which has implications for remodeling RNA networks in the context of signaling, disease, and evolution., Graphical abstract, Highlights • TDP-43 mutants affect condensation properties to a similar extent at multiple scales • Binding-region condensates form on long RNA regions with dispersed UG-rich motifs • RBPchimera-CLIP indicates homomeric interactions promote molecular-scale condensates • Condensation selectively tunes the regulatory capacity of TDP-43; e.g., autoregulation, The condensation propensity of an RNA-binding protein tunes its binding to specific RNA regions across the transcriptome and affects its RNA processing functions. Formation of these “binding-region condensates,” promoted by specific motif types that are dispersed across long RNA regions, expands the ways in which RNA binding can be selectively controlled beyond canonical RNA-binding domains.

Coxsackievirus B3 (CVB3) is a positive single-strand RNA virus causing myocarditis, pancreatitis and meningitis. During CVB3 infection, various host cellular components, including proteins and non-coding RNAs, interact with the virus and affect viral infection. Poly(rC) binding protein 1 (PCBP1) is a multifunctional RNA binding protein regulating transcription, translation and mRNA stability of a variety of genes. In this study, we observed a significant reduction of PCBP1 protein during CVB3 infection. By bioinformatic prediction and luciferase-assay verification, we confirmed that the expression of PCBP1 was directly inhibited by miR-21, a microRNA upregulated during CVB3 infection. Furthermore, we found that overexpression of PCBP1 promoted CVB3 infection and knocking down of PCBP1 inhibited it. In the subsequent mechanism study, our results revealed that PCBP1 blocked the translation of p62/SQSTM1 (sequestosome 1), an autophagy-receptor protein suppressing CVB3 replication, by interacting with the cis-element in the 5' untranslational region (5' UTR) of p62/SQSTM1. In summary, our studies have identified PCBP1 as a beneficial factor for CVB3 infection. These findings may deepen the understanding of host-virus interactions and provide a potential target for intervention of CVB3 infection.

Neurons provide a rich setting for studying post-transcriptional control. Here, we investigate the landscape of translational control in neurons and search for mRNA features that explain differences in translational efficiency (TE), considering the interplay between TE, mRNA poly(A)-tail lengths, microRNAs, and neuronal activation. In neurons and brain tissues, TE correlates with tail length, and a few dozen mRNAs appear to undergo cytoplasmic polyadenylation upon light or chemical stimulation. However, the correlation between TE and tail length is modest, explaining

Polyadenylation and deadenylation of mRNA are major RNA modifications associated with nucleus-to-cytoplasm translocation, mRNA stability, translation efficiency, and mRNA decay pathways. Our current knowledge of polyadenylation and deadenylation has been expanded due to recent advances in transcriptome-wide poly(A) tail length assays. Whereas these methods measure poly(A) length by quantifying the adenine (A) base stretch at the 3' end of mRNA, we developed a more cost-efficient technique that does not rely on A-base counting, called tail-end-displacement sequencing (TED-seq). Through sequencing highly size-selected 3' RNA fragments including the poly(A) tail pieces, TED-seq provides accurate measure of transcriptome-wide poly(A)-tail lengths in high resolution, economically suitable for larger scale analysis under various biologically transitional contexts.

Alternative polyadenylation (APA) enhances gene regulatory potential by increasing the diversity of mRNA transcripts. 3' UTR shortening through APA correlates with enhanced cellular proliferation and is a widespread phenomenon in tumor cells. Here, we show that the ubiquitously expressed transcription factor Sp1 binds RNA in vivo and is a common repressor of distal poly(A) site usage. RNA sequencing identified 2,344 genes (36% of the total mapped mRNA transcripts) with lengthened 3' UTRs upon Sp1 depletion. Sp1 preferentially binds the 3' UTRs of such lengthened transcripts and inhibits cleavage at distal sites by interacting with the subunits of the core cleavage and polyadenylation (CPA) machinery. The 3' UTR lengths of Sp1 target genes in breast cancer patient RNA-seq data correlate with Sp1 expression levels, implicating Sp1-mediated APA regulation in modulating tumorigenic properties. Taken together, our findings provide insights into the mechanism for dynamic APA regulation by unraveling a previously unknown function of the DNA-binding transcription factor Sp1.

Extracellular vesicles (EVs) are important for intercellular communication and act as vehicles for biological material, such as various classes of coding and non-coding RNAs, a few of which were shown to selectively target into vesicles. However, protein factors, mechanisms, and sequence elements contributing to this specificity remain largely elusive. Here, we use a reporter system that results in different types of modified transcripts to decipher the specificity determinants of RNAs released into EVs. First, we found that small RNAs are more efficiently packaged into EVs than large ones, and second, we determined absolute quantities for several endogenous RNA transcripts in EVs (U6 snRNA, U1 snRNA, Y1 RNA, and GAPDH mRNA). We show that RNA polymerase III (pol III) transcripts are more efficiently secreted into EVs compared to pol II-derived transcripts. Surprisingly, our quantitative analysis revealed no RNA accumulation in the vesicles relative to the total cellular levels, based on both overexpressed reporter transcripts and endogenous RNAs. RNA appears to be EV-associated only at low copy numbers, ranging between 0.02 and 1 molecule per EV. This RNA association may reflect internal EV encapsulation or a less tightly bound state at the vesicle surface.

Mitochondria have their own double-stranded DNA genomes and systems to regulate transcription, mRNA processing, and translation. These systems differ from those operating in the host cell, and among eukaryotes. In recent decades, studies have revealed several plant-specific features of mitochondrial gene regulation. The polyadenylation status of mRNA is critical for its stability and translation in mitochondria. In this short review, I focus on recent advances in understanding the mechanisms regulating mRNA polyadenylation in plant mitochondria, including the role of poly(A)-specific ribonuclease-like proteins (PARNs). Accumulating evidence suggests that plant mitochondria have unique regulatory systems for mRNA poly(A) status and that PARNs play pivotal roles in these systems.

Accurately detecting and quantifying tumor-related microRNAs (miRNAs) in living cells is of great value for early cancer diagnosis. Herein, we present poly-adenine (polyA)-mediated spherical nucleic acid (SNA) nanoprobes for intracellular miRNA imaging in living cells.polyA-mediated spherical nucleic acid (pASNA) nanoprobes consist of gold nanoparticles (AuNPs) anchored with fluorophore-labeled DNA molecules pre-hybridized with recognition sequences and polyA tails. The detection performance for miRNAs in vitro was studied to confirm the feasibility of pASNA nanoprobes for imaging live cell miRNAs. Before the pASNA nanoprobes were used for imaging intracellular miRNAs in MCF-7, HeLa, and LO2 cells, the stability and non-cytotoxicity were investigated using Dnase I and a standard colorimetric CCK8 assay. Flow cytometry, qRT-PCR analyses were conducted to confirm the different expression levels of miR-155 in live cells. Results showed that the pASNA nanoprobes had good detection sensitivity and specificity, excellent stability, and low toxicity. After incubating with pASNA nanoprobes, noticeable fluorescence signal enhancement could be clearly observed in MCF-7 and HeLa cells but not LO2 cells by confocal microscopy. Flow cytometry analysis and qRT-PCR indicated that MCF-7 and HeLa cells had higher miR-155 expression levels compared to LO2 cells.The pASNA nanoprobes we developed had good sensitivity and specificity, excellent nuclease stability and low toxicity, thus representing a new approach to exquisitely reveal the distribution of endogenous miRNAs in live cells.

Nucleic acid amplification tests (NAATs) are powerful tools for the Japanese encephalitis virus (JEV). We demonstrated highly sensitive, specific, and rapid detection of JEV by colorimetric reverse-transcription loop-mediated isothermal amplification (cRT-LAMP). Under optimized conditions, the RT-LAMP assay results showed that the limit of detection was approximately equivalent to 1 RNA genome copy/μL with an assay time of 30 min. The assay was highly specific to JEV when tested with other mosquito-borne virus panels (Zika virus and dengue virus types 2–4). The ability to detect JEV directly from crude human sample matrices (serum and urine) demonstrated the suitability of our JEV RT-LAMP for widespread clinical application. The JEV RT-LAMP provides combination of rapid colorimetric determination of true-positive JEV RT-LAMP amplicons with our recently developed JEV-nanobarcodes, measured at absorbance wavelenght of 530 (A530) and 650 (A650), which have a limit of detection of 23.3 ng/μL. The AuNP:polyA10-JEV RT-LAMP nanobarcodes exhibited superior capability for stabilizing the true-positive JEV RT-LAMP amplicons against salt-induced AuNP aggregation, which improved the evaluation of true/false positive signals in the assay. These advances enable to expand the use of RT-LAMP for point-of-care tests, which will greatly bolster JEV clinical programs.

PCOS as the most common endocrine disorder of women in their reproductive age affects between 5-15 % of the female population. Apart from its cardinal symptoms, like irregular and anovulatory cycles, hyperandrogenemia and a typical ultrasound feature of the ovary, obesity, and insulin resistance are often associated with the disease. Furthermore, PCOS represents a status of chronic inflammation with permanently elevated levels of inflammatory markers including IL-6 and IL-18, TNF-α, and CRP. Inflammation, as discovered only recently, consists of two processes occurring concomitantly: active initiation, involving "classical" mediators including prostaglandins and leukotrienes, and active resolution processes based on the action of so-called specialized pro-resolving mediators (SPMs). These novel lipid mediator molecules derive from the essential ω3-poly-unsaturated fatty acids (PUFAs) DHA and EPA and are synthesized via specific intermediates. The role and benefits of SPMs in chronic inflammatory diseases like obesity, atherosclerosis, and Diabetes mellitus has become a subject of intense research during the last years and since PCOS features several of these pathologies, this review aims at summarizing potential roles of SPMs in this disease and their putative use as novel therapeutics.

Short Interspersed Elements (SINEs) are eukaryotic non-autonomous retrotransposons transcribed by RNA polymerase III (pol III). The 3′-terminus of many mammalian SINEs has a polyadenylation signal (AATAAA), pol III transcription terminator, and A-rich tail. The RNAs of such SINEs can be polyadenylated, which is unique for pol III transcripts. Here, B2 (mice and related rodents), Dip (jerboas), and Ves (vespertilionid bats) SINE families were thoroughly studied. They were divided into subfamilies reliably distinguished by relatively long indels. The age of SINE subfamilies can be estimated, which allows us to reconstruct their evolution. The youngest and most active variants of SINE subfamilies were given special attention. The shortest pol III transcription terminators are TCTTT (B2), TATTT (Ves and Dip), and the rarer TTTT. The last nucleotide of the terminator is often not transcribed, accordingly, the truncated terminator of its descendant becomes nonfunctional. The incidence of complete transcription of the TCTTT terminator is twice higher compared to TTTT and thus functional terminators are more likely preserved in daughter SINE copies. Young copies have long poly(A) tails, however, they gradually shorten in host generations. Unexpectedly, the tail shortening below A10 increases the incidence of terminator elongation by Ts thus restoring its efficiency. This process can be critical for the maintenance of SINE activity in the genome.

International audience; "Would it be possible to analyze molecular mechanisms and structural organisation of polyribosome assemblies using cryo electron tomography?" - we asked through a longstanding collaboration between my research group and that of Alexander S. Spirin. Indeed, it was: we found that double-row polyribosomes can have both circular and linear arrangements of their mRNA [Afonina, Z. A., et al. (2013) Biochemistry (Moscow)], we figured out how eukaryotic ribosomes assemble on an mRNA to form supramolecular left-handed helices [Myasnikov, A. G., et al. (2014) Nat. Commun.], that the circularization of polyribosomes is poly-A and cap-independent [Afonina, Z. A., et al. (2014) Nucleic Acids Res.], and that intermediary polyribosomes with open structures exist after a transition from a juvenile phase to strongly translating polysomes of medium size [Afonina, Z. A., et al. (2015) Nucleic Acids Res.] until they form densely packed helical structures with reduced activity. Our joint fruitful exchanges, hence, led to major advances in the field, which are reviewed here from a personal and historical perspective in memory of Alexander S. Spirin.

Motivation Alternative polyadenylation (APA) has been widely recognized as a widespread mechanism modulated dynamically. Studies based on 3′ end sequencing and/or RNA-seq have profiled poly(A) sites in various species with diverse pipelines, yet no unified and easy-to-use toolkit is available for comprehensive APA analyses. Results We developed an R package called movAPA for modeling and visualization of dynamics of alternative polyadenylation across biological samples. movAPA incorporates rich functions for preprocessing, annotation and statistical analyses of poly(A) sites, identification of poly(A) signals, profiling of APA dynamics and visualization. Particularly, seven metrics are provided for measuring the tissue-specificity or usages of APA sites across samples. Three methods are used for identifying 3′ UTR shortening/lengthening events between conditions. APA site switching involving non-3′ UTR polyadenylation can also be explored. Using poly(A) site data from rice and mouse sperm cells, we demonstrated the high scalability and flexibility of movAPA in profiling APA dynamics across tissues and single cells. Availability and implementation https://github.com/BMILAB/movAPA. Supplementary information Supplementary data are available at Bioinformatics online.

A nature-inspired strategy is developed to build dual-network hydrogels made up of rigid graphene oxide-functionalized nanocellulose (GO@NC) network and flexible poly[acrylamide-co-(acrylic acid)] (poly(AAm-co-AAc)) network. A pre-stretching method is used to form a muscle-shape anisotropic architecture. The penetration of poly(AAm-co-AAc) flexible network relieves the stiffness of NC network, thus improving the average elongation at break from 86.2 % to 748.0 %. Compared with the poly(AAm-co-AAc), the average rupture tensile strength rises remarkably by 228.6 %. The dual-crosslinked strategy endows the GO@NC-poly(AAm-co-AAc) hydrogels with a fast, stable and repeatable self-healing ability, which can achieve 85.0 % of healing efficiency after only 600 s of self-healing and maintain 76.2 % of initial strength after 10 cycles of breaking-self-healing. The superb self-healing ability is similar to the muscle function. For potential applications, the hydrogels can achieve real-time, stable, and long-term sensing as smart wearable strain sensors (high gauge factor: 5.13), and can effectively purify Sudan IV wastewater as green recyclable adsorbents.

Recent years have seen an explosion of interest in the subject of alternative polyadenylation in plants. Connections between the polyadenylation complex and numerous developmental and stress responses are well-established. However, those that link stimuli with the functioning of the polyadenylation complex are less well understood. To this end, it is imperative to clearly delineate the roles of the polyadenylation complex in both plant growth AND alternative polyadenylation. It is also necessary to understand the ways by which other molecular processes may contribute to alternative polyadenylation. This review discusses these issues, with a focus on instances that reveal mechanisms by which mRNA polyadenylation may be regulated. Insights from from characterizations of mutants affected in the polyadenylation complex are discussed, as are the limitations of such characterizations when it comes to teasing out cause and effect. These limitations encourage explorations to other processes that are beyond the core polyadenylation complex. Two such processes that sculpt the plant transcriptome - transcription termination and the epigenetic control of transposon activity - also contribute to regulated poly(A) site choice. These subjects define "the right places" - molecular mechanisms that contribute to the wide-ranging control of gene expression via mRNA polyadenylation.

In our project herein, we use the case of farmworkers, an underserved and understudied population at high risk for Type-2 Diabetes Mellitus (T2DM), as a paradigm of an integrated action-oriented research, education and extension approach involving the development of long-term equitable strategies providing empowerment and tailored-made solutions that support practical decision-making aiming to reduce risk of T2DM and ensuing cardiovascular disease (CVD). A Technology-based Empowerment Didactic module (TEDm) and an Informed Decision-Making enhancer (IDMe) coupled in a smart application (app) for farmworkers aiming to teach, set goals, monitor, and support in terms of nutrition, hydration, physical activity, sleep, and circadian rhythm towards lowering T2DM risk, is to be developed and implemented considering the particular characteristics of the population and setting. In parallel, anthropometric, biochemical, and clinical assessments will be utilized to monitor risk parameters for T2DM and compliance to dietary and wellness plans. The app incorporating anthropometric/clinical/biochemical parameters, dietary/lifestyle behavior, and extent of goal achievement can be continuously refined and improved through machine learning and re-programming. The app can function as a programmable tool constantly learning, adapting, and tailoring its services to user needs helping optimization of practical informed decision-making towards mitigating disease symptoms and associated risk factors. This work can benefit apart from the direct beneficiaries being farmworkers, the stakeholders who will be gaining a healthier, more vibrant workforce, and in turn the local communities.

MicroRNAs (miRNAs) are small noncoding RNAs that posttranscriptionally regulate gene expression. The aberrant expression of miRNAs is related to many diseases. MiRNAs can serve as potential biomarkers for the prognosis and diagnosis of cancers and other human diseases. However, the short sequence and high sequence similarity of miRNAs impede detection. Herein, we propose a method to integrate polyA-tailing and CRISPR/Cas12a to amplify and detect all miRNAs with high specificity and sensitivity. PolyA-tailing enables efficient amplification of RNA and introduces a universal PAM sequence for Cas12a to unlock its PAM restriction. The CRISPR-Cas system guarantees the specific recognition of nucleic acid sequences with a single base mismatch. A limit of detection (LOD) as low as 50 fM was achieved. The practical application ability of polyA-CRISPR/Cas12a-based miRNA detection was validated by miRNA analyses in multiple cancer cell samples. With the increasing stability of RNA samples, low cost, excellent specificity, and sensitivity, this method demonstrates great potential to scale up to parallel diagnostic sets for miRNA-related disease.

Monitoring the level of matrix metalloproteinase-9 (MMP-9) and inhibiting its expression is important for the diagnosis and treatment of various diseases. However, the analysis of MMP-9 is challenging owing to its very low content in the blood, especially at the early stages of diseases. Therefore, we developed an ultrasensitive and easy-to-use immunosensor based on a three-dimensional (3D) bioplatform for the determination of the total MMP-9 concentration in plasma. The used 3D bioplatform (G2 poly(amidoamine) dendrimer; PAMAM) improved the sensitivity of the determination by significantly expanding the surface area of the receptor layer. The antigen-antibody recognition process was controlled by quartz crystal microbalance with dissipation (QCM-D) and electrochemical impedance spectroscopy (EIS). The effect of the orientation of antibody molecules in the sensing layer on the work parameters of the immunosensor was analyzed using unmodified PAMAM (PAMAM-NH

The genus Flavivirus comprises numerous emerging and re‐emerging arboviruses causing human illness. Vaccines are the best approach to prevent flavivirus diseases. But pathogen diversities are always one of the major hindrances for timely development of new vaccines when confronting unpredicted flavivirus outbreaks. We used West Nile virus (WNV) as a model to develop a new live‐attenuated vaccine (LAV), WNV‐poly(A), by replacing 5ʹ portion (corresponding to SL and DB domains in WNV) of 3ʹ‐UTR with internal poly(A) tract. WNV‐poly(A) not only propagated efficiently in Vero cells, but also was highly attenuated in mouse model. A single‐dose vaccination elicited robust and long‐lasting immune responses, conferring full protection against WNV challenge. Such “poly(A)” vaccine strategy may be promising for wide application in the development of flavivirus LAVs because of its general target regions in flaviviruses., West Nile virus (WNV) is one of the many members of the genus Flavivirus known to cause human diseases. Here we present a novel live attenuated WNV vaccine, WNV‐poly(A), that protects against WNV infections in a mouse model.

G-quadruplexes are the non-canonical nucleic acid structures that are preferentially formed in G-rich regions. This structure has been shown to be associated with many biological functions. Regardless of the broad efforts on DNA G-quadruplexes, we still have limited knowledge on RNA G-quadruplexes, especially in a transcriptome-wide manner. Herein, by integrating the DMS-seq and the bioinformatics pipeline, we profiled and depicted the RNA G-quadruplexes in the human transcriptome. The genes that contain RNA G-quadruplexes in their specific regions are significantly related to immune pathways and the COVID-19-related gene sets. Bioinformatics analysis reveals the potential regulatory functions of G-quadruplexes on miRNA targeting at the scale of the whole transcriptome. In addition, the G-quadruplexes are depleted in the putative, not the real, PAS-strong poly(A) sites, which may weaken the possibility of such sites being the real cleaved sites. In brief, our study provides insight into the potential function of RNA G-quadruplexes in post-transcription.

Alkaline phosphatase (ALP) is an important hydrolase with crucial roles in biological processes, and the dysregulation of ALP may cause various human diseases. The conventional ALP assays usually involve cumbersome procedures with poor sensitivity. Herein, taking advantage of intrinsic superiorities of molecular beacons (MBs) and unique features of terminal deoxynucleotidyl transferase (TdT), we demonstrate for the first time the 3'-terminal repair-powered dendritic nanoassembly of polyadenine (A) MBs for one-step quantification of ALP in human serum. When ALP is present, it catalyzes 3'-terminal dephosphorylation of poly-A MBs to induce TdT-mediated template-free polymerization, generating long chains of polythymidine (T) sequences. The long poly-T chains can function as the anchoring templates to hybridize with many poly-A MBs, leading to the unfolding of loop structures and the dissociation of FAM/BHQ1 pairs (the 1st amplification stage). Subsequently, all 3'-hydroxylated poly-A MBs can be extended with the assistance of TdT to generate the branched long poly-T chains, leading to the hybridization of more poly-A MBs and the dissociation of more FAM/BHQ1 pairs (the 2nd amplification stage). Through multiple rounds of extension, assembly, and activation of poly-A MBs, dendritic DNA nanostructures are automatically formed, resulting in the dissociation of abundant fluorophores from the FAM/BHQ1 pairs to generate an exponentially amplified fluorescence signal (the nth amplification stage). This strategy possesses high sensitivity and excellent specificity, and the detection limit can reach 1 cell. Moreover, it can evaluate kinetic parameters, screen inhibitors, estimate cellular inhibition effects, and measure ALP in human serums.