Smartphone-assisted fluorescent analysis of polyT-Cu-nanoprobes using nucleic acid amplification test for the diagnosis of tuberculosis.

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.

Conformational Dynamics of Poly(T) Single-Stranded DNA at the Single-Molecule Level.

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.

Interfacial Photo-Cross-Linking: Simple but Powerful Approach for Fabricating Capsule Polymer Particles with Tunable pH-Responsive Controlled Release Capability.

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.

DNA Translocation through Vertically Stacked 2D Layers of Graphene and Hexagonal Boron Nitride Heterostructure Nanopore.

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.

Rational Control of the Activity of a Cu2+-Dependent DNAzyme by Re-engineering Purely Entropic Intrinsically Disordered Domains.

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 Cu2+-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.

Click-Nucleic-Acid-Containing Codelivery System Inducing Collapse of Cellular Homeostasis for Tumor Therapy through Bidirectional Regulation of Autophagy and Glycolysis.

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.

Construction of a Novel Biosensor Based on the Self-assembly of Dual-Enzyme Cascade Amplification-Induced Copper Nanoparticles for Ultrasensitive Detection of MicroRNA153.

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.

Molecular inversion probe-rolling circle amplification with single-strand poly-T luminescent copper nanoclusters for fluorescent detection of single-nucleotide variant of SMN gene in diagnosis of spinal muscular atrophy.

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 CuSO4 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 Cu2+ 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.

High-sensitive sensing of plant microRNA by integrating click chemistry with an unusual on-bead poly(T)-promoted transcription amplification.

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.

Evaluation of hydrophilic interaction liquid chromatography, capillary zone electrophoresis and drift tube ion-mobility quadrupole time of flight mass spectrometry for the characterization of phosphodiester and phosphorothioate oligonucleotides.

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.

Visible light-induced thymine dimerisation based on large localised field gradient by non-uniform optical near-field.

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.

2D DNA lattice arrays assembled from DNA dumbbell tiles using poly(A-T)-rich stems.

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.

Exonuclease III-assisted signal amplification strategy for sensitive fluorescence detection of polynucleotide kinase based on poly(thymine)-templated copper nanoparticles.

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.

Compact quantum dot surface modification to enable emergent behaviors in quantum dot-DNA composites.

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., λem = 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.

Label-free detection of miRNA cancer markers based on terminal deoxynucleotidyl transferase-induced copper nanoclusters.

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.

Specific Delivery of Oligonucleotides to the Cell Nucleus via Gentle Compression and Attachment of Polythymidine.

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., 105 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 ß 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.

DNA-templated copper nanoclusters as a fluorescent probe for fluoride by using aluminum ions as a bridge.

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 Al3+ ions, the fluorescence of CuNCs is quenched because the interaction of Al3+ 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 Al3+ from the DNA and the formation of the Al(OH)3F- 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.

Directed Energy Transfer through DNA-Templated J-Aggregates.

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.

Theoretical Model for Solvent-Induced Base Stacking Interactions in Solvent-Free DNA Simulations.

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.

Fluorescence immunoassay based on the enzyme cleaving ss-DNA to regulate the synthesis of histone-ds-poly(AT) templated copper nanoparticles.

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.