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Rapid and sensitive detection of pathogenic bacteria is crucial for disease prevention and control. The CRISPR/Cas12a system with the DNA cleavage capability holds promise in pathogenic bacteria diagnosis. However, the sensitivity of CRISPR-based assays remains a challenge. Herein, we report a versatile and sensitive pathogen sensing platform (HTCas12a) based on the CRISPR/Cas12a system, hybridization chain reaction (HCR) and Poly T-copper fluorescence nanoprobe. The sensitivity is improved by HCR and the Poly-T-Cu reporter probe reduces the overall experiment cost to less than one dollar per sample. Our results demonstrate the specific recognition of target nucleic acid fragments from other pathogens. Furthermore, a good linear correlation between fluorescence intensity and target quantities were achieved with detection limits of 23.36 fM for Target DNA and 4.17 CFU/mL for S.aureus, respectively. The HTCas12a system offers a universal platform for pathogen detection in various fields, including environmental monitoring, clinical diagnosis, and food safety., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Tuberculosis is one of devastating infectious diseases in the world, and early diagnosis and treatment can help overcome this global burden. In this work, a new detection platform combining smartphone-assisted fluorescent analysis and highly sensitive fluorescent copper nanoprobes (CuNPs) in a specific nucleic acid amplification test (NAAT) for the diagnosis of tuberculosis (TB) was demonstrated and validated using clinical samples. To enhance the precision and accuracy of detection, polymerase chain reaction (PCR), padlock probe (PLP) ligation, and rolling circle amplification (RCA) were combined. Long poly(thymine) (polyT) single-stranded DNA was synthesized through RCA, and polyT-CuNPs were formed by adding copper(II) ions and sodium ascorbate as reducing agents; subsequently, the results were visualized through the excitation from a UV transilluminator and quantified with just a smartphone. After optimization, this proposed platform was validated by testing 18 residual DNA samples after TB PCR, including 8 TB-negative and 10 TB-positive samples, and exhibited a detection limit of 5 fg/μL. The findings indicate the potential of this platform for practical application, where it can be combined with a smartphone for image analysis to achieve accurate on-site detection of TB, especially in resource-limited settings., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The conformational dynamics of single-stranded DNA (ss-DNA) are implicated in the mechanisms of several key biological processes such as DNA replication and damage repair and have been modeled with those of semiflexible or flexible polymer. The high flexibility and customizability of ss-DNA also make it an excellent polymeric material for materials engineering. Polythymidine (poly(T)) is an excellent model ss-DNA as a flexible polymer since it does not form any secondary structure. However, only limited experimental results have been reported of poly(T) conformational dynamics with a very short length that is not relevant to the aforementioned processes and applications. Here, we provide the first experimental results of the conformational dynamics of poly(T) with lengths in the range of 130-170 nucleotides at the single-molecule level. Our experiments are based on single-molecule FRET and a DNA hairpin structure of which the folding kinetics are governed by the conformational dynamics of poly(T). We found that the folding kinetics deviate far from those of a flexible polymer model with a harmonic bending potential. To this end, we derived a simple model for the kinetics of DNA hairpin folding from the self-avoiding-walk (SAW). Our model describes the conformational dynamics of poly(T) very well and enables estimation of the conformational dimensionality. The estimated dimensionalities suggest that ss-DNA is completely flexible at 100 mM or a higher NaCl concentration, but not at 50 mM. These results will help understand the conformational dynamics of ss-DNA implicated in several key biological processes and maximize the utility of ss-DNA for materials engineering. Also, our system and model provide an excellent platform to investigate the conformational dynamics of ss-DNA.

Herein, we describe capsule polymer particles with precisely controlled pH-responsive release properties prepared directly via the interfacial photo-cross-linking of spherical poly(2-diethylaminoethyl methacrylate- co -2-cinnamoylethyl methacrylate) (P(DEAEMA-CEMA)) particles. In the interfacial photo-cross-linking, photoreactive cinnamoyl groups in the polymer particles were cross-linked via [2π + 2π] cycloaddition reactions at the polymer/water interface, showing that the shell-cross-linked hollow polymer particles can be directly prepared from spherical polymer particles. The approach has fascinating advantages such as using minimal components, simplicity, and not requiring sacrificial template particles and toxic solvents. The following important observations are made: (I) encapsulated materials were stably retained in the capsule particles under neutral pH conditions; (II) encapsulated materials were released from the capsule particles under acidic pH conditions; (III) the release kinetics of encapsulated materials were controlled by the pH conditions; i.e., immediate and sustained release was achieved by varying the acidity of the aqueous media; (IV) the photoirradiation time did not significantly affect the release kinetics under different pH conditions; and (V) the pH-responsive release properties were regulated by changing the polymer composition in P(DEAEMA-CEMA). Furthermore, by exploiting the pH-responsiveness, capsule particles are successfully obtained via an all-aqueous process from spherical polymer particles. The advantages of the all-aqueous encapsulation process allowed the water-soluble biomacromolecules such as DNA and saccharides to be successfully encapsulated in the P(DEAEMA-CEMA) hollow particles. With this simple interfacial photo-cross-linking strategy, we envision the ready synthesis of sophisticated particulate materials for broad application in advanced research fields.

The function and activity of many proteins is finely controlled by the modulation of the entropic contribution of intrinsically disordered domains that are not directly involved in any recognition event. Inspired by this mechanism, we demonstrate here that we could finely regulate the catalytic activity of a model DNAzyme (i.e., a synthetic DNA sequence with enzyme-like properties) by rationally introducing intrinsically disordered nucleic acid portions in its original sequence. More specifically, we have re-engineered here the well-characterized Cu 2+ -dependent DNAzyme that catalyzes a self-cleavage reaction by introducing a poly(T) linker domain in its sequence. The linker is not directly involved in the recognition event and connects the two domains that fold to form the catalytic core. We demonstrate that the enzyme-like activity of this re-engineered DNAzyme can be modulated in a predictable and fine way by changing the length, and thus entropy, of such a linker domain. Given these attributes, the rational design of intrinsically disordered domains could expand the available toolbox to achieve a control of the activity of DNAzymes and, in analogy, ribozymes through a purely entropic contribution.

Cost-effective, fast, and reliable DNA sequencing can be enabled by advances in nanopore-based methods, such as the use of atomically thin graphene membranes. However, strong interaction of DNA bases with graphene leads to undesirable effects such as sticking of DNA strands to the membrane surface. While surface functionalization is one way to counter this problem, here, we present another solution based on a heterostructure nanopore system, consisting of a monolayer of graphene and hexagonal boron nitride (hBN) each. Molecular dynamics studies of DNA translocation through this heterostructure nanopore revealed a surprising and crucial influence of the heterostructure layer order in controlling the base specific signal variability. Specifically, the heterostructure with graphene on top of hBN had nearly 3-10× lower signal variability than the one with hBN on top of graphene. Simulations point to the role of differential underside sticking of DNA bases as a possible reason for the observed influence of the layer order. Our studies can guide the development of experimental systems to study and exploit DNA translocation through two-dimensional heterostructure nanopores for single molecule sequencing and sensing applications.

As a rapid proliferating tissue, tumor cells have to optimize nutrient utilization to withstand harsh conditions. Several approaches have been explored to inhibit the growth and metastasis of tumor by disrupting the reprogrammed tumor metabolism. However, nutrient limitations within solid tumors may induce the metabolic flexibility of malignant cells to sustain growth and survival using one nutrient to fill metabolite pools normally supplied by the other. To overcome this predicament, a promising click-nucleic-acid-containing platform for codelivery of rapamycin, anti-PFKFB4 siRNA, and targeting ligand aptamer AS1411 was applied. PFKFB4 could act as a promising target for tumor therapy for being a molecular fulcrum that could couple glycolysis to autophagy by promoting aggressive metastatic tumors. The downregulation of PFKFB4 can help inhibit the SRC3/Akt/mTOR pathway, leading autophagy to the direction of promoting apoptosis of tumor cells, which is induced by the collapse of tumor cellular homeostasis, while low dosages of rapamycin could decrease surgery-induced immune dysfunction. Enhanced tumor autophagy, favorable in vivo antitumor efficacy, and effective systematic immune activation are observed after treatment, suggesting that autophagy and glycolysis can serve as an integrated target for tumor treatment.

In this study, a simple fluorescent detection of survival motor neuron gene (SMN) in diagnosis of spinal muscular atrophy (SMA) based on nucleic acid amplification test and the poly-T luminescent copper nanoclusters (CuNCs) was established. SMA is a severely genetic diseases to cause infant death in clinical, and detection of SMN gene is a powerful tool for pre- and postnatal diagnosis of this disease. This study utilized the molecular inversion probe for recognition of nucleotide variant between SMN1 (c.840 C) and SMN2 (c.840 C  >  T) genes, and rolling circle amplification with a universal primer for production of poly-T single-strand DNA. Finally, the fluorescent CuNCs were formed on the poly-T single-strand DNA template with addition of CuSO 4 and sodium ascorbate. The fluorescence of CuNCs was only detected in the samples with the presence of SMN1 gene controlling the disease of SMA. After optimization of experimental conditions, this highly efficient method was performed under 50 °C for DNA ligation temperature by using 2U Ampligase, 3 h for rolling circle amplification, and the formation of the CuNCs by mixing 500 μM Cu 2+ and 4 mM sodium ascorbate. Additionally, this highly efficient method was successfully applied for 65 clinical DNA samples, including 4 SMA patients, 4 carriers and 57 wild individuals. This label-free detection strategy has the own potential to not only be a general method for detection of SMN1 gene in diagnosis of SMA disease, but also served as a tool for detection of other single nucleotide polymorphisms or nucleotide variants in genetic analysis through designing the different sensing probes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

MicroRNAs (miRNAs) have received extensive attention because of their potential as biomarkers for cancer diagnosis and monitoring, and their effective detection is very significant. Here, a specific, one-pot, rapid, femtomolar sensitive miRNAs detection biosensor was developed based on the target-triggered three-way junction (3-WJ) and terminal deoxynucleotide transferase (TDT)/Nt.BspQI in combination with activated copper nanoparticles (CuNPs) self-assembly. To this end, a 3-WJ hairpin probe and helper probe were designed to selectively identify the target miRNA, so as to form a stable 3-WJ structure that further triggered the double-enzyme cycling to produce poly T to activate the self-assembly of CuNPs. Based on the simplicity of CuNPs generation, the poly T template fluorescence CuNPs can detect the minimum detection limit of 1 fm within 1.75 h. In addition, the applicability of this method in complex samples was demonstrated by analyzing the whole-blood RNA extraction from Parkinson patients, consisting of the results of commercial miRNA kits. The developed strategy performs powerful implications for miRNA detection, which may be beneficial for the effective diagnostic assays and biological research of Parkinson's disease.

MicroRNAs (miRNAs) act as pivotal regulators in plants. Therefore, sensing strategies with high specificity and high sensitivity are desired for plant miRNA analysis in order to unveil the exact biofunctions of miRNAs. Toward this goal, a fluorescent assay is developed based on a two-step signal amplification strategy. In the first step, target miRNA-templated cycling click nucleic acid ligation is employed for target recognition and amplification, the product of which can bind to magnetic microbeads (MBs) and introduce the T7 promoter sequence to the surface. In the second step, the poly(T) containing transcription template partially hybridizes with the T7 promoter sequence on the ligated strand and then regulates the on-bead transcription in a cycling manner with the participation of T7 RNA polymerase. Surprisingly, different from other reported templates, the poly(T) template improves the transcription efficiency to an unexpectedly high level by releasing ultra-long RNA chains in the reaction system. Finally, the RNA intercalating dye, RiboGreen, is utilized to specifically light up the as-produced RNA chains for low-background signal readout. Benefiting from the elaborate design, the detection limit of plant miRNA is down to ∼0.1 amol. This strategy provides a highly specific and highly sensitive platform for plant miRNA detection, which promises potential in the practical applications of miRNA-related biofunctions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Oligonucleotide-based medicines that can modulate gene expression have numerous potential applications in targeted therapies. Most of the commercialized therapeutic oligonucleotides are chemically modified to increase their in vivo lifetime. In this work, we studied poly-deoxy(thymidylic) acids (dT) and modified phosphorothioate oligonucleotides (PS). Several analytical techniques, including ion-pair reverse phase liquid chromatography, are described in the literature to assess their quality but most of them present significant drawbacks. In the present study, dT and PS mixtures were analyzed by hydrophilic interaction liquid chromatography (HILIC) and capillary zone electrophoresis (CZE) coupled to ultraviolet detection. In HILIC, the selectivities of three types of stationary phases (dihydroxypropane, phosphorylcholine and amide) were compared. Optimal conditions were determined and consisted of an amide stationary phase with a mobile phase made up of water, acetonitrile and 15 mM ammonium acetate (pH 5.5). In those conditions, high resolving power and good repeatability were achieved. In CZE, the effect of the background electrolyte (BGE), its pH and concentration were evaluated. A BGE made up of 300 mM ammonium acetate adjusted to pH 6.0 was selected. Finally, the two techniques were compared in terms of selectivity, repeatability and peak efficiency. In the second part of the study, HILIC and CZE were both coupled to a drift-tube ion-mobility quadrupole time-of-flight MS detector (DTIMS-QTOF) to assess the added value of this coupling for oligonucleotide characterization. Indeed, by using the measured collision cross section (CCS), the evaluation of the number of nucleotides was performed. Looking across the results, HILIC and CZE coupled to DTIMS-QTOF can be considered as promising tools for the quality control of oligonucleotides., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019. Published by Elsevier B.V.)

The localised excitations of several molecular reactions utilising optical irradiation have been studied in the field of molecular physics. In particular, deoxyribonucleic acid (DNA) strands organise the genetic information of all living matter. Therefore, artificial methods for freely controlling reactions using only light irradiation are highly desirable for reactions of these strands; this in regard with artificial protein synthesis, regional genetic curing, and stochastic analysis of several genetic expressions. Generally, DNA strands have strong absorption features in the deep ultra-violet (DUV) region, which are related to the degradation and reconstruction of the strand bonding structures. However, irradiation by DUV light unavoidably induces unintended molecular reactions which can damage and break the DNA strands. In this paper, we report a photo-induced molecular reaction initiated by the irradiation of DNA strands with visible light. We utilised photo-dissociation from the vibrational levels induced by non-uniform optical near-fields surrounding nanometric Au particles to which DNA strands were attached. The results were experimentally observed by a reduction in the DUV absorbance of the DNA strands during irradiation. There was a much higher yield of molecular reactions than expected due to the absorbance of visible light, and no defects were caused in the DNA strands.

Poly(A-T)-rich sequences have been applied as stems of DNA dumbbell tiles for construction of single crystalline 2D DNA lattice arrays in slightly acidic solutions. These arrays show much higher stability and better organised crystalline lattice structures than those assembled from DNA dumbbell tiles with randomly sequenced stems in slightly alkaline environments. DNA nanotechnology probably provides a useful platform to study the mechanical properties of DNA duplexes with specific sequences.

The variations in microRNA (miRNA) expression levels can be useful biomarkers for the diagnosis of different cancers. In this work, a label-free and sensitive fluorescent method for detection of miRNA-21 is described based on duplex-specific nuclease (DSN) assist target recycling and terminal deoxynucleotidyl transferase (TdT) induced copper nanoclusters (CuNCs). In the absence of target, the 3'-phosphorylated probe DNA cannot be hydrolyzed by DSN and extended by TdT, and failed to synthesizing fluorescent CuNCs. However, the target miRNA-21 can caused the digestion of probe DNA with DSN, releasing primer DNA with 3'-OH. After that, the primer DNA can forms long poly T with the assistance of TdT, leading to synthesize high fluorescent CuNCs. The fluorescence change of CuNCs can be used to identify the concentration of target miRNA-21. Under optimal experimental conditions, this strategy could quantitatively detect miRNA-21 down to 18.7 pM. We have also demonstrated the practical application of our proposed method for monitoring miRNA-21 expression levels in cancer cells. Moreover, this method show good specificity for miRNA-21 detection due to the strong preference of DSN for cutting perfectly matched DNA/RNA duplex, which holds great potential for highly specific quantification of biomarkers in bioanalysis and clinical diagnosis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

A sensitive and label-free fluorometric method has been developed for the determination of polynucleotide kinase (PNK) activity, by employing exonuclease III (Exo III)-assisted cyclic signal amplification and poly(thymine)-templated copper nanoparticles (polyT-CuNPs). In the presence of PNK, cDNA with 5'-hydroxyl termini was phosphorylated and then hybridized with tDNA to form the cDNA/tDNA duplex, which subsequently triggered the λ exonuclease cleavage reaction, eventually resulting in the release of tDNA. The released tDNA could unfold the hairpin structure of HP DNA to generate partially complementary duplex (tDNA/HP DNA), wherein the HP DNA possessed T-rich sequences (T30) and tDNA recognition sequence. With the help of Exo III digestion, the tDNA was able to initiate the cycle for the generation of T-rich sequences, the template for the formation of fluorescent CuNPs. Conversely, the cDNA could not be cleaved by λ exonuclease without PNK and individual HP DNA could not be hydrolyzed by Exo III. The T-rich sequence was caged in HP DNA, resulting in a weak fluorescence signal. Under optimized conditions, the fluorescence intensity was linearly correlated to a concentration range of 0.001 to 1 U mL-1 with a low detection limit of 2 × 10-4 U mL-1. Considering the intriguing analytical performance, this approach could be explored to screen T4 PNK inhibitors and hold promising applications in drug discovery and disease therapy.

Quantum dot (QD) biological imaging and sensing applications often require surface modification with single-stranded deoxyribonucleic acid (ssDNA) oligonucleotides. Furthermore, ssDNA conjugation can be leveraged for precision QD templating via higher-order DNA nanostructures to exploit emergent behaviors in photonic applications. Use of ssDNA-QDs across these platforms requires compact, controlled conjugation that engenders QD stability over a wide pH range and in solutions of high ionic strength. However, current ssDNA-QD conjugation approaches suffer from limitations, such as the requirement for thick coatings, low control over ssDNA labeling density, requirement of large amounts of ssDNA, or low colloidal or photostability, restraining implementation in many applications. Here, we combine thin, multidentate, phytochelatin-3 (PC3) QD passivation techniques with strain-promoted copper-free alkyne-azide click chemistry to yield functional ssDNA-QDs with high stability. This process was broadly applicable across QD sizes (i.e., λ em = 540, 560, 600 nm), ssDNA lengths (i.e., 10-16 base pairs, bps), and sequences (poly thymine, mixed bps). The resulting compact ssDNA-QDs displayed a fluorescence quenching efficiency of up to 89% by hybridization with complementary ssDNA-AuNPs. Furthermore, ssDNA-QDs were successfully incorporated with higher-order DNA origami nanostructure templates. Thus, this approach, combining PC3 passivation with click chemistry, generates ssDNA-PC3-QDs that enable emergent QD properties in DNA-based devices and applications.

Nonviral delivery of nucleic acids to the cell nucleus typically requires chemical methods that do not guarantee specific delivery (e.g., transfection agent) or physical methods that may require extensive fabrication (e.g., microfluidics) or an elevated pressure (e.g., 10 5 Pa for microneedles). We report a method of delivering oligonucleotides to the nucleus with high specificity (relative to the cytosol) by synergistically combining chemical and physical approaches. Particularly, we demonstrate that DNA oligonucleotides appended with a polythymidine [poly(T)] segment (chemical) profusely accumulate inside the nucleus when the cells are under gentle compression imposed by the weight of a single glass coverslip (physical; ∼2.2 Pa). Our "compression-cum-poly(T)" delivery method is simple, can be generalizable to three "hard-to-transfect" cell types, and does not induce significant levels of cytotoxicity or long-term oxidative stress to the treated cells when provided the use of suitable compression times and oligonucleotide concentrations. In bEnd.3 endothelial cells, compression-aided intranuclear delivery of poly(T) is primarily mediated by importin β and nucleoporin 62. Our method significantly enhances the intranuclear delivery of antisense oligonucleotides to bEnd.3 endothelioma cells and the inhibition of two target genes, including a reporter gene encoding the enhanced green fluorescent protein and an intranuclear lncRNA oncogene (metastasis-associated lung adenocarcinoma transcript 1), when compared with delivery without gentle compression or poly(T) attachment. Our data underscore the critical roles of pressure and nucleotide sequence on the intranuclear delivery of nucleic acids.

Strongly coupled molecular dye aggregates have unique optoelectronic properties that often resemble those of light harvesting complexes found in Nature. The exciton dynamics in coupled dye aggregates could enhance the long-range transfer of optical excitation energy with high efficiency. In principle, dye aggregates could serve as important components in molecular-scale photonic devices; however, rational design of these coupled dye aggregates with precise control over their organization, interactions, and dynamics remains a challenge. DNA nanotechnology has recently been used to build an excitonic circuit by organizing pseudoisocyanine (PIC) dyes forming J-aggregates on the templates of poly(dA)-poly(dT) DNA duplexes. Here, the excitonic properties of the PIC J-aggregates on DNA are characterized spectroscopically in detail using poly(dA)-poly(dT) tract lengths of 24 and 48 base pairs. The excitonic properties of these DNA templated dye assemblies depend on the length and sequence of the DNA template. The incorporation of a gap of two GC base pairs between two segments of poly(dA)-poly(dT) DNA markedly reduces the delocalization of excitation in the J-aggregates. With a quantum dot (QD) as the light absorber and energy donor and using Alexa Fluor 647 (AF647) as the energy acceptor, with a DNA-templated J-aggregate in between, significant energy transfer from QD to AF647 is observed over a distance far longer than possible without the aggregate bridge. By comparing the efficiency of energy transfer through a continuous J-aggregate with the efficiency when the aggregate has a discontinuity in the middle, the effects of energy transfer within the aggregate bridge between the donor and acceptor are evaluated.

A selective fluorescent on-off-on probe has have designed for the detection of fluoride (F - ) ions based on DNA-templated copper nanocluster (CuNCs) and by using aluminum(III) ions as a bridge. A 40-mer polythymine acts as a template for the reduction of Cu(II) to Cu(0) by ascorbic acid. This result is the formation of red fluorescent CuNCs, with excitation/emission peaks at 340/640 nm. After addition of Al 3+ ions, the fluorescence of CuNCs is quenched because the interaction of Al 3+ and DNA disturbs the formation of DNA-templated CuNCs. Fluorescence is restored on addition of fluoride to the system. This is due to the desorption of Al 3+ from the DNA and the formation of the Al(OH) 3 F - complex. This system displays a fast fluorometric response to fluoride, with high selectivity over other anions. Fluorescence increases linearly in the 2 to 150 μM F - concentration range, and the detection limit is 1.0 μM. This probe has been successfully used for the detection of F - ions in four brands of toothpaste. The method is rapid, cost-effective, selective, and does not require toxic solvents and reagents. Graphical abstract Schematic presentation of a method for fluorometric determination of fluoride by using DNA-templated copper nanoclusters (CuNCs) and using aluminum(III) as a bridge. The red fluorescence of the CuNCs is quenched in the presence of Al(III) ions but restored after addition of fluoride.

Ultrahigh-throughput conformational sampling of biopolymers like nucleic acids are most effectively carried out without explicit solvents, but the physical origins of almost all inter- and intramolecular interactions controlling nucleic acid structures are rooted in water. Single-stranded (ss) DNAs or RNAs in water are characterized by ensembles of diverse conformations. To properly capture solvent-induced nucleobase stacking interactions in an otherwise solvent-free Monte Carlo algorithm, theoretical models are developed here to describe the solvent entropy and dispersion terms in base stacking free energies. To validate these models, equilibrium ensembles of ss (dA) n and (dT) n sequences ( n = 30, 40, and 50) were simulated, and they quantitatively reproduced experimental small-angle X-ray scattering (SAXS) data. Simulated dA ensembles show substantial stacking. While less prevalent, stacking in dT chains is not negligible. Analysis of SAXS profiles suggests that excess features between wavevector 0.03 and 0.18 Å -1 correlate with stacking, and stacking in dA versus dT chains is chain length-dependent, where (dT) 30 and (dA) 30 chains have more similar structures, but longer dA chains show more stacking over dT. The average stack length in ss-dA chains is 5-10 nucleotides, yielding an estimate for the overall A|A stacking free energy at ∼1 kcal/mol.

We have performed a systematic study to analyze the effect of ssDNA length, nucleobase composition, and the type of two-dimensional nanoparticles (2D-nps) on the desorption response of 36 two-dimensional nanoassemblies (2D-NAs) against several proteins. The studies were performed using fluorescently labeled polyA, polyC, and polyT with 23, 18, 12, and 7 nucleotide-long sequences. The results suggest that the ssDNAs with polyC and longer sequences are more resistant to desorption, compared to their counterparts. In addition, 2D-NAs assembled using WS 2 were least susceptible to desorption by the proteins tested, whereas nGO 2D-NAs were the most susceptible nanoassemblies. Later, the results of these systematic studies were used to construct a sensor array for discrimination of seven model proteins (BSA, lipase, alkaline phosphatase, acid phosphatase, protease, β-galactosidase, and Cytochrome c). Neither the ssDNAs nor the 2D-nps have any specific interaction with the proteins tested. Only the displacement of the ssDNAs from the 2D-np surface was measured upon the disruption of the existing forces within 2D-NAs. A customized sensor array with five 2D-NAs was developed as a result of a careful screening/filtering process. The sensor array was tested against 200 nM of protein targets, and each protein was discriminated successfully. The results suggest that the systematic studies performed using various ssDNAs and 2D-nps enabled the construction of a sensor array without a bind-and-release sensing mechanism. The studies also demonstrate the significance of systematic investigations in the construction of two-dimensional DNA nanoassemblies for functional studies.

Herein, for the first time we report a novel competitive fluorescence immunoassay for the ultrasensitive detection of aflatoxin B1 (AFB1) using histone-ds-poly(AT) templated copper nanoparticles (His-pAT CuNPs) as the fluorescent indicator. In this immunoassay, glucose oxidase (Gox) was used as the carrier of the competing antigen to catalyze the formation of hydrogen peroxide (H2O2) from glucose. H2O2 was converted to a hydroxyl radical using Fenton's reagent, which further regulated the fluorescence signals of His-pAT CuNPs. Owing to the ultrahigh sensitivity of the ss-DNA to the hydroxyl radical, the proposed fluorescence immunoassay exhibited a favorable dynamic linear detection of AFB1 ranging from 0.46 pg mL-1 to 400 pg mL-1 with an good half maximal inhibitory concentration and limit of detection of 6.13 and 0.15 pg mL-1, respectively. The intra- and inter-assay showed that the average recoveries for AFB1 spiked corn samples ranged from 96.87% to 100.73% and 96.67% to 114.92%, respectively. The reliability of this method was further confirmed by adopting ultra-performance liquid chromatography coupled with the fluorescence detector method. In summary, this work offers a novel screening strategy with high sensitivity and robustness for the quantitative detection of mycotoxins or other pollutants for food safety and clinical diagnosis.

Exosomes, a class of small extracellular vesicles, play important roles in various physiological and pathological processes by serving as vehicles for transferring and delivering membrane and cytosolic molecules between cells. Since exosomes widely exist in various body fluids and carry molecular information on their originating cells, they are being regarded as potential noninvasive biomarkers. Nevertheless, the development of convenient and quantitative exosome analysis methods is still technically challenging. Here, we present a low-cost assay for direct capture and rapid detection of exosomes based on a copper-mediated signal amplification strategy. The assay involves three steps. First, bulk nanovesicles are magnetically captured by cholesterol-modified magnetic beads (MB) via hydrophobic interaction between cholesterol moieties and lipid membranes. Second, bead-binding nanovesicles of exosomes with a specific membrane protein are anchored with aptamer-modified copper oxide nanoparticles (CuO NPs) to form sandwich complexes (MB-exosome-CuO NP). Third, the resultant sandwich complexes are dissolved by acidolysis to turn CuO NP into copper(II) ions (Cu 2+ ), which can be reduced to fluorescent copper nanoparticles (CuNPs) by sodium ascorbate in the presence of poly(thymine). The fluorescence emission of CuNPs increases with the increase of Cu 2+ concentration, which is directly proportional to the concentration of exosomes. Our method allows quantitative analysis of exosomes in the range of 7.5 × 10 4 to 1.5 × 10 7 particles/μL with a detection of limit of 4.8 × 10 4 particles/μL in biological sample. The total working time is about 2 h. The assay has the potential to be a simple and cost-effective method for routine exosome analysis in biological samples.

Poly(thymine)-hosted copper nanoparticles (poly T-CuNPs) have emerged as a promising label-free fluorophore for bioanalysis, but its application in RNA-related studies is still rarely explored. Herein, by utilizing duplex-specific nuclease (DSN) as a convertor to integrate target recycling mechanism into terminal deoxynucleotidyl transferase (TdT)-mediated superlong poly T-CuNPs platform, a specific and sensitive method for microRNA detection has been developed. In this strategy, a 3'-phosphorylated DNA probe can hybridize with target RNA and then be cut by DSN to produce 3'-hydroxylated fragments, which can be further tailed by TdT with superlong poly T for fluorescent CuNPs synthesis. As proof of concept, an analysis of let-7d was achieved with a good linear correlation between 20 and 1000 pM (R 2  = 0.9965) and a detection limit of 20 pM. Moreover, both homologous and heterologous microRNAs were also effectively discriminated. This strategy might pave a brand-new way for designing label-free and sensitive microRNA assays., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Fluorescent copper nanoparticles (CuNPs) have received great attention due to their distinct characteristics of facile synthesis, tunable fluorescence emission, high photostability, and biological compatibility, and they have been widely used for chemical and biological analyses. Bleomycins (BLMs) are widely used antitumor agents for the clinical treatment of various cancers. Here, we develop a sensitive and label-free fluorescence method for the quantitative detection of BLM on the basis of BLM-initiated enzymatic polymerization-mediated synthesis of fluorescent CuNPs. We design two hairpin DNAs: one (Hp1) for the recognition of BLM and the other (Hp2) for signal amplification. In the presence of BLM, it may recognize and cleave the 5'-GC-3' site of the Hp1 stem, releasing the 8-17 DNAzyme fragment. The resultant 8-17 DNAzyme fragments may bind with the loop of Hp2 to form a partial double-stranded DNA (dsDNA) duplex, initiating the cyclic cleavage of Hp2 in the presence of Zn2+-dependent DNAzymes and generating numerous new DNA fragments with the free 3'-OH terminal, which can induce the formation of a poly(thymine) (poly-T) sequence with the assistance of terminal deoxynucleotidyl transferase (TdTase). Subsequently, the ploy-T sequence may function as the template for the synthesis of CuNPs with strong fluorescence emission. This method shows good selectivity and high sensitivity with a detection limit as low as 8.1 × 10-16 M, and it exhibits good performance in serum samples. Moreover, this method has distinct advantages of simplicity and low cost, holding great potential in clinical diagnosis and biomedical research.

Herein, we report a class of molecular spherical nucleic acid (SNA) nanostructures. These nano-sized single molecules are synthesized from T 8 polyoctahedral silsesquioxane and buckminsterfullerene C 60 scaffolds, modified with 8 and 12 pendant DNA strands, respectively. These conjugates have different DNA surface densities and thus exhibit different levels of nuclease resistance, cellular uptake, and gene regulation capabilities; the properties displayed by the C 60 SNA conjugate are closer to those of conventional and prototypical gold nanoparticle SNAs. Importantly, the C 60 SNA can serve as a single entity (no transfection agent required) antisense agent to efficiently regulate gene expression. The realization of molecularly pure forms of SNAs will open the door for studying the interactions of such structures with ligands and living cells with a much greater degree of control than the conventional polydisperse forms of SNAs., Competing Interests: The authors declare no conflict of interest.

Calcium phosphate (CaP) has long been used for DNA delivery, although its fundamental interaction with DNA, especially with single-stranded DNA oligonucleotides, remains to be fully understood. Using fluorescently labeled oligonucleotides, we herein studied DNA adsorption isotherm and the effect of DNA length and sequence. Longer DNAs are adsorbed more strongly, and at neutral pH, poly-C DNAs are adsorbed more than the other three DNA homopolymers. However, at near pH 11, the pH of CaP synthesis, T 30 DNA is adsorbed more strongly than C 30 or A 30 . This can explain why T 30 and G 30 can fully inhibit the growth of CaP, while A 30 and C 30 only retarded its growth kinetics. DNA adsorption also reduces aggregation of CaP. DNA desorption experiments were carried out using concentrated urea, thymidine, or inorganic phosphate as competitors, and desorption was observed only in the presence of phosphate, suggesting that DNA uses its phosphate backbone to interact with the CaP surface. Desorption was also promoted by raising the NaCl concentration suggesting the electrostatic nature of interaction. Finally, ten different metal phosphate materials were synthesized by co-precipitating each metal ion (Ce 3+ , Fe 3+ , Ca 2+ , Ni 2+ , Zn 2+ , Mn 2+ , Ba 2+ , Cu 2+ , Sr 2+ , Co 2+ ), and DNA adsorption by these phosphate precipitants was found to be related to their surface charge and metal chemistry. This work has revealed fundamental surface science of DNA adsorption by CaP and other metal phosphate salts, and this knowledge might be useful for gene delivery, biomineralization, and DNA-directed assembly of metal phosphate materials.

For the efficient surface plasmon resonance (SPR)-based DNA assay researching, signal amplification tactics were absolutely necessary. In this work, a sensitive SPR-DNA sensor was developed by employing in situ synthesis of copper nanoparticles (CuNPs) templated by poly-T sequences DNA from terminal deoxynucleotidyl transferase (TdT)-mediated extension, and synergistically with nano-effect deposition as the mass relay. The objective of this strategy was manifold: firstly, tDNA hybridized with the optimal designed probes to active the TdT-mediated DNA extension onto the surface of SPR chip, resulted a long poly-T sequences ssDNA chain in dsDNA terminal onto surface of gold chip and characterized by SPR signal amplitudes. Secondly, copper ion (Cu 2+ ) adsorbed into the skeleton of poly-T sequences DNA, with the aid of ascorbic acid (VC) to achieve the Cu 2+ reduction, copper nanostructures (CuNPs) was synchronously generated onto the single nucleotide chain anchoring in dsDNA derivatives and the formation was featured by transmission electron micrographs (TEM) and electrochemistry. Lastly, dsDNA-complexed CuNPs (CuNPs@dsDNA) triggered the final signal amplification via real-time conversion of the additive catechol violet (CV) into oligomer or chelation precipitation by CuNPs-tagged reporters. With the proposed setups, a precise and replicable DNA sensing platform for specific target oligo was obtained with a detection limit down to 3.21 femtomolar, demonstrating a beneficial overlapping exploitation of nanomaterials and biochemical reaction as unique SPR infrastructure. Such triple-amplification strategic setups, the possibility of various methods abutment and biocompatibility weight reactor was amassed and adapted to more biological detection field., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Aptamer affinity capillary electrophoresis-laser-induced fluorescence (CE-LIF) for protein detection takes advantage of aptamers for their ease of synthesis and labeling, small size, and having many negative charges. Its success relies on the high binding affinity of aptamers. One 15-mer DNA aptamer (5'-GGT TGG TGT GGT TGG-3', Apt15) shows desirable specificity for human α-thrombin, an important enzyme with multiple functions in blood. However, Apt15 has weak binding affinity, and the use of Apt15 in affinity CE-LIF analysis remains challenging. Here we reported that extension of Apt15 at the 3'-end with a polyT tail having length of 18 T or longer significantly enhanced its affinity and enabled a well-isolated and stable peak for thrombin-aptamer complex in affinity CE. It was likely that the improvement of binding affinity resulted from double binding, an additional interaction of the polyT tail with thrombin in addition to the Apt15 section binding to thrombin. With dye-labeled Apt15 having a T 25 tail, we achieved detection of thrombin at concentrations as low as 0.1 nM by affinity CE-LIF. This aptamer probe specifically bound to human α-thrombin, showing negligible affinity for human β- and γ-thrombin, which are proteolyzed derivatives of human alpha α-thrombin and share similar structure. This strategy of adding a polyT extension also enhanced the binding affinity of anti-immunoglobulin E aptamer in CE-LIF analysis, showing that the affinity enhancement approach is not limited to the thrombin-binding aptamer and has potential for more applications in bioanalysis.

The human mitochondrial ssDNA-binding protein (mtSSB) is a homotetrameric protein, involved in mtDNA replication and maintenance. Although mtSSB is structurally similar to SSB from Escherichia coli (EcoSSB), it lacks the C-terminal disordered domain, and little is known about the biophysics of mtSSB-ssDNA interactions. Here, we characterized the kinetics and thermodynamics of mtSSB binding to ssDNA by equilibrium titrations and stopped-flow kinetic measurements. We show that the mtSSB tetramer can bind to ssDNA in two distinct binding modes: (SSB) 30 and (SSB) 60 , defined by DNA binding site sizes of 30 and 60 nucleotides, respectively. We found that the binding mode is modulated by magnesium ion and NaCl concentration, but unlike EcoSSB, the mtSSB does not show negative intersubunit cooperativity. Global fitting of both the equilibrium and kinetic data afforded estimates for the rate and equilibrium constants governing the formation of (SSB) 60 and (SSB) 30 complexes and for the transitions between the two binding modes. We found that the mtSSB tetramer binds to ssDNA with a rate constant near the diffusion limit (2 × 10 9 m -1 s -1 ) and that longer DNA (≥60 nucleotides) rapidly wraps around all four monomers, as revealed by FRET assays. We also show that the mtSSB tetramer can directly transfer from one ssDNA molecule to another via an intermediate with two DNA molecules bound to the mtSSB. In conclusion, our results indicate that human mtSSB shares many physicochemical properties with EcoSSB and that the differences may be explained by the lack of an acidic, disordered C-terminal tail in human mtSSB protein., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Deoxyribonuclease I (DNase I) is an important physiological indicator and diagnostic biomarker, but traditional methods for assessing its activity are time-consuming, laborious, and usually radioactive. Herein, by effectively combining the special functions of DNase I and terminal deoxynucleotidyl transferase (TdT), a simple, green, cost-effective, label-free and ultrasensitive assay for DNase I activity has been constructed based on superlong poly(thymine)-hosted copper nanoparticles (poly T-CuNPs). In this strategy, a 3'-phosphorylated DNA primer is designed to block TdT polymerization. After addition of DNase I, the primer could be digested to release 3'-hydroxylated fragments, which could further be tailed by TdT in dTTP pool with superlong poly T ssDNA for CuNPs formation. Fluorescence measurements and gel electrophoresis demonstrated its feasibility for DNase I analysis. The results indicated that with a size of 3-4nm, the CuNPs templated by TdT-polymerized superlong poly T (>500 mer) had several advantages such as short synthetic time (<5min), large Stokes shift (~275nm) and intense red fluorescence emission. Under the optimal conditions, quantitative detection of DNase I was realized, showing a good linear correlation between 0.02 and 2.0U/mL (R 2 =0.9928) and a detection limit of 0.02U/mL. By selecting six other nucleases or proteins as controls, an excellent specificity was also verified. Then, the strategy was successfully applied to detect DNase I in diluted serum with a standard addition method, thus implying its reliability and practicability for biological samples. The proposed strategy might be promising as a sensing platform for related molecular biology and disease studies., (Copyright © 2017 Elsevier B.V. All rights reserved.)

We recently reported that a great variety of DNA oligonucleotides (ONs) used as chiral selectors in partial-filling capillary electrophoresis (CE) exhibited interesting enantioresolution properties toward low-affinity DNA binders. Herein, the sequence prerequisites of ONs for the CE enantioseparation process were studied. First, the chiral resolution properties of a series of homopolymeric sequences (Poly-dT) of different lengths (from 5 to 60-mer) were investigated. It was shown that the size increase-dependent random coil-like conformation of Poly-dT favorably acted on the apparent selectivity and resolution. The base-unpairing state constituted also an important factor in the chiral resolution ability of ONs as the switch from the single-stranded to double-stranded structure was responsible for a significant decrease in the analyte selectivity range. Finally, the chemical diversity enhanced the enantioresolution ability of single-stranded ONs. The present work could lay the foundation for the design of performant ON chiral selectors for the CE separation of weak DNA binder enantiomers., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In this study, the binding mode of porphyrin-free single-stranded poly[d(AT)] and trans-BMPyP was observed in the Z-form trans-BMPyP-poly[d(A-T) 2 ] complex induced by extensive stacking depending on the temperature and concentration through circular dichroism (CD). The Z-form trans-BMPyP-poly[d(A-T) 2 ] complex (R = 0.30) retained the Z-form DNA structure at a low temperature (20 °C) by the trans-BMPyP molecules. When the temperature was increased to 60 °C, the DNA was almost unfolded as a single-stranded poly[d(AT)], but the extensive stacking binding mode of trans-BMPyP was maintained and the shape of the porphyrin Soret band was symmetrically changed in comparison with the shape of the Z-form DNA. However, when the temperature was raised to 80 °C, the extensive stacking binding mode of trans-BMPyP was also unfolded almost completely. The binding mode of the trans-BMPyP-single-stranded poly[d(AT)] complex was very similar to the already known binding mode of porphyrins and a double-stranded DNA. The binding mode was dependent on the concentration ratio ([porphyrin]/[DNA]): a monomeric binding mode at a concentration ratio of 0.04, a moderate groove binding mode at a concentration ratio between 0.08 and 0.16, and extensive stacking at a concentration ratio between 0.20 and 0.30. The same result was obtained when the temperature of the Z-form DNA (R = 0.30) was increased to 60 °C. However, those binding modes were not found in cis-BMPyP, which was because, in the extensive stacking of trans-BMPyP along the DNA skeleton, the distance between the two positive methylpyridine ions at the trans site and thymine, one of the DNA bases, is decreased, creating a much more hydrophobic environment. In addition, the poly AT sequences found from the CD spectra for the binding of trans-BMPyP-poly[d(A-T) 2 ] and trans-BMPyP-poly[d(AT)] (R = 0.30) showed that both of them underwent effective extensive stacking and that the chirality of extensive stacking was dependent on the form of DNA.

A new strategy based on enzymatically engineered primer extension poly-thymine (EPEPT) and nanomaterials in situ generation technology is reported for direct detection of microRNA (miRNA) in a fluorescence turn-on format using the sequential and complementary reactions catalyzed by Klenow Fragment exo - (KFexo - ) and terminal deoxynucleotidyl transferase (TdTase). The short miRNA can be efficiently converted into long poly-thymine (polyT) sequences, which function as template for in situ formation of fluorescence copper nanoparticles (CuNPs) as nano-dye for detecting miRNA. The polyT-CuNPs can effectively form and emit intense red fluorescence under the 340nm excitation. For the proof of concept, microRNA-21 (miR-21) was selected as the model target to testify this strategy as a versatile assay platform. By directly using miR-21 as the primer, the simple, rapid and sensitive miRNA detection was successfully achieved with a good linearity between 1pM and 1nM and a detection limit of 100fM. Thus, the EPEPT strategy holds great potential in biochemical sensing research as an efficient and universal platform., (Copyright © 2016 Elsevier B.V. All rights reserved.)

trans-BMPyP induced the B-Z transition for alternated AT oligonucleotides as it was evident by inversed CD spectrum. The transition occurred simultaneously with appearance of the extensive stacking of porphyrin. Complete B-Z transition required at least 14 base-pairs long. Insertion of one or two GC base pairs prevented the B-Z transition., (Copyright © 2016. Published by Elsevier B.V.)

DNA building blocks consisting of up to four nucleobases are investigated using the EOM-CCSD and CC2-LR methods in two B-DNA-like arrangements of a poly-adenine:poly-thymine (poly-A:poly-T) system. Excitation energies and oscillator strengths are presented and the characteristics of the excited states are discussed. Excited states of single-stranded poly-A systems are highly delocalized, especially the spectroscopically bright states, where delocalization over up to four fragments can be observed. In the case of poly-T systems, the states are somewhat less delocalized, extending to maximally about three fragments. A single A:T Watson-Crick pair has highly localized states, while delocalization over base pairs can be observed for some excited states of the (A)2:(T)2 system, but intrastrand delocalization is more pronounced in this case, as well. As for the characteristics of the simulated UV absorption spectra, a significant decrease of intensity can be observed in the case of single strands with increasing chain length; this is due to the stacking interactions and is in accordance with previous results. On the other hand, the breaking of H-bonds between the two strands does not alter the spectral intensity considerably, it only causes a redshift of the absorption band, thus it is unable to explain the experimentally observed DNA hyperchromism on its own, and stacking interactions need to be considered for the description of this effect as well.

Gold nanoparticle (AuNP)-templated spherical nucleic acids (SNAs) have been demonstrated as an important functional material in bionanotechnology. Fabrication of SNAs having high hybridization capacity to their complementary sequences is critical to ensure their applicability in areas such as antisense gene therapy and cellular sensing. The traditional salt-aging procedure is effective but tedious, requiring 1-3 days to complete. The rapid low-pH assisted protocol is efficient, but causes concerns related to nonspecific DNA adsorption to the AuNP core. To address these issues, we systematically compared the SNAs prepared by these two methods (salt-aging method and low-pH protocol). In terms of the number of complementary DNA that each SNA can bind and the average binding affinity of each thiolated DNA probe to its complementary strand, both methods yielded comparable hybridizability, although higher loading capacity was witnessed with SNAs made using the low-pH method. Additionally, it was found that nonspecific DNA binding could be eliminated almost completely by a simple glutathione (GSH) treatment of SNAs. Compared to conventional methods using toxic mercapto-hexanol or alkanethiols to remove nonspecific DNA adsorption, GSH is mild, cost-effective, and technically easy to use. In addition, GSH-passivated SNAs minimize the toxicity concerns related to AuNP-induced GSH depletion and therefore offer a more biocompatible alternative to previously reported SNAs. Moreover, rational design of probe sequences through inclusion of a polythymine spacer into the DNA sequences resulted in enhanced DNA loading capacity and stability against salt-induced aggregation. This work provides not only efficient and simple technical solutions to the issue of nonspecific DNA adsorption, but also new insights into the hybridizability of SNAs.

The binding of distamycin dimeric analog (Pt-bis-Dst) to poly[d(A-T)] x poly[d(A-T)1, poly(dA) x poly(dT) and duplex O23 with the sequence 5'-GCCAATATATATATATTATTAGG-3' which is present at the origin of replication of herpes simplex virus OriS is investigated with the use of UV and CD spectroscopy. The distinction of the synthetic polyamide from a natural antibiotic lies in the fact that in the synthetic polyamide there are two distamycin moieties bound via a glycine cis-diamino platinum group. It was shown that the binding of Pt-bis-Dst to poly[d(A-T)] x poly[d(A-T)] and poly(dA) x poly(dT) reaches saturation if one molecule of the ligand occurs at approximately every 8 bp. With further increase in the ratio of the added ligand to the base pairs in CD spectra of complexes with poly[d(A-T)] x poly[d(A-T)], we observed that the maximum wavelength band tend to be shifted towards longer wavelengths, while in the spectral region of 290-310 nm a "shoulder", that was absent in the spectra of the complexes obtained at low polymer coverages by the ligand, appeared. At high molar concentration ratios of ligand to oligonucleotide Pt-bis-Dst can bind to poly[d(A-T)] x poly[d(A-T)] in the form of hairpins or may form associates by the interaction between the distamycin moieties of neighboring molecules of Pt-bis-Dst. The structure of the complexes is stabilized by interactions between pirrolcarboxamide moieties of two molecules of Pt-bis-Dst adsorbed on adjacent overlapping binding sites. These interactions are probably also responsible for the concentration-dependent spectral changes observed during the formation of a complex between Pt-bis-Dst and poly[d(A-T)] x poly[d(A-T)]. Spectral changes are almost absent in binding of Pt-bis-Dst to poly(dA) x poly(dT). Binding of Pt-bis-Dst to duplex O23 reaches saturation if two ligand molecules occur in a duplex that contains a cluster of 18 AT pairs. With increasing the molar concentration ratio of the ligand to the duplex CD spectra undergo concentration-dependent changes similar to those observed during binding of Pt-bis-Dst to poly [d(A-T)] x poly[d(A-T)]. Testing for antiviral efficacy of Pt-bis-Dst showed that the concentration, at which the cytopathic effect produced by the herpes simplex virus in cell culture Vero E6 halved, is equal to 1.5 μg/ml and the selectivity index for evaluating antiviral activity is 65 at a relatively low cytotoxicity. The concentration of Pt-bis-Dst, at which approximately half the cells are killed, is equal to 100 μg/ml.

It is well-known that even small perturbations of the DNA sequence can drastically and unpredictably disrupt or alter the fluorescence of DNA-stabilized silver nanoclusters (DNA-AgNCs). Understanding how the structure of DNA affects the nanocluster that it stabilizes is the key to rationalizing such effects. We approach this challenge by strategically modifying the stem sequence of a hairpin DNA that hosts a spectrally pure, red-emitting nanocluster. Most of our modifications (base composition, sequence orientation, and loop location) reduce AgNC fluorescence in purity and shift it in wavelength, but one modification (appending poly(thymidine) to the 3' end of the stem) is inert with respect to fluorescence. Microfluidic capillary electrophoresis reveals that all of the modifications induce conformational changes of the DNA and that the original, spectrally pure nanocluster exists in two structurally distinct conformations. Interestingly, appending five or more thymidines, despite having no effect on fluorescence, eliminates this structural degeneracy. To explain this result, we propose that the original spectrally pure cluster is stabilized by a pair of hairpins whose stems can arrange in either a cis or trans orientation. Finally, we quantify the extent to which thymidine appendages of different lengths can be used to fine-tune the electrophoretic mobility of DNA-AgNC.

RAG complex consisting of RAG1 and RAG2 is a site-specific endonuclease responsible for the generation of antigen receptor diversity. It cleaves recombination signal sequence (RSS), comprising of conserved heptamer and nonamer. Nonamer binding domain (NBD) of RAG1 plays a central role in the recognition of RSS. To investigate the DNA binding properties of the domain, NBD of murine RAG1 was cloned, expressed and purified. Electrophoretic mobility shift assays showed that NBD binds with high affinity to nonamer in the context of 12/23 RSS or heteroduplex DNA. NBD binding was specific to thymines when single stranded DNA containing poly A, C, G or T were used. Biolayer interferometry studies showed that poly T binding to NBD was robust and comparable to that of 12RSS. More than 23 nt was essential for NBD binding at homothymidine stretches. On a double-stranded DNA, NBD could bind to A:T stretches, but not G:C or random sequences. Although NBD is indispensable for sequence specific activity of RAGs, external supplementation of purified nonamer binding domain to NBD deleted cRAG1/cRAG2 did not restore its activity, suggesting that the overall domain architecture of RAG1 is important. Therefore, we define the sequence requirements of NBD binding to DNA.

Absent in melanoma 2 (AIM2) is an intracellular pattern-recognition receptor, which is a member of the PYHIN protein family, consisting of a PYD domain and an IFN-inducible nuclear localization (HIN) domain. AIM2 is reported to oligomerize with adaptor protein ASC upon sensing bacterial and viral cytosolic DNA in order to form the AIM2 inflammasome, which activates caspase-1 leading to IL-1β secretion. Dysregulation of AIM2 inflammasome is supposed to result in autoinflammatory and autoimmune diseases. Thus, the development of new targeted drugs against AIM2 inflammasome would be important for the treatment of these diseases. However, since AIM2 inflammasome is an intracellular receptor, enforced internalization of both ligands and candidate molecules is necessary for the screening of AIM2-inflammasome-targeted molecules. We developed a reconstituted AIM2 inflammasome in a cell-free system with amplified luminescent proximity homogeneous assay (Alpha). Strong Alpha signal was detected upon incubation with poly-deoxyadenylic-deoxythymidylic acid, poly(dA:dT), whereas no Alpha signal was detected upon incubation with muramyl dipeptide, one of the NLR ligands of Nod2 ligand. The interaction between AIM2 and ASC was disrupted by an anti-human ASC monoclonal antibody, CRID3, a class of diarylsulfonylurea-containing compounds, and glycyrrhizin, a substance found in liquorice root. Thus, the reconstituted AIM2 inflammasome in a cell-free system is useful for screening AIM2-inflammasome-targeted therapeutic molecules., (Copyright © 2015 Elsevier B.V. All rights reserved.)

An electrochemical biosensor was developed for Hg(2+) determination based on DNA hybridization. In the presence of Hg(2+), the target and probe DNAs with thymine-thymine (T-T) mismatches could hybridize by forming T-Hg(2+)-T complex. This induced DNA hybridization led to the decrease in reduction peak currents of ethyl green (EG) as electroactive label, which could be used for determination of Hg(2+). The difference in the value of the peak currents of EG before and after DNA hybridization (ΔI) was linear with the concentration of Hg(2+) in the range of 9.0 × 10(-11)-1.0 × 10(-9) M. The detection limit was 3.08 × 10(-11) M., (Copyright © 2015 Elsevier Inc. All rights reserved.)

In this work, poly-thymine stabilized copper nanoclusters have been used as a fluorescence probe for melamine sensing for the first time. Melamine can bind to thymine through hydrogen bond, which could dramatically enhance the fluorescence intensity of poly-thymine stabilized copper nanoclusters. The enhancement factors (I-I0)/I0 increase linearly with the lgCmelamine over the melamine concentration range of 0.1 µM to 6 µM. The detection limit of melamine is 95 nM, which is 200 times lower than the US Food and Drug Administration estimate melamine safety limit 20 µM. Melamine in milk was detected with good recovery, which suggested that this novel fluorescence probe has great potential in practical application., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Biomineralized fluorescent metal nanoparticles have attracted considerable interest in many fields by virtue of their excellent properties in synthesis and application. Poly(thymine)-templated fluorescent copper nanoparticles (T-CuNPs) as a promising nanomaterial has been exploited by us recently and displays great potential for signal transducing in biochemical analysis. However, the application of T-CuNPs is rare and still at an early stage. Here, a new fluorescent analytical strategy has been developed for H2O2 and oxidase-based biosensing by exploiting T-CuNPs as an effective signal indicator. The mechanism is mainly based on the poly(thymine) length-dependent formation of T-CuNPs and the probe's oxidative cleavage. In this assay, the probe T40 can effectively template the formation of T-CuNPs by a fast in situ manner in the absence of H2O2, with high fluorescent signal, while the probe is cleaved into short-oligonucleotide fragments by hydroxyl radical (·OH) which is formed from the Fenton reaction in the presence of H2O2, leading to the decline of fluorescence intensity. By taking advantage of H2O2 as a mediator, this strategy is further exploited for oxidase-based biosensing. As the proof-of-concept, glucose in human serum has been chosen as the model system and has been detected, and its practical applicability has been investigated by assay of real clinical blood samples. Results demonstrate that the proposed strategy has not only good detection capability but also eminent detection performance, such as simplicity and low-cost, holding great potential for constructing effective sensors for biochemical and clinical applications.

A bottom-up strategy was developed for the enzyme mediated synthesis of Cu nanoparticles, which showed good sensing performance.

Cyromazine (CYR) can cause serious damage to the organs of animals or human beings, and it was found to bind to polythymine (polyT10) via multiple hydrogen bonding interactions. Based on this novel finding, a highly sensitive and simple colorimetric method was developed for CYR detection by using label-free gold nanoparticles (AuNPs) and polyT10. Under the optimized conditions, excellent linearity was acquired for CYR within the range of 1-500 ng mL(-1). In addition, the spectra and color changes of the AuNP solution were measured by spectrophotometry and observed by the naked eye, and the results showed that as low as 1 and 5 ng mL(-1) of CYR could be detected, depending upon the measurement methods. Afterwards, cucumber was selected to investigate the sample matrix effect and a sample pretreatment procedure was developed with simple homogenization and filtration. Even after 200 times dilution, the limit of detection (LOD) and limit of quantitation (LOQ) reached 252 ng g(-1) and 500 ng g(-1), respectively. The LOD and LOQ satisfied the Chinese requirement for the maximum residue limit (MRL), which is 0.5-1 μg g(-1) of CYR in most vegetables. The assay also showed a good average recovery of 83.7-104.8% with the RSD of less than 7% and good selectivity for cyromazine over other pesticides that may exist in vegetable samples. The method proposed in this study was simple, fast, and highly sensitive and accurate, and the test result with this method was visible to the naked eye. Therefore, it could be used for routine determination of CYR residues in cucumber samples.

A new, simple and sensitive fluorescence strategy was developed for the trypsin assay based on copper nanoparticles (CuNPs) and its different fluorescence response toward trypsin-catalyzed hydrolysis of cytochrome c (Cyt c). Polythymine (poly T)-templated CuNPs served as effective fluorescent probes. Cyt c is well-known to act as a quencher. However, herein, a low concentration of Cyt c was designed specially to act as the substrate of trypsin to avoid the quenching effects by electron transfer from Cyt c to CuNPs. In the presence of trypsin, Cyt c hydrolyzes to small peptides, releasing free cysteine residues. Nonfluorescent coordination complexes were formed upon exposure to free cysteine residues by a metal-ligand bond between Cu atoms and sulfur atoms, leading to a decreased fluorescence response to CuNPs. This novel method for the quantitative determination of trypsin has a linear detection range from 0.25 μg mL(-1) to 1000 μg mL(-1) and a relatively low detection limit of 42 ng mL(-1). To the best of our knowledge, this is the first application of the trypsin-catalyzed hydrolysis reaction of Cyt c to produce quenching effect in bioanalysis, which provided a novel approach for the biochemical sensing strategy.

Pyrimidine polyphosphates were first detected in cells 5 decades ago; however, their biological significance remains only partially resolved. Such nucleoside polyphosphates are believed to be produced nonspecifically by promiscuous enzymes. Herein, synthetically prepared deoxythymidine 5'-tetraphosphate (p4dT) was evaluated with a thymidylyltransferase, Cps2L. We have identified p4dT as a substrate for Cps2L and evaluated the reaction pathway by analysis of products using high-performance liquid chromatography, liquid chromatography and tandem mass spectrometry, and 31P nuclear magnetic resonance spectroscopy. Product analysis confirmed production of dTDP-Glc and triphosphate (P3) and showed no trace of dTTP-Glc and PPi, which could arise from alternative pathways for the reaction mechanism.

Ethanol affects unequally the thermal stability of DNA and RNA. It stabilizes RNA, while destabilizing DNA. The variation of the relative viscosity (η/η0) of [poly(dA-dT)]2 with temperature unveils transitions close to the respective denaturation temperature, calculated spectrophotometrically and calorimetrically. From the raw data densities and speeds of sound, the volumetric observables were calculated. In all cases studied, a change in sign from low to high ethanol content occurred for both partial molar volume (ϕV) and partial molar adiabatic compressibility (ϕK(S)). The minima, close to 10%, should correspond to the highest solvation and the maxima, close to 30%, to the lowest solvation. For 40-50% ethanol, the solvation increases again. The complex structure of ethanol-water, for which changes are observed in regions close to such critical concentrations, justifies the observed behaviour. The variation of ϕV and ϕK(S) was sharper for RNA compared with respect to DNA, indicating that the solvation sequence is poly(rA)·poly(rU) < ct-DNA < [poly(dA-dT)]2.

Due to its importance to develop strategies for copper(II) (Cu(2+)) detection, we here report a visual and portable strategy for Cu(2+) detection based on designing and using a strip-like hydrogel. The hydrogel is functionalized through caging poly(thymine) as probes, which can effectively template the formation of fluorescent copper nanoparticles (CuNPs) in the presence of the reductant (ascorbate) and Cu(2+). On the hydrogel's surface, uniform wells of microliter volume (microwells) are printed for sample-injection. When the injected sample is stained by Cu(2+), fluorescent CuNPs will be in situ templated by poly T in the hydrogel. With ultraviolet (UV) irradiation, the red fluorescence of CuNPs can be observed by naked-eye and recorded by a common camera without complicated instruments. Thus, the strategy integrates sample-injection, reaction and indication with fast signal response, providing an add-and-read manner for visual and portable detection of Cu(2+), as well as a strip-like strategy. Detection ability with a detectable minimum concentration of 20 μM and practically applicable properties have been demonstrated, such as resistance to environmental interference and good constancy, indicating that the strategy holds great potential and significance for popular detection of Cu(2+), especially in remote regions. We believe that the strip-like hydrogel-based methodology is also applicable to other targets by virtue of altering probes.