RESUMEN
Toehold-mediated strand displacement and its regulatory tools are fundamental for DNA nanotechnology. However, current regulatory tools all need to change the original sequence of reactants, making the regulation inconvenient and cumbersome. More importantly, the booming development of DNA nanotechnology will soon promote the production of packaged and batched devices or circuits with specified functions. Regarding standardized, packaged DNA nanodevices, access to personalized post-modification will greatly help users, whereas none of the current regulatory tools can provide such access, which has greatly constrained DNA nanodevices from becoming more powerful and practical. Herein, we developed a novel regulation tool named Cap which has two basic functions of subtle regulation of the reaction rate and erasability. Based on these functions, we further developed three advanced functions. Through integration of all functions of Cap and its distinct advantage of working independently, we finally realized personalized tailor-made post-modification on pre-fabricated DNA circuits. A pre-fabricated dual-output DNA circuit was successfully transformed into an equal-output circuit, a signal-antagonist circuit and a covariant circuit according to our requirements. Taken together, Cap is easy to design and generalizable for all strand displacement-based DNA nanodevices. We believe the Cap tool will be widely used in regulating reaction networks and personalized tailor-made post-modification of DNA nanodevices.
Asunto(s)
ADN , Nanotecnología , ADN/genética , Recombinación GenéticaRESUMEN
Sperm DNA fragmentation is a sign of sperm nuclear damage. The sperm chromatin dispersion (SCD) test is a reliable and economical method for the evaluation of DNA fragmentation. However, the cut-off value for differentiation of DNA fragmented sperms is fixed at 1/3 with limited statistical justification, making the SCD test a semi-quantitative method that gives user-dependent results. We construct a collection of deep neural networks to automate the evaluation of bright-field images for SCD tests. The model can detect valid sperm nuclei and their locations from the input images captured with a 20× objective and predict the geometric parameters of the halo ring. We construct an annotated dataset consisting of N = 3120 images. The ResNet 18 based network reaches an average precision (AP50) of 91.3%, a true positive rate of 96.67%, and a true negative rate of 96.72%. The distribution of relative halo radii is fit to the multi-peak Gaussian function (p > 0.99). DNA fragmentation is regarded as those with a relative halo radius 1.6 standard deviations smaller than the mean of a normal cluster. In conclusion, we have established a deep neural network based model for the automation and quantification of the SCD test that is ready for clinical application. The DNA fragmentation index is determined using Gaussian clustering, reflecting the natural distribution of halo geometry and is more tolerable to disturbances and sample conditions, which we believe will greatly improve the clinical significance of the SCD test.
Asunto(s)
Cromatina , Semen , Masculino , Humanos , Espermatozoides , ADN/genética , Núcleo Celular , Fragmentación del ADNRESUMEN
Seesaw circuits are essential for molecular computing and biosensing. However, a notable limitation of seesaw circuits lies in the irreversible depletion of components, precluding the attainment of system recovery and rendering nucleic acid circuits non-reusable. We developed a brand-new method for creating controllable and reusable seesaw circuits. By using the nicking endonucleases Nt.BbvCI and Nt.Alwi, we removed "functional components" while keeping the "skeletal components" for recurrent usage. T-inputs were introduced, increasing the signal-to-noise ratio of AND logic from 2.68 to 11.33 and demonstrating compatibility. We identified the logic switching feature and verified that it does not impair circuit performance. We also built intricate logic circuits, such as OR-AND gate, to demonstrate the versatility of our methodology. This controllable reusability extends the applications of nanotechnology and bioengineering, enhancing the practicality and efficiency of these circuits across various domains.
Asunto(s)
ADN , Ácidos Nucleicos , Endonucleasas , BioingenieríaRESUMEN
Although CRISPR-Cas12a [clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 12a] combining pre-amplification technology has the advantage of high sensitivity in biosensing, its generality and specificity are insufficient, which greatly restrains its application range. Here, we discovered a new targeting substrate for LbaCas12a (Lachnospiraceae bacterium Cas12a), namely double-stranded DNA (dsDNA) with a sticky-end region (PAM-SE+ dsDNA). We discovered that CRISPR-Cas12a had special enzymatic properties for this substrate DNA, including the ability to recognize and cleave it without needing a protospacer adjacent motif (PAM) sequence and a high sensitivity to single-base mismatches in that substrate. Further mechanism studies revealed that guide RNA (gRNA) formed a triple-stranded flap structure with the substrate dsDNA. We also discovered the property of low-temperature activation of CRISPR-Cas12a and, by coupling with the unique DNA hybridization kinetics at low temperature, we constructed a complete workflow for low-abundance point mutation detection in real samples, which was fast, convenient and free of single-stranded DNA (ssDNA) transformation. The detection limits were 0.005-0.01% for synthesized strands and 0.01-0.05% for plasmid genomic DNA, and the mutation abundances provided by our system for 28 clinical samples were in accordance with next-generation sequencing results. We believe that our work not only reveals novel information about the target recognition mechanism of the CRISPR-Cas12a system, but also greatly broadens its application scenarios.
Asunto(s)
Proteínas Asociadas a CRISPR , Sistemas CRISPR-Cas , Proteínas Asociadas a CRISPR/metabolismo , Proteínas Bacterianas/metabolismo , ADN/química , ADN de Cadena Simple/genéticaRESUMEN
DNA methylation is closely related to cancer. It is generally accepted that DNA methylation detection is crucial in cancer diagnosis, prognosis, and treatment monitoring. Therefore, there is an urgent demand for developing a simple, rapid, highly sensitive, and highly specific methylation detection method to detect DNA methylation at specific sites quantitatively. In this work, we introduce a DNA methylation detection method based on MutS and methylation-specific PCR, named MutS-based methylation-specific PCR (MB-MSP), which has the advantages of simplicity, speed, high specificity, sensitivity, and broad applicability. Utilizing the MutS's ability to bind mismatched base pairs, we inhibit not only the amplification of unmethylated DNA but also nonspecific primer amplification. We achieved a detection sensitivity of 0.5% for the methylated genes of ACP1, CLEC11A, and SEPT9 by MB-MSP. It has a good linear relationship and a detection time of only 1.5 h. To validate the feasibility of the MB-MSP method in clinical application, we conducted methylation detection on plasma-circulating tumor DNA samples from 10 liver cancer patients and 5 healthy people, achieving a 100% accuracy rate. In conclusion, MB-MSP, as a novel and reliable DNA methylation detection tool, holds significant application value and potential for advancing early cancer diagnosis.
Asunto(s)
Metilación de ADN , Neoplasias , Humanos , Proteínas MutS , ADN/genética , Reacción en Cadena de la Polimerasa/métodosRESUMEN
The CRISPR-Cas12a system is a new type of genome editing tool with high efficiency and targeting. However, other sequences in the genome may also be cleaved nonspecifically, resulting in unavoidable off-target effects. Therefore, it is necessary to learn more about the mechanism of CRISPR-Cas12a to recognize target sequences to avoid its off-target effects. Here, we show that insertion (DNA bubble) or deletion (RNA bubble) of the target dsDNA sequence compared with the crRNA sequence, the CRISPR-Cas12a system can still recognize and cleave the target dsDNA sequence. We conclude that the tolerance of CRISPR-Cas12a to the bubbles is closely related to the location and size of the bubble and the GC base content of crRNA. In addition, we used the unique property of CRISPR-Cas12a to invent a new method to detect mutations and successfully detect the CD41-42(-CTTT) mutation. The detection limit of this method is 0.001%. Overall, our results strongly indicate that in addition to considering off-target effects caused by base mismatches, a comprehensive off-target analysis of the insertion and deletion of the target dsDNA sequence is required, and specific guidelines for effectively reducing potential off-target cleavage are proposed, to improve the safety manual of CRISPR-Cas12a biological application.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Sistemas CRISPR-Cas/genética , ADN/genética , Edición Génica/métodos , Mutación , ARN/genéticaRESUMEN
BACKGROUND: Sperm DNA integrity is crucial for normal fertilization, implantation, and embryo development. Several assays are available to assess sperm DNA fragmentation but are limited by high price, complicated processes, and low accuracy. METHODS: We developed a secondary amplification detection system based on terminal deoxynucleotidyl transferase and endonuclease IV, which could efficiently measure the number of 3'-OH (equivalent to the number of breakpoints). We applied this detection system in single stranded DNA with standard concentrations to obtain the standard curve. We then broke the double stranded genomic DNA by ultrasound and enzyme digestion and used the detection system to monitor the increase of DNA breakpoints. Finally, we used this method to measure the mean number of sperm DNA breakpoints (MDB) in 80 sperm samples. RESULTS: We successfully measured the number of 3'-OH in single stranded DNA with standard concentration and obtained the standard curve. The linear range for the number of DNA breakpoints was from 0.1 nM to 15 nM. The detection method was successfully validated on λ DNA and 80 human sperm samples. The results of real clinical samples revealed that the mean number of DNA breakpoints (MDB) had a stronger relevance with the sperm motility and clinical pregnancy outcomes than the commonly used parameter of DNA fragmentation index (DFI). CONCLUSION: We have developed a straight-forward method for direct measurement of the mean number of DNA breakpoints in sperms. The method has advantages of short time-consumption, simple operation, high analytical sensitivity, and low requirement for instrumentation, which makes it conducive to clinical application. The proposed new parameter (MDB) could be a more direct, accurate and clinically significant indicator for evaluating the sperm DNA integrity.
Asunto(s)
Motilidad Espermática , Espermatozoides , ADN/genética , Roturas del ADN , Fragmentación del ADN , Femenino , Humanos , Masculino , EmbarazoRESUMEN
Single base mutations are closely related to cancer diagnosis and treatment. The fluorescent probe method is one of the important methods to detect single-base mutations. We constructed a universal probe detection system based on endonuclease IV and the DNA strand displacement reaction. The system uses two toehold strand displacement reactions to relay the mutation information to the universal strand. There is no need to design the probe one-by-one for each mutation point during multi-site detection. It has the advantages of simple operation, rapid detection, and low cost. We used this method to detect common clinical mutation sites (PTEN R130Q/EGFR L858R/PTEN rs1473918395), and the detection limit can reach 0.1%-1%. The detection system can provide a new rapid and economical method for clinical single-base mutation detection, and has broad application prospects in diagnosis and prognostic evaluation.
Asunto(s)
Técnicas Biosensibles , Mutación Puntual , Técnicas Biosensibles/métodos , ADN/genética , Desoxirribonucleasa IV (Fago T4-Inducido)/genética , Colorantes Fluorescentes , MutaciónRESUMEN
Single-nucleotide variation (SNV) detection plays significant roles in disease diagnosis and treatment. Generally, auxiliary probe, restricted design rules, complicated detection system, and repeated experimental parameter optimization are needed to obtain satisfactory tradeoff between sensitivity and selectivity for SNV discrimination, especially when different mutant sites need to be distinguished. To overcome these limitations, we developed a universal, straightforward, and relatively cheap SNV discrimination strategy, which simultaneously possessed high sensitivity and selectivity. The excellent performance of this strategy was ascribed to the SNV discrimination property of endonuclease IV (Endo IV) and the different hydrolysis behavior between free deoxyribozyme (DNAzyme) and the trapped DNAzyme to the substrates modified on gold nanoparticles (AuNPs). When Endo IV recognized the mutant-type target (MT), free DNAzyme was released from the probe, and the DNAzyme motor was activated with the help of cofactor Mn2+ to generate an amplified fluorescence signal. On the contrary, the wild-type target (WT) could not effectively trigger the DNAzyme motor. Moreover, for different SNV types, the corresponding probe could be designed by simply changing the sequence hybridized with the target and retaining the DNAzyme sequence. Thus, the fluorescence signal generation system does not need to change for different SNV targets. Five clinical-related SNVs were determined with the limit of detection (LOD) ranging from 0.01 to 0.05%, which exhibited competitive sensitivity over existing SNV detection methods. This strategy provided another insight into the properties of Endo IV and DNAzyme, expanded the applications of DNAzyme motor, and has great potential to be used for precision medicine.
Asunto(s)
Técnicas Biosensibles , ADN Catalítico , Nanopartículas del Metal , Desoxirribonucleasa IV (Fago T4-Inducido) , Oro , Humanos , Límite de Detección , NucleótidosRESUMEN
Fluorescent aptamer beacons (FABs) are a major category of biosensors widely used in environmental analysis. However, due to their low compatibility, it is difficult to use the common FABs for biological samples. To overcome this challenge, construction of FABs with complex structures to adapt the nature of biological samples is currently in progress in this field. Unlike previous works, we moved our range of vision from the FAB itself to the biological sample. Inspired by this idea, in this work, flat membrane-based liquid-phase microextraction (FM-LPME) with sufficient sample cleanup and preconcentration capacities was integrated with FABs. With the merits of both FM-LPME and FABs, the integrated LPME-FAB system displayed a clear synergistic enhancement for target analysis. Specifically, LPME in the LPME-FAB system provided purified and enriched Hg2+ for the FAB recognition, while the FAB recognition event promoted the extraction efficiency of LPME. Due to superior performances, the LPME-FAB system achieved highly sensitive analysis of Hg2+ in urine samples with a detection limit of 27 nM and accuracies in the range of 98-113%. To the best of our knowledge, this is the first time that an integrated LPME-FAB system was constructed for target analysis in biological samples. We believe that this study will provide a new insight into the next generation of biosensors, where the integration of sample preparation with detection probes is as important as the design of complex probes in the field of bioanalysis.
Asunto(s)
Microextracción en Fase LíquidaRESUMEN
Nucleases are powerful tools in various biomedical applications, such as genetic engineering, biosensing, and molecular diagnosis. However, the commonly used nucleases (endonuclease IV, apurinic/apyrimidinic endonuclease-1, and λ exonuclease) are prone to the nonspecific cleavage of single-stranded DNA, making the desired reactions extremely low-yield and unpredictable. Herein, we have developed guiding-strand-controlled nuclease systems and constructed theoretical kinetic models to explain their mechanisms of action. The models displayed excellent agreement with the experimental results, making the kinetics highly predictable and tunable. Our method inhibited the nonspecific cleavage of single-stranded probes while maintaining highly efficient cleavage of double-stranded DNA. We also demonstrated the clinical practicability of the method by detecting a low-frequency mutation in a genomic DNA sample extracted from the blood of a patient with cancer. The limit of detection could be 0.01% for PTEN rs121909219. We believe that our findings provide a powerful tool for the field and the established model provides us a deeper understanding of the enzymatic activities of DNA nucleases.
Asunto(s)
ADN-(Sitio Apurínico o Apirimidínico) Liasa , Desoxirribonucleasas , ADN/genética , Reparación del ADN , ADN de Cadena Simple/genética , Desoxirribonucleasa IV (Fago T4-Inducido)/genética , Desoxirribonucleasa IV (Fago T4-Inducido)/metabolismo , Desoxirribonucleasas/metabolismo , Humanos , Cinética , Mutación , Especificidad por SustratoRESUMEN
Gene mutations are important biomarkers for the diagnosis, classification, monitoring, and prognosis evaluation of cancers and genetic diseases. Both personalized cancer treatment and noninvasive prenatal testing require methods to accurately determine the abundance of mutation. At present, the widely adopted and convenient methods for measuring mutation abundance are mainly based on relative quantification, which requires negative samples and strict control of the analyte amounts. The development of DNA-probe-based methods that can determine the mutation abundance without negative samples nor control of analyte amount is highly preferred. The key to solving this bottleneck lies in whether the probe's response to mutation abundance can be completely independent of the number of targeted DNA strands. Herein, we propose the design of a self-internal-reference probe system. We established a theoretical model of this system and used the model to guide the design of probes. In this model, we provided quantitative corrections to the test results from the internal reference, thereby eliminating the influence of substrate amount. Therefore, the purification and quantification processes toward polymerase chain reaction (PCR) amplicons can be omitted. We applied this system to analyze unquantified PCR products aimed at cancer mutation detection and noninvasive prenatal testing.
Asunto(s)
MutaciónRESUMEN
Determination of the methylation levels of genes of interest is fundamental for biological and medical research that involves DNA methylation. Using the average methylation levels of multiple CpG sites to represent the methylation levels of the whole gene is much more accurate than using that of only one CpG site. However, current methods that can provide the average methylation levels of several CpG sites are all expensive, time-consuming (several weeks), and labor-intensive. Herein, guided by the unique thermodynamics of the DNA strand-displacement process, we constructed a DNA fluorescent probe for determination of the average methylation levels of multiple CpG sites. Theoretical calculations of the reaction process and proof-of-concepts experiments on two to three CpG sites of synthesized DNA validated the basic principles of our probe. Taking two CpG sites in the promotor regions of SF-1 (steroidogenic factor 1) gene and VDR (vitamin D receptor) gene as the targets, we successfully measured their average methylation levels in nine endometrial cancer patients and two healthy persons. We believe our probe will be a very useful tool in the field, and we anticipate it being widely adopted by biological and medical investigators.
Asunto(s)
Islas de CpG , Metilación de ADN , ADN/genética , ADN/química , Sondas de ADN/análisis , Sondas de ADN/genética , Neoplasias Endometriales/genética , Femenino , Colorantes Fluorescentes/análisis , Humanos , Regiones Promotoras Genéticas , Receptores de Calcitriol/genética , Espectrometría de Fluorescencia/métodos , Factor Esteroidogénico 1/genética , TermodinámicaRESUMEN
Sensitive detection of low-abundance point mutations in blood or tissue may provide a great opportunity for the minimally invasive diagnosis of cancer and other related diseases. We demonstrate a novel method for ultra-sensitive detection of point mutations at low abundance by combination of branch migration-based PCR with endonuclease IV-assisted target recycling probe/blocker system. The method is able to identify the point mutations at abundances down to 0.01-0.02%. We anticipate this method to be widely adopted in clinical diagnosis and molecular research.
Asunto(s)
Análisis Mutacional de ADN/métodos , Sondas de ADN/metabolismo , Desoxirribonucleasa IV (Fago T4-Inducido)/metabolismo , Mutación Puntual , Reacción en Cadena de la Polimerasa/métodos , Sondas de ADN/química , Colorantes Fluorescentes/química , Humanos , Fosfohidrolasa PTEN/genética , Sensibilidad y EspecificidadRESUMEN
Sensitive detection of the single nucleotide variants in cell-free DNA (cfDNA) may provide great opportunity for minimally invasive diagnosis and prognosis of cancer and other related diseases. Here, we demonstrate a facile new strategy for quantitative measurement of cfDNA mutations at low abundance in the cancer patients' plasma samples. The method takes advantage of a novel property of lambda exonuclease which effectively digests a 5'-fluorophore modified dsDNA with a 2-nt overhang structure and sensitively responds to the presence of mismatched base pairs in the duplex. It achieves a limit of detection as low as 0.02% (percentage of the mutant type) for BRAFV600E mutation, NRASQ61R mutation and three types of EGFR mutations (G719S, T790M and L858R). The method enabled identification of BRAFV600E and EGFRL858R mutations in the plasma of different cancer patients within only 3.5 h. Moreover, the terminal structure-dependent reaction greatly simplifies the probe design and reduces the cost, and the assay only requires a regular real-time PCR machine. This new method may serve as a practical tool for quantitative measurement of low-abundance mutations in clinical samples for providing genetic mutation information with prognostic or therapeutic implications.
Asunto(s)
ADN Tumoral Circulante/sangre , Análisis Mutacional de ADN/métodos , Exodesoxirribonucleasas , Proteínas Virales , ADN Tumoral Circulante/química , ADN/química , ADN/metabolismo , Receptores ErbB/genética , Humanos , Neoplasias/genética , Proteínas Proto-Oncogénicas B-raf/genéticaRESUMEN
Lambda exonuclease (λ exo) plays an important role in the resection of DNA ends for DNA repair. Currently, it is also a widely used enzymatic tool in genetic engineering, DNA-binding protein mapping, nanopore sequencing and biosensing. Herein, we disclose two noncanonical properties of this enzyme and suggest a previously undescribed hydrophobic interaction model between λ exo and DNA substrates. We demonstrate that the length of the free portion of the substrate strand in the dsDNA plays an essential role in the initiation of digestion reactions by λ exo. A dsDNA with a 5' non-phosphorylated, two-nucleotide-protruding end can be digested by λ exo with very high efficiency. Moreover, we show that when a conjugated structure is covalently attached to an internal base of the dsDNA, the presence of a single mismatched base pair at the 5' side of the modified base may significantly accelerate the process of digestion by λ exo. A detailed comparison study revealed additional π-π stacking interactions between the attached label and the amino acid residues of the enzyme. These new findings not only broaden our knowledge of the enzyme but will also be very useful for research on DNA repair and in vitro processing of nucleic acids.
Asunto(s)
Disparidad de Par Base , ADN/metabolismo , Exodesoxirribonucleasas/metabolismo , Proteínas Virales/metabolismo , Secuencia de Bases , Biocatálisis , ADN/química , ADN/genética , Exodesoxirribonucleasas/química , Exodesoxirribonucleasas/genética , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Conformación de Ácido Nucleico , Dominios Proteicos , Especificidad por Sustrato , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
Methylmercury (MeHg) is an extremely toxic environmental pollutant that can cause serious male reproductive developmental dysplasia in humans and animals. However, the molecular mechanisms underlying MeHg-induced male reproductive injury are not fully clear. The purpose of this study was to explore whether mitophagy and lysosome dysfunction contribute to MeHg-induced apoptosis of germ cell and to determine the potential mechanism. First, we confirmed the exposure of GC2-spd cells to mercury. In GC2-spd cells (a mouse spermatocyte cell line), we found that MeHg treatment led to an obvious increase of cell apoptosis accompanied by a marked rise of LC3-II expression and an elevated number of autophagosomes. These results were associated with the induction of oxidative stress and mitophagy. Interestingly, we found that MeHg did not promote but prevented autophagosome-lysosome fusion by impairing the lysosome function. Furthermore, as a lysosome inhibitor, chloroquine pre-treatment obviously enhanced LC3-II expression and mitophagy formation in MeHg-treated cells. This further proved that the induction of mitophagy and the injury of the lysosome played an important role in the GC2-spd cell apoptosis induced by MeHg. Our findings indicate that MeHg caused apoptosis in the GC2-spd cells, which were dependent on oxidative stress-mediated mitophagy and the lysosome damaging-mediated inhibition of autophagic flux induced by MeHg.
Asunto(s)
Autofagosomas/efectos de los fármacos , Autofagia/efectos de los fármacos , Sustancias Peligrosas/toxicidad , Compuestos de Metilmercurio/toxicidad , Animales , Apoptosis , Autofagosomas/metabolismo , Células Germinativas/metabolismo , Lisosomas/metabolismo , Ratones , Mitofagia , Estrés OxidativoRESUMEN
Genetic mutations are important biomarkers for cancer diagnostics and surveillance. Preferably, the methods for mutation detection should be straightforward, highly specific and sensitive to low-level mutations within various sequence contexts, fast and applicable at room-temperature. Though some of the currently available methods have shown very encouraging results, their discrimination efficiency is still very low. Herein, we demonstrate a branch-migration based fluorescent probe (BM probe) which is able to identify the presence of known or unknown single-base variations at abundances down to 0.3%-1% within 5 min, even in highly GC-rich sequence regions. The discrimination factors between the perfect-match target and single-base mismatched target are determined to be 89-311 by measurement of their respective branch-migration products via polymerase elongation reactions. The BM probe not only enabled sensitive detection of two types of EGFR-associated point mutations located in GC-rich regions, but also successfully identified the BRAF V600E mutation in the serum from a thyroid cancer patient which could not be detected by the conventional sequencing method. The new method would be an ideal choice for high-throughput in vitro diagnostics and precise clinical treatment.
Asunto(s)
Receptores ErbB/genética , Colorantes Fluorescentes/química , Sondas Moleculares/química , Mutación Puntual , Proteínas Proto-Oncogénicas B-raf/genética , Composición de Base , Receptores ErbB/metabolismo , Colorantes Fluorescentes/metabolismo , Expresión Génica , Humanos , Sondas Moleculares/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Proteínas Proto-Oncogénicas B-raf/metabolismo , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/patologíaRESUMEN
Detection of heavy metals is of great importance for food safety and environmental analysis. Among various heavy metal ions, mercury ion is one of the most prevalent species. The methods for detection of mercury were numerous, and the T-Hg-T based assay was promising due to its simplicity and compatibility. However, traditional T-Hg-T based methods mainly relied on multiple T-Hg-T to produce enough conformational changes for further detection, which greatly restrained the limit of detection. Hence, we established a branch-migration based fluorescent probe and found that single T-Hg-T could produce strong signals. The sensing mechanism of our method in different reaction modes was explored, and the detection limits were determined to be 18.4 and 14.7 nM in first-order reaction mode and mixed reaction mode, respectively. Moreover, coupled with Endonuclease IV assisted signal amplification, the detection limit could be 1.2 nM, lower than most DNA based fluorometric assays. For practicability, the specificity of our assay toward different interfering ions was investigated and detection of Hg2+ in deionized water and lake water was also achieved with similar recoveries compared to those of atomic fluorescence spectrometry, which demonstrated the practicability of our method in real samples. Definitely, the proposed branch migration probe would be a promising substitution for current DNA probes based on recognition of multiple T-Hg-T and we anticipate it to be widely adopted in food and environmental analysis.
Asunto(s)
Colorantes Fluorescentes/análisis , Colorantes Fluorescentes/química , Mercurio/análisis , Sondas de ADN , Fluorometría , Mercurio/químicaRESUMEN
We have disclosed the unique inhibition effect of small molecule-protein interactions toward the DNA branch migration process and constructed a complete thermodynamic model for it. The disclosed effect was further coupled with the steric hindrance effect to establish a homogeneous assay for proteins and small molecules with ultra-high inhibition factors and sensitivity.