Stem-loop Hairpin Research Articles

Deciphering the evolutionary landscape of severe fever with thrombocytopenia syndrome virus across East Asia.

Severe fever with thrombocytopenia syndrome virus (SFTSV) poses a significant public health challenge in East Asia, necessitating a deeper understanding of its evolutionary dynamics to effectively manage its spread and pathogenicity. This study provides a comprehensive analysis of the genetic diversity, recombination patterns, and selection pressures across the SFTSV genome, utilizing an extensive dataset of 2041 sequences from various hosts and regions up to November 2023. Employing maximum likelihood and Bayesian evolutionary analysis by sampling trees (BEAST), we elucidated the phylogenetic relationships among nine distinct SFTSV genotypes (A, B1, B2, B3, B4, C, D, E, and F), revealing intricate patterns of viral evolution and genotype distribution across China, South Korea, and Japan. Furthermore, our analysis identified 34 potential reassortments, underscoring a dynamic genetic interplay among SFTSV strains. Genetic recombination was observed most frequently in the large segment and least in the small segment, with notable recombination hotspots characterized by stem-loop hairpin structures, indicative of a structural propensity for genetic recombination. Additionally, selection pressure analysis on critical viral genes indicated a predominant trend of negative selection, with specific sites within the RNA-dependent RNA polymerase and glycoprotein genes showing positive selection. These sites suggest evolutionary adaptations to host immune responses and environmental pressures. This study sheds light on the intricate evolutionary mechanisms shaping SFTSV, offering insights into its adaptive strategies and potential implications for vaccine development and therapeutic interventions.

Stimulus-Responsive Four-Stranded DNA Nanoring Assembly to Host Multiple Nanosilver Clusters for Cooperatively Enhanced Fluorescence Biosensing.

Exploring the ability of four-stranded DNA nanorings (fsDNRs) to host multiple nanosilver clusters (NAgCs) for cooperatively amplifiable fluorescence biosensing to a specific initiator (tI*) is fascinating. By designing three DNA single strands and three analogous stem-loop hairpins, we developed a functional fsDNR through sequential cross-opening and overlapped hybridization. Note that a substrate strand (SS) was programmed with six modules: two severed splits (sT and sT') of NAgCs template, two sequestered segments by a middle unpaired spacer, and a partition for tI*-recognizable displacement, while sT and sT' were also tethered in two ends of three hairpins. At first, a triple dsDNA complex with stimulus-responsiveness was formed to guide the specific binding to tI*, while the exposed toehold of the SS activated the forward cascade hybridization of three hairpins, until the ring closure in the tailored self-assembly pathway for forming the fsDNR. The resulting four duplexes forced each pair of sT/sT' to be merged as the parent template in four nicks, guiding the preferential synthesis of four clusters in the shared fsDNR, thereby cooperatively amplifying the green fluorescence signal for sensitive assay of tI*. Meanwhile, the topological conformation of fsDNR can be stabilized by the as-formed cluster adducts to rivet the pair of two splits in the nicks. Benefitting from the self-enhanced effect of multiple emitters, this label-free fluorescent sensing strategy features simplicity, rapidity, and high on-off contrast, without involving complicated nucleic acid amplifiers.

Engineering smart thioflavin T binders with switchable DNA topologies for constructing multiple label-free biosensors

Thioflavin T (ThT) has been used as a sensitive fluorescence probe for lighting up nucleic acids. The binding of ThT is dependent on the structural topologies of DNA sequences. To date, most oligonucleotides interact with ThT through only a single topology, and this limits the applications of ThT as a sensing agent. Here, we modulated an HIV-1 integrase aptamer, 93del, to have switchable topologies by optimizing its 3′ flanking residues to enhance its ThT-binding capacity. Our data revealed that the sequences formed stem-loop hairpins and I-motif/G-quadruplex (G4) hybrids in Na+ buffer under alkaline and acidic conditions, respectively. Both topologies could bind ThT with high affinity (Kd ∼ 0.5 μM) and dramatically enhance the fluorescent signal of ThT. However, in the presence of K+, the sequences could be assembled into interlocked G4s, which had a low affinity for ThT but a high affinity for N-methyl mesoporphyrin IX (NMM). This allowed us to build highly sensitive fluorescent K+ sensors that were insusceptible to interference from Na+. Additionally, we found that the stacking of homologous base pairs in the hairpin loops was essential for ThT recognition and could be used as an indicator in the construction of label-free molecular beacons, allowing monitoring of microRNAs effectively in human serum spiked test. By employing external stimuli to induce the transformation of these topologies, we were able to sensitively detect adenosine triphosphate (ATP) molecules or Hg2+in vitro under specific acidic conditions. Our study provides a strategy for exploiting undiscovered functions of existing aptamers.

Molecular Compressive Force Sensor for Mapping Forces at the Cell-Substrate Interface.

Mechanical forces are crucial for biological processes such as T cell antigen recognition. A suite of molecular tension probes to measure pulling forces have been reported over the past decade; however, there are no reports of molecular probes for measuring compressive forces, representing a gap in the current mechanobiology toolbox. To address this gap, we report a molecular compression reporter using pseudostable hairpins (M-CRUSH). The design principle was based on a pseudostable DNA structure that folds in response to an external compressive force. We created a library of DNA stem-loop hairpins with varying thermodynamic stability, and then used Förster Resonance Energy Transfer (FRET) to quantify hairpin folding stability as a function of temperature and crowding. We identified an optimal pseudostable DNA hairpin highly sensitive to molecular crowding that displayed a shift in melting temperature (Tm) of 7 °C in response to a PEG crowding agent. When immobilized on surfaces, this optimized DNA hairpin showed a 29 ± 6% increase in FRET index in response to 25% w/w PEG 8K. As a proof-of-concept demonstration, we employed M-CRUSH to map the compressive forces generated by primary naïve T cells. We noted dynamic compressive forces that were highly sensitive to antigen presentation and coreceptor engagement. Importantly, mechanical forces are generated by cytoskeletal protrusions caused by acto-myosin activity. This was confirmed by treating cells with cytoskeletal inhibitors, which resulted in a lower FRET response when compared to untreated cells. Furthermore, we showed that M-CRUSH signal is dependent on probe density with greater density probes showing enhanced signal. Finally, we demonstrated that M-CRUSH probes are modular and can be applied to different cell types by displaying a compressive signal observed under human platelets. M-CRUSH offers a powerful tool to complement tension sensors and map out compressive forces in living systems.

Structural Dynamics Role of AGG Interruptions in Inhibition CGG Repeat Expansion Associated with Fragile X Syndrome.

Abnormal expansion of trinucleotide CGG repeats is responsible for Fragile X syndrome. AGG interruptions in CGG repeat tracts were found in most healthy individuals, suggesting a crucial role in preventing disease-prone repeat expansion. Previous biophysics studies emphasize a difference in the secondary structure affected by AGG interruptions. However, the mechanism of how AGG interruptions impede repeat expansion remains elusive. We utilized single-molecule fluorescence resonance energy transfer spectroscopy to investigate the structural dynamics of CGG repeats and their AGG-interrupted variants. Tandem CGG repeats fold into a stem-loop hairpin structure with the capability to undergo a conformational rearrangement to modulate the length of the overhang. However, this conformational rearrangement is much more retarded when two AGG interruptions are present. Considering the significance of hairpin slippage in repeat expansion, we present a molecular basis suggesting that the internal loop created by two AGG interruptions acts as a barrier, obstructing the hairpin slippage reconfiguration. This impediment potentially plays a crucial role in curbing abnormal expansion, thereby contributing to the genomic stability.

Programmable SARS-CoV-2 mutation detection platform based on ligation-triggered isothermal exponential amplification coupled with self-priming amplification

In this work, we developed a programmable mutation detection platform with a dual amplification strategy for universal detection of SARS-CoV-2 mutations based on ligation-triggered isothermal exponential amplification reaction(L-EXPAR) and phosphorothioate modified self-priming amplification (PS-SPA). In the presence of mutant target (MT) sequence, it complements with hairpin probe (H1) and hairpin probe (H2) and triggered L-EXPAR at lower temperature. A large number of single-strand DNA (ssDNA) is produced in the circle of ligation, nicking, polymerization, and strand displacement. The ssDNA products containing target sequences can feed back and initiate another L-EXPAR. The ligated H1-H2 complex triggers another isothermal process termed PS-PSA at higher temperature. 20 bases of H1-H2 complex on the 5′ end modified with PS can form a short stem-loop hairpin due to the lower Tm of PS modification. The resulting overhanging 3′ end can also fold back as a new primer to initiate the next round of polymerization. In the circle of PS-SPA, the H1-H2 chain can be extended to be a long hairpin concatemer containing a great number of tandem target sequences. The resulting DNA products can be monitored in real-time fluorescent signaling. In the presence of the SARS-CoV-2 wild type (WT) sequence, the ligation between H1 and H2 cannot happen due to the mismatch of the last base on the 3′ end of H1. The proposed assay displayed a good linear range from 1 fM to 1 nM with a detection limit of femtomole level for the detection of SARS-CoV-2 spike gene D614G mutation and Del246-252 + D253N deletion.

The return of the "Mistigri" (virus adaptative gain by gene loss) through the SARS-CoV-2 XBB.1.5 chimera that predominated in 2023.

Severe acute respiratory syndrome coronavirus 2 XBB.1.5 is the first recombinant lineage to predominate at the country and global scales. Very interestingly, like the Marseille-4B subvariant (or B.1.160) and the pandemic variant B.1.1.7 (or Alpha) previously, it has its ORF8 gene inactivated by a stop codon. We aimed here to study the distribution of stop codons in ORF8 of XBB.1.5 and non-XBB.1.5 genomes. We identified that a stop codon was present at 89 (74%) ORF8 codons in ≥1 of 15 222 404 genomes available in GISAID. The mean proportion of genomes with a stop codon per codon was 0.11% (range, 0%-7.8%). In addition, a stop codon was detected at 15 (12%) codons in at least 1000 genomes. These 15 codons are notably located on seven stem-loop hairpin regions and in the signal peptide region for the case of the XBB.1.5 lineage (codon 8). Thus, it is very likely that stop codons in ORF8 gene contributed on at least three occasions and independently during the pandemic to the evolutionary success of a lineage that became transiently predominant. Such association of gene loss with evolutionary success, which suits the recently described Mistigri rule, is an important biological phenomenon very unknown in virology while largely described in cellular organisms.

Target-responsive DNA translator for amplified and sensitive sensing of Myc-Max transcription factor dimer

Myc-Max is a central eukaryotic transcription factor dimer that plays key roles in regulating cellular activities such as cell growth, proliferation, and differentiation. Selective and sensitive quantification of Myc-Max thus has valuable significances for biological studies and disease diagnosis. On the basis of a new DNA translator probe, we construct an amplified FRET sensing strategy for sensitively detecting Myc-Max in human serums via DNAzyme and clamped hybridization chain reaction (cHCR)-integrated signal amplifications. Myc-Max binds the DNA translator and switches its conformation to activate the DNAzyme and trigger cHCR for the assembly generation of DNA networks from two respective dye-labeled dual stem-loop and palindromic sequence-containing hairpin structures. Such DNA networks pull the two dyes into proximity position to exhibit highly magnified FRET responses for sensitively detecting Myc-Max with a 1.2 pM low detection limit in the dynamic range between 10 pM and 1 nM. Besides, sensitive discrimination of low levels of Myc-Max in diluted human serums has been verified by the developed method. Moreover, the sequence of the DNA translator can be tuned to accommodate different biomolecules, making the developed sensing strategy a versatile tool for detecting various trace biomarkers for diagnosing diseases at their early stages.

A highly sensitive electrochemical biosensor via short-stranded DNA activating LAMP(H+) to regulate i-motif folds for signal transduction

Exploring loop-mediated isothermal amplification (LAMP)-based electrochemical assay for short-stranded DNA biomarkers is intriguing. Herein, a HIV-specific DNA segment (tDNA) was introduced to implement a variant LAMP reaction, where the generated by-products H+, vLAMP(H+), acted as signal transducers for developing a sensitive electrochemical biosensor. As proof-of-concept, the tDNA-recognizable complement was programmed in a template hairpin (TH) that was ended with a 3′-terminus phosphate group to avoid non-specific elongation. Upon TH simultaneously hybridizing with tDNA and a precursor hairpin (PH), the PH underwent a loop-mediated extension with Bst polymerase and dNTPs, forming a new stem-loop hairpin in another terminus. Then the elongated product containing a double stem-loop dumbbell-like DNA structure (dDDS) was displaced and released. Using dDDS as an initiator, the vLAMP reaction occurred via consecutively cycled strand displacement for self-primed DNA incorporation. The resulting tDNA-dependent vLAMP(H+) was introduced in the modified electrode surface to guide the pH-sensitive i-motif (iM) folding. Thus, a signaling hairpin labeled with electroactive ferrocene (Fc) was dehybridized and closed, thereby bringing Fc close to the electrode interface for increased current signal readout that was highly sensitive to tDNA down to 0.034 fM. Our electrochemical biosensor would be applicable for detecting various short-stranded DNA or miRNA.

Structure-Guided Design of a Potent and Specific Inhibitor against the Genomic Mutator APOBEC3A.

Nucleic acid structure plays a critical role in governing the selectivity of DNA- and RNA-modifying enzymes. In the case of the APOBEC3 family of cytidine deaminases, these enzymes catalyze the conversion of cytosine (C) to uracil (U) in single-stranded DNA, primarily in the context of innate immunity. DNA deamination can also have pathological consequences, accelerating the evolution of viral genomes or, when the host genome is targeted by either APOBEC3A (A3A) or APOBEC3B (A3B), promoting tumor evolution leading to worse patient prognosis and chemotherapeutic resistance. For A3A, nucleic acid secondary structure has emerged as a critical determinant of substrate targeting, with a predilection for DNA that can form stem loop hairpins. Here, we report the development of a specific nanomolar-level, nucleic acid-based inhibitor of A3A. Our strategy relies on embedding the nucleobase 5-methylzebularine, a mechanism-based inhibitor, into a DNA dumbbell structure, which mimics the ideal substrate secondary structure for A3A. Structure-activity relationship studies using a panel of diverse inhibitors reveal a critical role for the stem and position of the inhibitor moiety in achieving potent inhibition. Moreover, we demonstrate that DNA dumbbell inhibitors, but not nonstructured inhibitors, show specificity against A3A relative to the closely related catalytic domain of A3B. Overall, our work demonstrates the feasibility of leveraging secondary structural preferences in inhibitor design, offering a blueprint for further development of modulators of DNA-modifying enzymes and potential therapeutics to circumvent APOBEC-driven viral and tumor evolution.

Conformational Heterogeneity of RNA Stem-Loop Hairpins Bound to FUS-RNA Recognition Motif with Disordered RGG Tail Revealed by Unbiased Molecular Dynamics Simulations.

RNA-protein complexes use diverse binding strategies, ranging from structurally well-defined interfaces to completely disordered regions. Experimental characterization of flexible segments is challenging and can be aided by atomistic molecular dynamics (MD) simulations. Here, we used an extended set of microsecond-scale MD trajectories (400 μs in total) to study two FUS-RNA constructs previously characterized by nuclear magnetic resonance (NMR) spectroscopy. The FUS protein contains a well-structured RNA recognition motif domain followed by a presumably disordered RGG tail that binds RNA stem-loop hairpins. Our simulations not only provide several suggestions complementing the experiments but also reveal major methodological difficulties in studies of such complex RNA-protein interfaces. Despite efforts to stabilize the binding via system-specific force-field adjustments, we have observed progressive distortions of the RNA-protein interface inconsistent with experimental data. We propose that the dynamics is so rich that its converged description is not achievable even upon stabilizing the system. Still, after careful analysis of the trajectories, we have made several suggestions regarding the binding. We identify substates in the RNA loops, which can explain the NMR data. The RGG tail localized in the minor groove remains disordered, sampling countless transient interactions with the RNA. There are long-range couplings among the different elements contributing to the recognition, which can lead to allosteric communication throughout the system. Overall, the RNA-FUS systems form dynamical ensembles that cannot be fully represented by single static structures. Thus, albeit imperfect, MD simulations represent a viable tool to investigate dynamic RNA-protein complexes.

AND-gated ratiometric fluorescence biosensing of dual-emissive Ag nanoclusters based on transient recycling amplification of bivariate targeting DNA

Development of logic AND-gated multivariate assay systems of DNA-scaffolded green- and red-fluorescent Ag nanoclusters (g-AgNC and r-AgNC) is promising and remains challenging. By using two DNA segments (To and Tr) as dual-targeting inputs, herein we report an innovative strategy that utilizes targets-recycled amplification to create a sensitive AND-gated light-up ratiometric sensor with reversely changed fluorescence of g-AgNC and r-AgNC. The design is based on that: two hairpins (Ho and Hr) are individually recognizable to To and Tr, and two hybridized duplexes are resultantly implemented for transient dissipative base pairing, i.e. Ho·Hr, releasing To and Tr for repeated recycling. In an original stem-loop hairpin reporter (HR), one exposed overhang is to host g-AgNC. When HR is unfolded by the cooperative sticky toeholds in Ho∙Hr, its liberated stem merging with the g-AgNC template is for r-AgNC clustering. Only when meeting simultaneously the overexpressed To and Tr in SARS-CoV-2, the AND-gated ratio fluorescence of r-AgNC over g-AgNC can be lighted-up in a typical assay, enabling highly specific-inputs binding and sensitive dose-dependence down to 0.29 pM. This enzyme-free and label-free AND-logic ratiometirc strategy illustrates the potential of using multicolor AgNCs emitters for more accurate multivariate assay.

Evolution of small and large ribosomal RNAs from accretion of tRNA subelements

tRNAs presumably accreted into modern ribosomal RNAs. Previous analyses showed similar secondary structures for ancient rRNA subelements and theoretical minimal RNA rings, candidate tRNA ancestors rationally designed from tRNA-unrelated principles. Here, analyses test which tRNA secondary structure subelements resemble ancient/recent rRNA subelements. Results show that ribosomal RNA subelements evolved from structures resembling 1. Upper half part of the tRNA secondary structure; and 2. Towards structures resembling (a) tRNA 5′ stem-loop hairpins in large rRNA subunit and (b) tRNA lower half part in small rRNA subunit (stop and start codons conservation model). tRNAs and rRNAs presumably originated from the tRNA upper half part including the acceptor stem. Modern split 5′ and 3′ tRNA genes (spliced at anticodons) apparently reproduce ancestral-like states, because the acceptor stem protocode suggests acceptor stems evolved from spliced anticodon-like stem-loop hairpins, strengthening central roles for acceptor stem CCA-addition at translation origins. The Root-Bernstein hypothesis on the existence of tRNA structural symmetries presumably reflects late 5’ tRNA stem-loop hairpin duplications, some integrating rRNAs. Analyses of tRNA subelements similarities with rRNA subelements suggest tRNAs evolved and re-evolved by different duplication-fusions, along different structural subdivision models. Hence, sequential/parallel processes, perhaps in the same ancestral organism(s) produced polyphyletic tRNAs. Results confirm RNA ring usefulness for understanding prebiotic and early life evolution, and their similarities with primordial protein coding and tRNA genes.

Target DNA-Activating Proximity-Localized Catalytic Hairpin Assembly Enables Forming Split-DNA Ag Nanoclusters for Robust and Sensitive Fluorescence Biosensing.

Proximity-localized catalytic hairpin assembly (plCHA) is intriguing for rapid and sensitive assay of an HIV-specific DNA segment (T*). Using template-integrated green Ag nanoclusters (igAgNCs) as emitters, herein, we report the first design of a T*-activated plCHA circuit that is confined in a three-way-junction architecture (3WJA) for the fluorescence sensing of T*. To this end, the T*-recognizable complement is programmed in a stem-loop hairpin (H1), and two split template sequences of igAgNCs are separately overhung contiguous to the paired stems of H1 and another hairpin (H2). The hybridization among H1, H2, and two single-stranded linkers (L1 and L2) allows the stable construction of 3WJA. Upon presenting the input T*, the 3WJA-localized plCHA is operated through toehold-mediated strand displacements of H1 and H2 reactants, and T* is rationally displaced and repeatably recycled, analogous to a specific catalyst, inducing more hairpin assembly events. Resultantly, the hybridized products enable the collective combination of two splits in the parent scaffold for hosting igAgNCs, outputting T*-dependent fluorescence response. Because of 3WJA structural confinement, the spatial proximity of two reactive hairpins yielded high local concentrations to manipulate the plCHA operation, achieving rapider reaction kinetics via T*-catalyzed recycling than typical catalytic hairpin assembly (CHA). This simple assay strategy would open the arena to develop various plCHA-based circuits capable of modulating the fluorescence emission of igAgNCs for applicable biosensing and bioanalysis.

Effects of Local Sequence, Reaction Conditions, and Various Additives on the Formation and Stability of Interstrand Cross-Links Derived from the Reaction of an Abasic Site with an Adenine Residue in Duplex DNA.

The experiments described here examined the effects of reaction conditions, various additives, and local sequence on the formation and stability interstrand cross-links (ICLs) derived from the reaction of an apurinic/apyrimidinic (AP) site with the exocyclic amino group of an adenine residue on the opposing strand in duplex DNA. Cross-link formation was observed in a range of different buffers, with faster formation rates observed at pH 5. Inclusion of the base excision repair enzyme alkyladenine DNA glycosylase (hAAG) which binds tightly to AP-containing duplexes decreased, but did not completely prevent, formation of the dA-AP ICL. Formation of the dA-AP ICL was not altered by the presence of the biological metal ion Mg2+ or the biological thiol, glutathione. Several organocatalysts of imine formation did not enhance the rate of dA-AP ICL formation. Duplex length did not have a large effect on dA-AP yield, so long as the melting temperature of the duplex was not significantly below the reaction temperature (the duplex must remain hybridized for efficient ICL formation). Formation of the dA-AP ICL was examined in over 40 different sequences that varied the neighboring and opposing bases at the cross-linking site. The results indicate that ICL formation can occur in a wide variety of sequence contexts under physiological conditions. Formation of the dA-AP ICL was strongly inhibited by the aldehyde-trapping agents methoxyamine and hydralazine, by NaBH3CN, by the intercalator ethidium bromide, and by the minor groove-binding agent netropsin. ICL formation was inhibited to some extent in bicarbonate and Tris buffers. The dA-AP ICL showed substantial inherent stability under a variety of conditions and was not a substrate for AP-processing enzymes APE1 or Endo IV. Finally, we characterized cross-link formation in a small (11 bp) stem-loop (hairpin) structure and in DNA-RNA hybrid duplexes.

Modulating the Fluorescence of Silver Nanoclusters Wrapped in DNA Hairpin Loops via Confined Strand Displacement and Transient Concatenate Ligation for Amplifiable Biosensing.

It is intriguing to modulate the fluorescence emission of DNA-scaffolded silver nanoclusters (AgNCs) via confined strand displacement and transient concatenate ligation for amplifiable biosensing of a DNA segment related to SARS-CoV-2 (s2DNA). Herein, three stem-loop structural hairpins for signaling, recognizing, and assisting are designed to assemble a variant three-way DNA device (3WDD) with the aid of two linkers, in which orange-emitting AgNC (oAgNC) is stably clustered and populated in the closed loop of a hairpin reporter. The presence of s2DNA initiates the toehold-mediated strand displacement that is confined in this 3WDD for repeatable recycling amplification, outputting numerous hybrid DNA-duplex conformers that are implemented for a transient "head-tail-head" tandem ligation one by one. As a result, the oAgNC-hosted hairpin loops are quickly opened in loose coil motifs, bringing a significant fluorescence decay of multiple clusters dependent on s2DNA. Demonstrations and understanding of the tunable spectral performance of a hairpin loop-wrapped AgNC via switching 3WDD conformation would be highly beneficial to open a new avenue for applicable biosensing, bioanalysis, or clinical diagnostics.

Genome-Wide Investigation of the MiR166 Family Provides New Insights into Its Involvement in the Drought Stress Responses of Tea Plants (Camellia sinensis (L.) O. Kuntze)

MicroRNA166 (miR166) is a highly conserved plant miRNA that plays a crucial role in plant growth and the resistance to various abiotic stresses. However, the miR166s in tea (Camellia sinensis (L.) O. Kuntze) have not been comprehensively identified and analyzed. This study identified 30 mature miR166s and twelve pre-miR166s in tea plants. An evolutionary analysis revealed that csn-miR166s originating from the 3′ arm of their precursors were more conserved than the csn-miR166s derived from the 5′ arm of their precursors. The twelve pre-miR166s in tea were divided into two groups, with csn-MIR166 Scaffold364-2 separated from the other precursors. The Mfold-based predictions indicated that the twelve csn-MIR166s formed typical and stable structures comprising a stem-loop hairpin, with minimum free energy ranging from −110.90 to −71.80 kcal/mol. An analysis of the CsMIR166 promoters detected diverse cis-acting elements, including those related to light responses, biosynthesis and metabolism, abiotic stress defenses, and hormone responses. There was no one-to-one relationship between the csn-miR166s and their targets, but most csn-miR166s targeted HD-Zip III genes. Physiological characterization of tea plants under drought stress showed that leaf water content proportionally decreased with the aggravation of drought stress. In contrast, tea leaves’ malondialdehyde (MDA) content proportionally increased. Moreover, the cleavage site of the ATHB-15-like transcript was identified according to a modified 5′ RNA ligase-mediated rapid amplification of cDNA ends. The RT-qPCR data indicated that the transcription of nine csn-miR166s was negatively correlated with their target gene.

Amplifiable ratiometric fluorescence biosensing of nanosilver multiclusters populated in three-way-junction DNA branches

To explore the fluorescence bio-responsiveness of emissive silver nanoclusters (AgNCs) populated in DNA-branched scaffolds is intriguing yet challenging. In response to a desired targeting model (T*) as a vehicle, herein a customized three-way-junction DNA construct (TWJDC) is assembled via competitive hybridizing cascade among three stem-loop hairpins with specific base sequences, where the repeated recycling of T* enables the exponentially amplifiable output of rigid TWJDC. As designed, these stable hybridization products are highly T*-stimulated responsive and constructing-directional. In the three branched-arms, the unpaired sticky ends provide isotropic binding sites for a signaling hairpin encoded with two C-rich templates of green- and red-AgNCs clustering. The identical ligation of signal probe with three arms of TWJDC liberates its locked stem, enabling the separate growth of red-clusters in three branches. As demonstrated, three clusters of red-AgNCs possess advantageous self-enhancing fluorescent performance relative to single or two cluster(s), good biocompatibility and low cytotoxicity. Utilizing the bicolor AgNCs as dual-emitters with reversely changed emission intensity, we developed an innovative ratiometric strategy displaying sensitively linear dose-dependence on variable T* down to 1.9 pM, which can afford a promising platform for biosensing, bioanalysis, cell imaging, or even clinical theranostics.

RNA regulation in immunity.

RNA regulation in immunity.

Characterizing Fluorescence Emissions of Hairpin DNA-Templated Silver Nanoclusters by cDNA Hybridization.

Stem-loop hairpin DNA probes have high hybridization specificity and unique selectivity to target molecules such as DNA and small molecules. DNA-templated silver nanoclusters (DNA-AgNCs) has been widely used to detect biomolecules of interest due to the photostable, bright, and efficient methods. In this study, we measured fluorescence emission of hairpin DNA upon hybridization with cDNA and mutant cDNA (cDNA-1) or mutant cDNA containing mismatched bases in the stem region (cDNA-2). Fluorescence intensity of hairpin DNA-AgNCs in the presence of cDNA was 1.80 times higher than that of hairpin DNA-AgNCs alone, but decreased to 66% in the presence of cDNA-1 containing mismatched base corresponding to the hairpin stem region. This study demonstrated that fluorescence intensities of hairpin DNA-AgNCs were dependent on hybridization with either wild-type and mutant cDNAs.