Real-time label-free detection of dynamic aptamer-small molecule interactions using a nanopore nucleic acid conformational sensor.

Nucleic acids can undergo conformational changes upon binding small molecules. These conformational changes can be exploited to develop new therapeutic strategies through control of gene expression or triggering of cellular responses and can also be used to develop sensors for small molecules such as neurotransmitters. Many analytical approaches can detect dynamic conformational change of nucleic acids, but they need labeling, are expensive, and have limited time resolution. The nanopore approach can provide a conformational snapshot for each nucleic acid molecule detected, but has not been reported to detect dynamic nucleic acid conformational change in response to small -molecule binding. Here we demonstrate a modular, label-free, nucleic acid-docked nanopore capable of revealing time-resolved, small molecule-induced, single nucleic acid molecule conformational transitions with millisecond resolution. By using the dopamine-, serotonin-, and theophylline-binding aptamers as testbeds, we found that these nucleic acids scaffolds can be noncovalently docked inside the MspA protein pore by a cluster of site-specific charged residues. This docking mechanism enables the ion current through the pore to characteristically vary as the aptamer undergoes conformational changes, resulting in a sequence of current fluctuations that report binding and release of single ligand molecules from the aptamer. This nanopore tool can quantify specific ligands such as neurotransmitters, elucidate nucleic acid-ligand interactions, and pinpoint the nucleic acid motifs for ligand binding, showing the potential for small molecule biosensing, drug discovery assayed via RNA and DNA conformational changes, and the design of artificial riboswitch effectors in synthetic biology.

Generation and Single-Molecule Characterization of a Sequence-Selective Covalent Cross-Link Mediated by Mechlorethamine at a C-C Mismatch in Duplex DNA for Discrimination of a Disease-Relevant Single Nucleotide Polymorphism.

Many strategies for the detection of nucleic acid sequence rely upon Watson-Crick hybridization of a probe strand to the target strand, but the reversible nature of nucleic acid hybridization presents an inherent challenge: short probes that provide high target specificity have relatively low target affinity resulting in signal losses. Sequence-specific covalent cross-linking reactions have the potential to provide both selective target capture and durable signal. We explore a novel approach involving sequence-specific covalent cross-linking of a probe to target DNA combined with single-molecule nanopore detection of the cross-linked DNA. Here, we exploited the selective reaction of mechlorethamine at a C-C mismatch for covalent capture of a target DNA sequence corresponding to a cancer-driving mutation at position 1799 of the human BRAF kinase gene. We then demonstrated that the α-hemolysin protein nanopore can be employed for the unambiguous, single-molecule detection of the cross-linked probe-target complex. Cross-linked DNA generates an unmistakable deep and persistent current block (≥5 s) that is easily distinguished from the microsecond and millisecond blocks generated by translocation of single-stranded DNA and uncross-linked duplexes through the nanopore.

Remote Activation of a Nanopore for High-Performance Genetic Detection Using a pH Taxis-Mimicking Mechanism.

Aerolysin protein pore has been widely used for sensing peptides and proteins. However, only a few groups explored this nanopore for nucleic acids detection. The challenge is the extremely low capture efficiency for nucleic acids (>10 bases), which severely lowers the sensitivity of an aerolysin-based genetic biosensor. Here we reported a simple and easy-to-operate approach to noncovalently transform aerolysin into a highly nucleic acids-sensitive nanopore. Through a remote pH-modulation mechanism, we simply lower the pH on one side of the pore, then aerolysin is immediately "activated" and enabled to capture target DNA/RNA efficiently from the opposite side of the pore. This mechanism also decelerates DNA translocation, a desired property for sequencing and gene detection, allowing temporal separation of DNAs in different lengths. This method provides insight into the nanopore engineering for biosensing, making aerolysin applicable in genetic and epigenetic detections of long nucleic acids.

Sequence-Specific Covalent Capture Coupled with High-Contrast Nanopore Detection of a Disease-Derived Nucleic Acid Sequence.

Hybridization-based methods for the detection of nucleic acid sequences are important in research and medicine. Short probes provide sequence specificity, but do not always provide a durable signal. Sequence-specific covalent crosslink formation can anchor probes to target DNA and might also provide an additional layer of target selectivity. Here, we developed a new crosslinking reaction for the covalent capture of specific nucleic acid sequences. This process involved reaction of an abasic (Ap) site in a probe strand with an adenine residue in the target strand and was used for the detection of a disease-relevant T→A mutation at position 1799 of the human BRAF kinase gene sequence. Ap-containing probes were easily prepared and displayed excellent specificity for the mutant sequence under isothermal assay conditions. It was further shown that nanopore technology provides a high contrast-in essence, digital-signal that enables sensitive, single-molecule sensing of the cross-linked duplexes.

Mimicking Ribosomal Unfolding of RNA Pseudoknot in a Protein Channel.

Pseudoknots are a fundamental RNA tertiary structure with important roles in regulation of mRNA translation. Molecular force spectroscopic approaches such as optical tweezers can track the pseudoknot's unfolding intermediate states by pulling the RNA chain from both ends, but the kinetic unfolding pathway induced by this method may be different from that in vivo, which occurs during translation and proceeds from the 5' to 3' end. Here we developed a ribosome-mimicking, nanopore pulling assay for dissecting the vectorial unfolding mechanism of pseudoknots. The pseudoknot unfolding pathway in the nanopore, either from the 5' to 3' end or in the reverse direction, can be controlled by a DNA leader that is attached to the pseudoknot at the 5' or 3' ends. The different nanopore conductance between DNA and RNA translocation serves as a marker for the position and structure of the unfolding RNA in the pore. With this design, we provided evidence that the pseudoknot unfolding is a two-step, multistate, metal ion-regulated process depending on the pulling direction. Most notably, unfolding in both directions is rate-limited by the unzipping of the first helix domain (first step), which is Helix-1 in the 5' → 3' direction and Helix-2 in the 3' → 5' direction, suggesting that the initial unfolding step in either pulling direction needs to overcome an energy barrier contributed by the noncanonical triplex base-pairs and coaxial stacking interactions for the tertiary structure stabilization. These findings provide new insights into RNA vectorial unfolding mechanisms, which play an important role in biological functions including frameshifting.

p53-induced gene 3 mediates cell death induced by glutathione peroxidase 3.

Expression of glutathione peroxidase 3 (GPx3) is down-regulated in a variety of human malignancies. Both methylation and deletion of GPx3 gene underlie the alterations of GPx3 expression in prostate cancer. A strong correlation between the down-regulation of GPx3 expression and progression of prostate cancer and the suppression of prostate cancer xenografts in SCID mice by forced expression of GPx3 suggests a tumor suppression role of GPx3 in prostate cancer. However, the mechanism of GPx3-mediated tumor suppression remains unclear. In this report, GPx3 was found to interact directly with p53-induced gene 3 (PIG3). Forced overexpression of GPx3 in prostate cancer cell lines DU145 and PC3 as well as immortalized prostate epithelial cells RWPE-1 increased apoptotic cell death. Expression of GPx3(x73c), a peroxidase-negative OPAL codon mutant, in DU145 and PC3 cells also increased cell death. The induced expression of GPx3 in DU145 and PC3 cells resulted in an increase in reactive oxygen species and caspase-3 activity. These activities were abrogated by either knocking down PIG3 or mutating the PIG3 binding motif in GPx3 or binding interference from a peptide corresponding to PIG3 binding motif in GPx3. In addition, UV-treated RWPE-1 cells underwent apoptotic death, which was partially prevented by knocking down GPx3 or PIG3, suggesting that GPx3-PIG3 signaling is critical for UV-induced apoptosis. Taken together, these results reveal a novel signaling pathway of GPx3-PIG3 in the regulation of cell death in prostate cancer.

Single-molecule investigation of G-quadruplex using a nanopore sensor.

This review article introduces the nanopore single-molecule method for the study of G-quadruplex nucleic acid structures. Single G-quadruplexes can be trapped into a 2 nm protein pore embedded in the lipid bilayer membrane. The trapped G-quadruplex specifically blocks the current through the nanopore, creating a signature event for quantitative analysis of G-quadruplex properties, from cation-determined folding and unfolding kinetics to the interactions with the protein ligand. The nanopore single-molecule method is simple, accurate, and requires no labels. It can be used to evaluate G-quadruplex mechanisms and it may have applications in G-quadruplex-based biosensors, nanomachines, and nanostructure assembly.

Molecular bases of cyclodextrin adapter interactions with engineered protein nanopores.

Engineered protein pores have several potential applications in biotechnology: as sensor elements in stochastic detection and ultrarapid DNA sequencing, as nanoreactors to observe single-molecule chemistry, and in the construction of nano- and micro-devices. One important class of pores contains molecular adapters, which provide internal binding sites for small molecules. Mutants of the alpha-hemolysin (alphaHL) pore that bind the adapter beta-cyclodextrin (betaCD) approximately 10(4) times more tightly than the wild type have been obtained. We now use single-channel electrical recording, protein engineering including unnatural amino acid mutagenesis, and high-resolution x-ray crystallography to provide definitive structural information on these engineered protein nanopores in unparalleled detail.

Single-molecule detection of folding and unfolding of the G-quadruplex aptamer in a nanopore nanocavity.

Guanine-rich nucleic acids can form G-quadruplexes that are important in gene regulation, biosensor design and nano-structure construction. In this article, we report on the development of a nanopore encapsulating single-molecule method for exploring how cations regulate the folding and unfolding of the G-quadruplex formed by the thrombin-binding aptamer (TBA, GGTTGGTGTGGTTGG). The signature blocks in the nanopore revealed that the G-quadruplex formation is cation-selective. The selectivity sequence is K(+) > NH(4)(+) approximately Ba(2+) > Cs(+) approximately Na(+) > Li(+), and G-quadruplex was not detected in Mg(2+) and Ca(2+). Ba(2+) can form a long-lived G-quadruplex with TBA. However, the capability is affected by the cation-DNA interaction. The cation-selective formation of the G-quadruplex is correlated with the G-quadruplex volume, which varies with cation species. The high formation capability of the K(+)-induced G-quadruplex is contributed largely by the slow unfolding reaction. Although the Na(+)- and Li(+)-quadruplexes feature similar equilibrium properties, they undergo radically different pathways. The Na(+)-quadruplex folds and unfolds most rapidly, while the Li(+)-quadruplex performs both reactions at the slowest rates. Understanding these ion-regulated properties of oligonucleotides is beneficial for constructing fine-tuned biosensors and nano-structures. The methodology in this work can be used for studying other quadruplexes and protein-aptamer interactions.

Clinical associations of the genetic variants of CTLA-4, Tg, TSHR, PTPN22, PTPN12 and FCRL3 in patients with Graves' disease.

OBJECTIVE: Graves' disease (GD) is an organ-specific autoimmune disorder. Both immune-modulating genes and thyroid-specific genes are involved in its genetic pathogenesis. It remains unclear, however, how the interactions of various susceptibility genes contribute to the pathogenesis and clinical severity of the disease. The purpose of this study was to investigate the relationships between GD and single nucleotide polymorphisms (SNPs) from CTLA-4, PTPN22, PTPN12, FCRL3 (general autoimmunity genes regulating T and B cells) and the TSHR and Tg genes (disease-specific genes). Furthermore, we evaluated the influences these SNPs have on the risk and severity of GD. DESIGN AND METHODS: This cross-sectional clinical study was performed in 436 GD patients and 316 healthy, gender-matched individuals. Twenty-eight SNPs from CTLA-4, PTPN22, PTPN12, FCRL3, TSHR and Tg genes were genotyped and their associations with the risk and severity of GD were analysed. RESULTS: The CTLA-4 rs231779, Tg rs2069550 and PTPN22 rs3789604 SNPs were associated with GD, with additive risk effects present in rs231779 and rs2069550. The ACACC and ACGCT haplotypes, composed of five SNPs in the CTLA-4 gene (rs4553808, rs5472909, rs231775, rs231777 and rs231779), were protective and risk haplotypes respectively. The AA genotype of PTPN22 rs3789604 and AA genotype of FCRL3 rs7528684 were correlated with a reduced risk of GD, while the CC genotype of TSHR rs2239610 was associated with higher serum concentrations of FT4 and TRAb. Logistic analysis confirmed the contribution of CTLA-4 rs231779 to the development of GD. CONCLUSIONS: These preliminary results demonstrate that the immune-regulatory gene CTLA-4 and the thyroid-specific gene Tg contribute to the risk of Graves' disease with additive effects, while PTPN22 rs3789604 and FCRL3 rs7528684 polymorphisms are protective against the disease. In addition, the TSHR rs2239610 SNP is related to the severity of Graves' disease.

Single molecule sensing by nanopores and nanopore devices.

Molecular-scale pore structures, called nanopores, can be assembled by protein ion channels through genetic engineering or be artificially fabricated on solid substrates using fashion nanotechnology. When target molecules interact with the functionalized lumen of a nanopore, they characteristically block the ion pathway. The resulting conductance changes allow for identification of single molecules and quantification of target species in the mixture. In this review, we first overview nanopore-based sensory techniques that have been created for the detection of myriad biomedical targets, from metal ions, drug compounds, and cellular second messengers to proteins and DNA. Then we introduce our recent discoveries in nanopore single molecule detection: (1) using the protein nanopore to study folding/unfolding of the G-quadruplex aptamer; (2) creating a portable and durable biochip that is integrated with a single-protein pore sensor (this chip is compared with recently developed protein pore sensors based on stabilized bilayers on glass nanopore membranes and droplet interface bilayer); and (3) creating a glass nanopore-terminated probe for single-molecule DNA detection, chiral enantiomer discrimination, and identification of the bioterrorist agent ricin with an aptamer-encoded nanopore.

Capturing single molecules of immunoglobulin and ricin with an aptamer-encoded glass nanopore.

Nanopore-based single-molecule biosensors have been extensively studied. Protein pores that have receptors attached to them are target-selective, but their real-time applications are limited by the fragility of the lipid membrane into which the protein pores are embedded. Synthetic nanopores are more stable and provide flexible pore sizes, but the selectivity is low when detecting in the translocation mode. In spite of modifications with probing molecules, such as antibodies, to potentiate specific targeting, these nanopores fail to bind individual target molecules. Distinguishing between binding and translocation blocks remains unsolved. Here, we propose an aptamer-encoded nanopore that overcomes these challenges. Aptamers are well-known probing oligonucleotides that have high sensitivity and selectivity. In contrast to antibodies, aptamers are much smaller than their targets, rendering target blockades in the nanopore much more distinguishable. We used aptamer-encoded nanopores to detect single molecules of immunoglobulin E and the bioterrorist agent ricin, sequentially captured by the immobilized aptamer in the sensing zone of the pore. The functional nanopore also probed sequence-dependent aptamer-protein interactions. These findings will facilitate the development of a universal nanopore for multitarget detection.

Method of creating a nanopore-terminated probe for single-molecule enantiomer discrimination.

Nanopores are increasingly utilized as tools for single-molecule detection in biotechnology. Many nanopores are fabricated through procedures that require special materials, expensive facilities and experienced operators, which limit their usefulness on a wider scale. We have developed a simple method of fabricating a robust, low-noise nanopore by externally penetrating a nanocavity enclosed in the terminal of a capillary pipet. The nanocavity was shown to have a pore size on the scale of a single molecule, verified by translocation of molecules of known sizes, including double-stranded DNA (2 nm), gold nanoparticles (10 nm), and ring-shaped cyclodextrin (1.5 nm). The small pore size allows entrapment of a single cyclodextrin molecule. The glass nanopore with a trapped cyclodextrin proves useful in single-molecule discrimination of chiral enantiomers.

Apolipoprotein E polymorphism in normal Han Chinese population: frequency and effect on lipid parameters.

Apolipoprotein E (ApoE) genotypes were studied in order to determine the prevalence and effect on lipid parameters in normal Han Chinese population. Fragments of ApoE gene forth exon containing codon 112 and 158 polymorphic locus were amplified by PCR, and then digested with Cfo I endonuclease. Genotypes and alleles frequencies of 168 healthy Han Chinese were calculated. The frequency of genotypes epsilon3/3, epsilon3/4, and epsilon2/3 was found to be 75.00, 10.70, and 11.90%, respectively, and 0.60, 1.20, and 0.60% for epsilon2/2, epsilon2/4, and epsilon4/4. The effects of ApoE genotypes and alleles on lipid parameters were analyzed. The effects of ApoE alleles on TC, LDL-C, ApoB was: along a decreasing gradient epsilon4 > epsilon3 > epsilon2. The effect of epsilon4 allele was to increase serum levels of TC, LDL-C and ApoB, and epsilon2 allele had an effect opposite to that of epsilon4 allele. Results obtained in this study indicate that ApoE polymorphism is an independent genetic factor on individual serum levels of lipids and apolipoproteins.

Selective covalent capture of a DNA sequence corresponding to a cancer-driving C>G mutation in the KRAS gene by a chemically reactive probe: optimizing a cross-linking reaction with non-canonical duplex structures.

Covalent reactions are used in the detection of various biological analytes ranging from low molecular weight metabolites to protein-protein complexes. The detection of specific nucleic acid sequences is important in molecular biology and medicine but covalent approaches are less common in this field, in part, due to a deficit of simple and reliable reactions for the covalent capture of target sequences. Covalent anchoring can prevent the denaturation (melting) of probe-target complexes and causes signal degradation in typical hybridization-based assays. Here, we used chemically reactive nucleic acid probes that hybridize with, and covalently capture, a target sequence corresponding to a cancer-driving variant of the human KRAS gene. Our approach exploits a reductive amination reaction to generate a stable covalent attachment between an abasic site in the probe strand and a guanine mutation at position 35 in the KRAS gene sequence. Importantly, systematic variation of the probe sequence in a manner that formally introduces non-canonical structures such as bulges and mispairs into the probe-target duplex led to probes with dramatically improved cross-linking properties. An optimized abasic site-containing probe enabled simultaneous quantitative detection of both mutant and wild-type KRAS sequences in mixtures.

Encapsulating a single G-quadruplex aptamer in a protein nanocavity.

The alpha-hemolysin (alphaHL) protein pore has many applications in biotechnology. This article describes a single-molecule manipulation system that utilizes the nanocavity enclosed by this pore to noncovalently encapsulate a guest molecule. The guest is the thrombin-binding aptamer (TBA) that folds into the G-quadruplex in the presence of cations. Trapping the G-quadruplex in the nanocavity resulted in characteristic changes to the pore conductance that revealed important molecular processes, including spontaneous unfolding of the quartet structure and translocation of unfolded DNA in the pore. Through detection with Tag-TBA, we localized the G-quadruplex near the entry of the beta-barrel inside the nanocavity, where the molecule vibrates and rotates to different orientations. This guest-nanocavity supramolecular system has potential for helping to understand single-molecule folding and unfolding kinetics.

[Prognostic factors in the relapse of Graves disease].

OBJECTIVE: To evaluate the variables which can be used as prognostic factors in predicting the outcome of Graves disease (GD) after treatment with antithyroid drugs. METHODS: We performed a retrospective audit of 204 patients with newly diagnosed Graves disease consecutively at the Ruijin Hospital. RESULTS: Overall, 110 patients (53.9%) were considered to be treatment failures. Age at the time of diagnosis was (31.0 +/- 12.2)years in the successful group and (36.3 +/- 14.0) years in the failure group. Free T3 (FT3) was (25.60 +/- 9.52) pmol/L and (19.16 +/- 6.38) pmol/L in the failure and the successful group (P = 0.001). FT3 to FT4 ratio and thyrotrophin receptor antibody (TRAb) levels were higher in the failure group (P = 0.001). Logistic regression analysis showed that thyroid size, FT3 to FT4 ratio and TRAb at the time of diagnosis were associated with failure outcome. The patients reached euthyroid state at 3, 6, 9 and 12 months respectively and in the failure group the patients with continued thyrotropin suppression were more than those in the successful group (P = 0.001). CONCLUSIONS: Graves disease patients with large thyroid size, high levels of TRAb and FT3 to FT4 ratio before drug treatment are more likely to fail to respond to antithyroid drug treatment. We also found that patients with continuing thyrotropin suppression and attainment of euthyroid state in the course of treatment had low remission rate and prolonged therapy.

The aerolysin nanopore: from peptidomic to genomic applications.

The aerolysin pore (ARP) is a newly emerging nanopore that has been extensively used for peptide and protein sensing. Recently, several groups have explored the application of ARP in detecting genetic and epigenetic markers. This brief review summarizes the current applications of ARP, progressing from peptidomic to genomic detection; the recently reported site-directed mutagenesis of ARP; and new genomic DNA sensing approaches, and their advantages and disadvantages. This review will also discuss the perspectives and future applications of ARP for nucleic acid sequencing and biomolecule sensing.

Single Locked Nucleic Acid-enhanced nanopore genetic discrimination of pathogenic serotypes and cancer driver mutations.

Rapid and accurate detection of single-nucleotide polymorphism (SNP) in pathogenic mutants is crucial for broad fields from food safety monitoring to disease diagnostics and prognosis. Here, we developed a nanopore single-molecule sensor, coupled with the locked nucleic acid (LNA) technique, to accurately discriminate SNPs for detection of Shiga toxin producing Escherichia coli (STEC) O157:H7 pathogen serotype, and cancer-derived driver mutations EGFR L858R and KRAS G12D. This sensitive method, with a simplified, low cost, easy-to-operate LNA design, can be applied in food science and medical detection that need rapid and accurate determination of genetic variations.

Single Locked Nucleic Acid-Enhanced Nanopore Genetic Discrimination of Pathogenic Serotypes and Cancer Driver Mutations.

Accurate and rapid detection of single-nucleotide polymorphism (SNP) in pathogenic mutants is crucial for many fields such as food safety regulation and disease diagnostics. Current detection methods involve laborious sample preparations and expensive characterizations. Here, we investigated a single locked nucleic acid (LNA) approach, facilitated by a nanopore single-molecule sensor, to accurately determine SNPs for detection of Shiga toxin producing Escherichia coli (STEC) serotype O157:H7, and cancer-derived EGFR L858R and KRAS G12D driver mutations. Current LNA applications that require incorporation and optimization of multiple LNA nucleotides. But we found that in the nanopore system, a single LNA introduced in the probe is sufficient to enhance the SNP discrimination capability by over 10-fold, allowing accurate detection of the pathogenic mutant DNA mixed in a large amount of the wild-type DNA. Importantly, the molecular mechanistic study suggests that such a significant improvement is due to the effect of the single-LNA that both stabilizes the fully matched base-pair and destabilizes the mismatched base-pair. This sensitive method, with a simplified, low cost, easy-to-operate LNA design, could be generalized for various applications that need rapid and accurate identification of single-nucleotide variations.