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Analytical Biochemistry Jun 1995A quantitative DNA probe assay process has been developed that uses exonuclease III. The fluorophore-labeled DNA probe is hybridized with specific sequences of the...
A quantitative DNA probe assay process has been developed that uses exonuclease III. The fluorophore-labeled DNA probe is hybridized with specific sequences of the target DNA and then enzymatically digested. As these probe hybridization and digestion cycle reactions are repeated at a fixed temperature, digested probes (shortened probes) accumulate in the reaction mixture in a manner similar to a DNA polymerase chain reaction. Investigation of the digestion characteristics of the DNA probe showed that a slight digestion of a free single-stranded probe produces a large background signal, which results in low detection sensitivity. The digestion of single-stranded DNA probes is caused by double-stranded formations in the molecules. This digestion decreases and the double-stranded-specific digestion increases with increasing reaction temperature. When the reaction occurs at 45 degrees C, the association rate of the enzyme on the double-stranded DNA is 700 times faster than that on single-stranded DNA. This enables selective digestion of double-stranded DNA. The detection limit is 9 x 10(-19) mol for a M13-phage DNA.
Topics: Animals; Base Sequence; DNA; DNA Primers; DNA, Single-Stranded; Exodeoxyribonucleases; Humans; Molecular Probe Techniques; Molecular Sequence Data
PubMed: 8572265
DOI: 10.1006/abio.1995.1320 -
Analytical Biochemistry Oct 2019DNA-templated silver nanoclusters (DNA-AgNCs) is a kind of fluorescent nanoclusters in-situ synthesized on DNA, thereby giving the DNA probe an inherent function of...
DNA-templated silver nanoclusters (DNA-AgNCs) is a kind of fluorescent nanoclusters in-situ synthesized on DNA, thereby giving the DNA probe an inherent function of label-free signal output. Herein, the DNA-AgNCs with a GCC-loop-structure which has a quantum field of 51.6% was firstly proposed. It was proved that the addition of the double-stranded structure on the GCC-loop drastically enhanced the fluorescence intensity of the AgNCs. Through the further studies of the relationship between DNA secondary structure and AgNCs, a kind of DNA-AgNCs-probe was designed based on the change of the secondary structure which was induced by the target strand. By this, the fluorescence signal of probe was in "turn on" mode. The probe system could be directly used for the detection of Norovirus RNA, which had a linearity of 20 nM to 1.8 μM with a detection limit of 18 nM. Moreover, this detection platform was also selective to differentiate mismatched RNA. It was expected to be a universal method for different RNA detection by changing the recognition sequence of the probe.
Topics: Biosensing Techniques; DNA Probes; Fluorescence; Limit of Detection; Metal Nanoparticles; Norovirus; RNA; Silver; Spectrometry, Fluorescence
PubMed: 31325417
DOI: 10.1016/j.ab.2019.113365 -
Bioorganic & Medicinal Chemistry Letters Mar 2022A novel Dansyl-nucleoside surrogate (Dns) based on (±)-trans-4-(hydroxymethyl) piperidin-3-ol was designed and synthesized. The Dns exhibited excellent solvatochromic...
A novel Dansyl-nucleoside surrogate (Dns) based on (±)-trans-4-(hydroxymethyl) piperidin-3-ol was designed and synthesized. The Dns exhibited excellent solvatochromic properties. About 90 nm of red-shift accompanied color change from green to orange could be achieved with an increase of solvent polarity. The Dns was incorporated into oligodeoxynucleotide by phosphoroamidite chemistry. Two kinds of Dns-incorporated fluorescent DNA probes were designed and synthesized for sensing variation of DNA duplexes based on color-changing manner. As a result, the color-changing DNA probe not only can detect complementary oligonucleotide, but also can distinguish mismatch flanked in Dansyl/nucleobase pair by naked eye. Moreover, the change of fluorescence color of sample solutions could be captured by smartphone, and the photographs could be digitalized by image-processing software. Thus, the Dns-incorporated fluorescent DNA probe is expected to open the way to point-of-care assays in the future.
Topics: Color; DNA; DNA Probes; Fluorescent Dyes; Molecular Structure; Nucleosides; Piperidines
PubMed: 35051579
DOI: 10.1016/j.bmcl.2022.128551 -
Nature Cell Biology Jun 2021In the last decade, DNA-based tension sensors have made significant contributions to the study of the importance of mechanical forces in many biological systems. Albeit...
In the last decade, DNA-based tension sensors have made significant contributions to the study of the importance of mechanical forces in many biological systems. Albeit successful, one shortcoming of these techniques is their inability to reversibly measure receptor forces in a higher regime (that is, >20 pN), which limits our understanding of the molecular details of mechanochemical transduction in living cells. Here, we developed a reversible shearing DNA-based tension probe (RSDTP) for probing molecular piconewton-scale forces between 4 and 60 pN transmitted by cells. Using these probes, we can easily distinguish the differences in force-bearing integrins without perturbing adhesion biology and reveal that a strong force-bearing integrin cluster can serve as a 'mechanical pivot' to maintain focal adhesion architecture and facilitate its maturation. The benefits of the RSDTP include a high dynamic range, reversibility and single-molecule sensitivity, all of which will facilitate a better understanding of the molecular mechanisms of mechanobiology.
Topics: Animals; Biosensing Techniques; Cell Adhesion; Cell Movement; DNA Probes; Fluorescent Dyes; Focal Adhesions; Integrins; Mechanotransduction, Cellular; Mice; Microscopy, Fluorescence; Microscopy, Video; NIH 3T3 Cells; Nanotechnology; Nucleic Acid Conformation; Stress, Mechanical; Time Factors
PubMed: 34059812
DOI: 10.1038/s41556-021-00691-0 -
Angewandte Chemie (International Ed. in... Oct 2019Both protease overexpression and local pH changes are key signatures of cancer. However, the sensitive detection of protease activities and the accurate measurement of...
Both protease overexpression and local pH changes are key signatures of cancer. However, the sensitive detection of protease activities and the accurate measurement of pH in a tumor environment remain challenging. Here, we develop a dual-response DNA probe that can simultaneously monitor protease activities and measure the local pH by translocation through α-hemolysin (αHL). The DNA probe bears a short peptide containing phenylalanine at a pre-designed position. Enzymatic cleavage of the peptide either exposes or removes the N-terminal phenylalanine that can form a complex with cucurbit[7]uril. Translocation of the DNA hybrid through αHL generates current signatures that can be used to quantify protease activities. Furthermore, the current signatures possess a pH-dependent pattern that reflects the local pH. Our results demonstrate that the versatile DNA probe may be further explored for simultaneously measuring multiple parameters of a complex system such as single cells in the future.
Topics: Biosensing Techniques; Cathepsin B; DNA Probes; Hemolysin Proteins; Hydrogen-Ion Concentration; Leucyl Aminopeptidase; Limit of Detection; Nanopores
PubMed: 31442357
DOI: 10.1002/anie.201907816 -
Coarse-grained model simulation-guided localized DNA signal amplification probe for miRNA detection.Biosensors & Bioelectronics Nov 2023DNA-based enzyme-free signal amplification strategies are widely employed to detect biomarkers in low abundance. To enhance signal amplification, localized DNA reaction...
DNA-based enzyme-free signal amplification strategies are widely employed to detect biomarkers in low abundance. To enhance signal amplification, localized DNA reaction units which increases molecular collision probability is commonly utilized. However, the current understanding of the structure-function relationships in localized DNA signal amplification probes is limited, leading to unsatisfied performance. In this study, we introduced a coarse-grained molecular model to simulate the dynamic behavior of two DNA reaction units within a DNA enzyme-free signal amplification circuit called Localized Catalytic Hairpin Assembly (LCHA). We investigated the impact of localized distance and flexibility on reaction performance. The most efficient LCHA probe guided by simulation exhibits sensitivity 28 times greater that of free CHA, with a detection limit of miR-21 reaching 16 pM, while the least effective LCHA probe demonstrated a modest improvement of only 7 times. We successfully employed the optimized probe to differentiate cancer cells from normal cells based on their miR-21 expression levels, showcasing its quantification ability. By elucidating the mechanistic insights and structure-function relationship in our work, we aim to contribute valuable information that can save users' time and reduce costs when designing localized DNA probes. With a comprehensive understanding of how the localization affects probe performance, researchers can now make more informed and efficient decisions during the design process. This work would find broad applications of DNA nanotechnology in biosensing, biocomputing, and bionic robots.
Topics: Biosensing Techniques; DNA Probes; Anilides; MicroRNAs
PubMed: 37611449
DOI: 10.1016/j.bios.2023.115622 -
The Analyst Nov 2022A simple, reliable, and cost-effective method for nucleic acid detection is of increasing interest in clinical diagnostics of infectious and genetic diseases. Currently,...
A simple, reliable, and cost-effective method for nucleic acid detection is of increasing interest in clinical diagnostics of infectious and genetic diseases. Currently, enzyme-mediated methods such as polymerase chain reactions and loop-mediated isothermal amplification are the most widely used methods in the qualitative and quantitative diagnosis of long nucleic acid sequences with high sensitivity. However, a high detection sensitivity for short-length sequences remains a significant challenge because it is difficult for the primers to bind to their sequences. Our previous study demonstrated a simple, enzyme-free, and sequence-specific colorimetric detection of 24-nucleotide short target DNA at room temperature using a developed assay that combines catalytic hairpin assembly (CHA) and enzyme-linked immunosorbent assay (ELISA)-mimicking methods. In this follow-up study, we aim to design and develop DNA-based signal amplifiers, or DNA dendrons, to improve the colorimetric detection of short target cDNA in the CHA-mediated ELISA-mimicking assay. DNA dendrons are highly branched conformations synthesized by the molecular self-assembly of three DNA oligomers. The assay using DNA dendrons demonstrates an enhanced detection sensitivity with the detection of approximately 50 pM of 24-nucleotide short target cDNA, which is a 16.4-fold higher detection limit compared to that obtained without DNA dendrons under the same conditions. Thus, applications of the developed DNA dendrons as an effective signal amplifier in DNA probe-based chemiluminescence assays have the potential to improve the colorimetric detection of short target cDNA with high sensitivity for the diagnosis of different diseases.
Topics: Colorimetry; DNA, Complementary; Dendrimers; Follow-Up Studies; Luminescence; DNA; Nucleic Acid Amplification Techniques; DNA Probes; Nucleotides; Limit of Detection; Biosensing Techniques
PubMed: 36239244
DOI: 10.1039/d2an01137a -
Mikrochimica Acta Mar 2020Herein, we report a rapid and sensitive colorimetric detection of Hg by designing a specific DNA probe with phosphorothioate RNA modification (PS-probe) for Hg...
Herein, we report a rapid and sensitive colorimetric detection of Hg by designing a specific DNA probe with phosphorothioate RNA modification (PS-probe) for Hg recognition and utilizing DNA-modified gold nanoparticles (DNA-AuNPs) as the transducer. The distance between two DNA-AuNPs is controlled by a linker DNA, providing the linker DNA-regulated aggregation or dispersion status of AuNPs in solution. Exonuclease III (Exo III) can trigger the recycled digestion of linker DNA strands, inhibiting the reformation of aggregated nanoparticles and hence leading to a color shift from purple to red. However, the Hg-induced cleavage of the PS-probe can efficiently prevent the digestion of linker DNA strands by Exo III and hence reassemble the modified AuNPs to form aggregates in purple color. Thus, a positive correlation between the linker DNA strands left and the addition of Hg provides a quantitative basis for Hg sensing. A linear range of A/A versus Hg concentration is achieved in the range 2-100 nM associated with a detection limit as low as 1.30 ± 0.04 nM. Moreover, the biosensor exhibits excellent selectivity for Hg. The strong selectivity behavior was confirmed by recoveries ranging from 96 to 114% in real water samples. Graphical abstractSchematic representation of sensing mechanism of Hg using a DNA probe with phosphorothioate RNA modification (PS-probe) and Exo III-assisted signal amplification.
Topics: Biosensing Techniques; Colorimetry; DNA Probes; Exodeoxyribonucleases; Gold; Mercury; Metal Nanoparticles; Nucleic Acid Amplification Techniques; Phosphates; RNA
PubMed: 32162015
DOI: 10.1007/s00604-020-4184-0 -
Analytica Chimica Acta Sep 2022Abnormal DNA methylation is closely related to the occurrence and development of many diseases. The determination of human DNA methyltransferase activity and the...
Abnormal DNA methylation is closely related to the occurrence and development of many diseases. The determination of human DNA methyltransferase activity and the screening of its inhibitors are extreme important for the diagnosis and the treatment of methylation-related diseases in clinic. Most of the current detection methods have the disadvantages of sophisticated design, high cost and low detection limit. By combining T7 promoter-contained DNA probe as the substrate for methyltransferase with CRISPR/Cas13a sensing strategy, a novel fluorescent sensing platform is designed to achieve simple, specific, sensitive detection of bacteria DNA methyltransferase (DNA-(N-6-adenine)-methyltransferase, Dam MTase) and also human methyltransferase (DNA (cytosine-5)-methyltransferase 1, Dnmt1). A hairpin DNA probe designed for Dam MTase and a double strand DNA probe for Dnmt1 are both methylated followed by the methylation-dependent site-specific cleavage, which result a T7 promoter-contained product and a T7 promoter-free one to respectively open and close the transcription and subsequent CRISPR/Cas13a target-initiated cleavage of fluorescence-labeled reporter RNA. In virtue of the specificity of methylation-dependent cleavage of probe, the efficient transcription amplification and CRISPR/Cas13a sequence-specific sensing, this strategy exhibited remarkable specificity and sensitivity, with the limit of detection of 3.10 × 10 U/mL for Dam MTase. Moreover, Dnmt1 activity in MCF-7 cells was detected and the inhibition of Apt. #9 was evaluated. This strategy for methyltransferase detection is convenient and efficient for inhibitor discovery and early cancer diagnosis.
Topics: Bacteria; Biosensing Techniques; Clustered Regularly Interspaced Short Palindromic Repeats; DNA; DNA Probes; Humans; Methyltransferases; Site-Specific DNA-Methyltransferase (Adenine-Specific)
PubMed: 36089307
DOI: 10.1016/j.aca.2022.340266 -
Analytica Chimica Acta May 2022DNA hydrogels are powerful candidates for stable and sensitive detection of disease-related nucleic acids. However, the ability to accurately detect is the cornerstone...
DNA hydrogels are powerful candidates for stable and sensitive detection of disease-related nucleic acids. However, the ability to accurately detect is the cornerstone of disease diagnosis. To improve the accuracy of DNA hydrogels for detecting targets, we herein reported the design of pH-responsive DNA hydrogels with ratiometric fluorescence. The DNA hydrogels were prepared from the pH-sensitive ZnO-NH and CO-Y-DNA probe assembled by the three complementary strands. With the use of miRNA-21 as the model analyte, the DNA hydrogels were applied to fluorescence ratio detection. Under acidic conditions, the ZnO-NH was decomposed, thereby releasing the CO-Y-DNA probe. Target miRNA-21 hybridized to the CO-Y-DNA probe, causing the change of fluorescence ratio between TAMRA and Cy5 that both modified in the CO-Y-DNA probe. The developed DNA hydrogels exhibited high accuracy and sensitivity with a low detection limit to 83 pM. In addition, the DNA hydrogels showed long-term stability against DNase I and GSH.
Topics: Biosensing Techniques; DNA; DNA Probes; Hydrogels; Hydrogen-Ion Concentration; MicroRNAs; Zinc Oxide
PubMed: 35491037
DOI: 10.1016/j.aca.2022.339795