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Methods in Molecular Biology (Clifton,... 2024RNA in situ hybridization reveals the abundance and location of gene expression in cells or tissues, providing a technical basis for the clinical diagnosis of diseases....
RNA in situ hybridization reveals the abundance and location of gene expression in cells or tissues, providing a technical basis for the clinical diagnosis of diseases. In this chapter, we show a "V" shape probe-mediated single-molecule chromogenic in situ hybridization (vsmCISH) technique for bright-field visualization of individual RNA molecules. In our method, several pairs of target hybridization probes are hybridized to RNA molecules and each probe pair forms a "V" shape overhang. The overhang oligonucleotides then mediated the proximity ligation to form DNA circles, followed by rolling circle amplification for signal enhancement and enzyme-catalyzed chromogenic reaction-based readout. The colorimetric assay avoids problems such as photobleaching and autofluorescence of current fluorescent in situ hybridization-based single-molecule RNA detection techniques. Furthermore, the relatively straightforward protocol makes the method useful for biological research and clinical diagnosis applications.
Topics: In Situ Hybridization; RNA; Humans; Chromogenic Compounds; Colorimetry; Single Molecule Imaging
PubMed: 38907917
DOI: 10.1007/978-1-0716-3918-4_11 -
Analytical Chemistry Jul 2024The quantitative detection of antibodies is crucial for the diagnosis of infectious and autoimmune diseases, while the traditional methods experience high background...
The quantitative detection of antibodies is crucial for the diagnosis of infectious and autoimmune diseases, while the traditional methods experience high background signal noise and restricted signal gain. In this work, we have developed a highly efficient electrochemical biosensor by constructing a programmable DNA nanomachine integrated with electrochemically controlled atom transfer radical polymerization (eATRP). The sensor works by binding the target antidigoxin antibody (anti-Dig) to the epitope of the recognization probe, which then initiates the cascaded strand displacement reaction on a magnetic bead, leading to the capture of cupric oxide (CuO) nanoparticles through magnetic separation. After CuO was dissolved, the eATRP initiators were attached to the electrode based on the Cu-catalyzed azide-alkyne cycloaddition. The subsequent eATRP reaction results in the formation of long electroactive polymers (poly-FcMMA), producing an amplified current response for sensitive detection of anti-Dig. This method achieved a detection limit at clinically relevant picomolar concentration in human serum, offering a sensitive, convenient, and cost-effective tool for detecting various biomarkers in a wide range of applications.
Topics: Polymerization; DNA; Electrochemical Techniques; Biosensing Techniques; Humans; Antibodies; Copper; Limit of Detection
PubMed: 38904276
DOI: 10.1021/acs.analchem.4c01176 -
Inorganic Chemistry Jul 2024As a typical RNA virus, the genetic information on HIV-1 is entirely stored in RNA. The reverse transcription activity of HIV-1 reverse transcriptase (RT) plays a...
As a typical RNA virus, the genetic information on HIV-1 is entirely stored in RNA. The reverse transcription activity of HIV-1 reverse transcriptase (RT) plays a crucial role in the replication and transmission of the virus. Non-nucleoside RT inhibitors (NNRTIs) block the function of RT by binding to the RNA binding site on RT, with very few targeting viral RNA. In this study, by transforming planar conjugated ligands into a spiro structure, we convert classical Ru(II) DNA intercalators into a nonintercalator. This enables selective binding to HIV-1 transactivation response (TAR) RNA on the outer side of nucleic acids through dual interactions involving hydrogen bonds and electrostatic attraction, effectively inhibiting HIV-1 RT and serving as a selective fluorescence probe for TAR RNA.
Topics: HIV Reverse Transcriptase; Reverse Transcriptase Inhibitors; Ligands; HIV-1; Ruthenium; RNA, Viral; Spiro Compounds; Coordination Complexes; Intercalating Agents; Molecular Structure; Humans; Anti-HIV Agents; HIV Long Terminal Repeat; Binding Sites
PubMed: 38904258
DOI: 10.1021/acs.inorgchem.4c01815 -
Analytical Chemistry Jul 2024Tumor-derived extracellular vesicles (TEVs) are rich in cellular information and hold great promise as a biomarker for noninvasive cancer diagnosis. However, accurate...
Ultrasensitive and Wash-Free Detection of Tumor Extracellular Vesicles by Aptamer-Proximity-Ligation-Activated Rolling Circle Amplification Coupled to Single Particle ICP-MS.
Tumor-derived extracellular vesicles (TEVs) are rich in cellular information and hold great promise as a biomarker for noninvasive cancer diagnosis. However, accurate measurement of TEVs presents challenges due to their low abundance and potential interference from a high number of EVs derived from normal cells. Herein, an aptamer-proximity-ligation-activated rolling circle amplification (RCA) method for EV membrane recognition, coupled with single particle inductively coupled plasma mass spectrometry (sp-ICP-MS) for the quantification of TEVs, is developed. When DNA-labeled ultrasmall gold nanoparticle (AuNP) probes bind to the long chains formed by RCA, they aggregate to form large particles. Notably, small AuNPs scarcely produce pulse signals in sp-ICP-MS, thereby detecting TEVs in a wash-free manner. By leveraging the strong binding affinity of aptamers, dual aptamers for EpCAM and PD-L1 recognition, and the sp-ICP-MS technique, this method offers remarkable sensitivity and selectivity in tracing TEVs. Under optimized conditions, the present method shows a favorable linear relationship between the pulse signal frequency of sp-ICP-MS and TEV concentration within the range of 10-10 particles/mL, along with a detection limit of 1.1 × 10 particles/mL. The pulse signals from sp-ICP-MS combined with machine learning algorithms are used to discriminate cancer patients from healthy donors with 100% accuracy. Due to its simple and fast operation and excellent sensitivity and accuracy, this approach holds significant potential for diverse applications in life sciences and personalized medicine.
Topics: Humans; Aptamers, Nucleotide; Extracellular Vesicles; Nucleic Acid Amplification Techniques; Metal Nanoparticles; Gold; Mass Spectrometry; Neoplasms; Epithelial Cell Adhesion Molecule; Limit of Detection
PubMed: 38904228
DOI: 10.1021/acs.analchem.4c02066 -
Spectrochimica Acta. Part A, Molecular... Jun 2024Herein, two simple fluorescent signal-on sensing strategies for detecting lead ions (Pb) were established based on structure-switching aptamer probes and...
Herein, two simple fluorescent signal-on sensing strategies for detecting lead ions (Pb) were established based on structure-switching aptamer probes and exonuclease-assisted signal amplification strategies. Two hairpin-structure fluorescent probes with blunt-ended stem arms were designed by extending the base sequence of Pb aptamer (PS2.M) and labelling the probes with FAM (in probe 1) and 2-aminopurine (2-AP) (in probe 2), respectively. In method 1, graphene oxide (GO) was added to adsorb probe 1 and quench the fluorescence emission of FAM to achieve low fluorescent background. In method 2, fluorescent 2-AP molecule inserted into the double-stranded DNA of probe 2 was quenched as a result of base stacking interactions, leading to a simplified, quencher-free approach. The addition of Pb can induce the probes to transform into G-quadruplex structures, exposing single DNA strands at the 3' end (the extended sequences). This exposure enables the activation of exonuclease I (Exo I) on the probes, leading to the cleavage effect and subsequent release of free bases and fluorophores, thereby resulting in amplified fluorescence signals. The two proposed methods exhibit good specificity and sensitivity, with detection limits of 0.327 nM and 0.049 nM Pb for method 1 and method 2, respectively, and have been successfully applied to detect Pb in river water and fish samples. Both detection methods employ the structure-switching aptamer probes and can be completed in two or three steps without the need for complex analytical instruments. Therefore, they have a broad prospect in the sensitive and simple detection of lead ion contamination in food and environmental samples.
PubMed: 38901233
DOI: 10.1016/j.saa.2024.124643 -
Journal of the American Chemical Society Jun 2024Folding thermodynamics, quantitatively described using parameters such as Δ, Δ, and Δ, is essential for characterizing the stability and functionality of noncanonical...
Folding thermodynamics, quantitatively described using parameters such as Δ, Δ, and Δ, is essential for characterizing the stability and functionality of noncanonical nucleic acid structures but remains difficult to measure at the molecular level. Leveraging the programmability of dynamic deoxyribonucleic acid (DNA) chemistry, we introduce a DNA-based molecular tool capable of performing a free energy shift assay (FESA) that directly characterizes the thermodynamics of noncanonical DNA structures in their native environments. FESA operates by the rational design of a reference DNA probe that is energetically equivalent to a target noncanonical nucleic acid structure in a series of toehold-exchange reactions, yet is structurally incapable of folding. As a result, a free energy shift (ΔΔ) is observed when plotting the reaction yield against the free energy of each toehold-exchange. We mathematically demonstrated that Δ, Δ, and Δ of the analyte can be calculated based on ΔΔ. After validating FESA using six DNA hairpins by comparing the measured Δ, Δ, and Δ values against predictions made by NUPACK software, we adapted FESA to characterize noncanonical nucleic acid structures, encompassing DNA triplexes, G-quadruplexes, and aptamers. This adaptation enabled the successful characterization of the folding thermodynamics for these complex structures under various experimental conditions. The successful development of FESA marks a paradigm shift and a technical advancement in characterizing the thermodynamics of noncanonical DNA structures through molecular tools. It also opens new avenues for probing fundamental chemical and biophysical questions through the lens of molecular engineering and dynamic DNA chemistry.
PubMed: 38899479
DOI: 10.1021/jacs.4c04721 -
Langmuir : the ACS Journal of Surfaces... Jul 2024Optical tweezers (OT) have evolved into powerful single molecule force spectroscopy tools to investigate protein folding-unfolding dynamics. To stretch a protein of...
Optical tweezers (OT) have evolved into powerful single molecule force spectroscopy tools to investigate protein folding-unfolding dynamics. To stretch a protein of interest using OT, the protein must be flanked with two double stranded DNA (dsDNA) handles. However, coupling dsDNA handles to the protein is often of low yield, representing a bottleneck in OT experiments. Here, we report a handle-free, all-protein-based OT method for investigating protein folding/unfolding dynamics. In this new method, we employed disordered elastin-like polypeptides (ELPs) as a molecular linker and the mechanically stable cohesin-dockerin (Coh-Doc) pair as the prey-bait system to enable the efficient capture and stretching of individual protein molecules. This novel approach was validated by using model proteins NuG2 and RTX-v, yielding experimental results comparable to those obtained by using the dsDNA handle approach. This new method provides a streamlined and efficient OT approach to investigate the folding-unfolding dynamics of proteins at the single molecule level, thus expanding the toolbox of OT-based single molecule force spectroscopy.
Topics: Optical Tweezers; Protein Folding; DNA; Protein Unfolding; Peptides; Proteins
PubMed: 38899455
DOI: 10.1021/acs.langmuir.4c01711 -
Luminescence : the Journal of... Jun 2024We aimed to develop a novel diagnostic method called multiplex fluorescence of loop primer upon self-dequenching loop-mediated isothermal amplification (mFLOS-LAMP) for...
We aimed to develop a novel diagnostic method called multiplex fluorescence of loop primer upon self-dequenching loop-mediated isothermal amplification (mFLOS-LAMP) for the rapid detection of Mycobacterium tuberculosis complex (MTBC). A set of specific primers was designed to target the detection of IS1081 and IS6110 genes, which are insertion sequences within the MTBC. The 110 sputum specimens collected were assessed using the established mFLOS-LAMP method, multiplex polymerase chain reaction, Xpert MTB/RIF, and smear microscopy. The optimal reaction temperature and duration for mFLOS-LAMP were determined to be 65°C and 30 min, respectively, by optimizing the entire system. The detection sensitivity of mFLOS-LAMP was 6.0 × 10 CFU/mL, by Bacillus Calmette-Guerin, and the mFLOS-LAMP sensitivity of M. tuberculosis H37Rv genomic DNA was 500 fg, and the specificity was 100%. The sensitivity of mFLOS-LAMP was 94.2% and the specificity was 96.6%, when Xpert MTB/RIF was used as the reference method. There was no statistically significant difference in their detection rate (χ = 0, P = 1.000), and the consistency was good (kappa = 0.909, P < 0.001). The receiver operating characteristic analysis yielded the maximum area under the curve of 0.954. The mFLOS-LAMP method demonstrated high sensitivity and specificity, allowing for swift and accurate detection of MTBC.
Topics: Mycobacterium tuberculosis; Nucleic Acid Amplification Techniques; Fluorescent Dyes; Humans; DNA, Bacterial; Sensitivity and Specificity; Molecular Diagnostic Techniques
PubMed: 38899381
DOI: 10.1002/bio.4795 -
Mikrochimica Acta Jun 2024A smartphone-based electrochemical aptasensing platform was developed for the point-of-care testing (POCT) of carcinoembryonic antigen (CEA) based on the ferrocene (Fc)...
A smartphone-based electrochemical aptasensing platform was developed for the point-of-care testing (POCT) of carcinoembryonic antigen (CEA) based on the ferrocene (Fc) and PdPt@PCN-224 dual-signal labeled strategy. The prepared PdPt@PCN-224 nanocomposite showed a strong catalytic property for the reduction of HO. Phosphate group-labeled aptamer could capture PdPt@PCN-224 by Zr-O-P bonds to form PdPt@PCN-224-P-Apt. Therefore, a dual signal labeled probe was formed by the hybridization between Fc-DNA and PdPt@PCN-224-P-Apt. The presence of CEA forced PdPt@PCN-224-P-Apt to leave the electrode surface due to the specific affinity, leading to the decrease of the reduction current of HO. At the same time, the Fc-DNA strand changed to hairpin structure, which made Fc closer to the electrode and resulted in the increase of the oxidation current of Fc. Thus, CEA can be accurately determined through both signals: the decrease of HO reduction current and the increase of Fc oxidation current, which could avoid the false positive signal. Under the optimal conditions, the prepared aptasensor exhibited a wide linear range from 1 pg·mL to 100 ng·mL and low detection limits of 0.98 pg·mL and 0.27 pg·mL with Fc and PdPt@PCN-224 as signal labels, respectively. The aptasensor developed in this study has successfully demonstrated its capability to detect CEA in real human serum samples. These findings suggest that the proposed sensing platform will hold great potential for clinical tumor diagnosis and monitoring.
Topics: Carcinoembryonic Antigen; Aptamers, Nucleotide; Smartphone; Electrochemical Techniques; Humans; Biosensing Techniques; Hydrogen Peroxide; Limit of Detection; Palladium; Point-of-Care Testing; Ferrous Compounds; Metallocenes; Platinum
PubMed: 38898338
DOI: 10.1007/s00604-024-06493-z -
Scientific Reports Jun 2024This study probes the utility of biomarkers for microsatellite instability (MSI) detection and elucidates the molecular dynamics propelling colorectal cancer (CRC)...
This study probes the utility of biomarkers for microsatellite instability (MSI) detection and elucidates the molecular dynamics propelling colorectal cancer (CRC) progression. We synthesized a primer panel targeting 725 MSI loci, informed by The Cancer Genome Atlas (TCGA) and ancillary databases, to construct an amplicon library for next-generation sequencing (NGS). K-means clustering facilitated the distillation of 8 prime MSI loci, including activin A receptor type 2A (ACVR2A). Subsequently, we explored ACVR2A's influence on CRC advancement through in vivo tumor experiments and hematoxylin-eosin (HE) staining. Transwell assays gauged ACVR2A's role in CRC cell migration and invasion, while colony formation assays appraised cell proliferation. Western blotting illuminated the impact of ACVR2A suppression on CRC's PI3K/AKT/mTOR pathway protein expressions under hypoxia. Additionally, ACVR2A's influence on CRC-induced angiogenesis was quantified via angiogenesis assays. K-means clustering of NGS data pinpointed 32 MSI loci specific to tumor and DNA mismatch repair deficiency (dMMR) tissues. ACVR2A emerged as a pivotal biomarker, discerning MSI-H tissues with 90.97% sensitivity. A curated 8-loci set demonstrated 100% sensitivity and specificity for MSI-H detection in CRC. In vitro analyses corroborated ACVR2A's critical role, revealing its suppression of CRC proliferation, migration, and invasion. Moreover, ACVR2A inhibition under CRC-induced hypoxia markedly escalated MMP3, CyclinA, CyclinD1, and HIF1α protein expressions, alongside angiogenesis, by triggering the PI3K/AKT/mTOR cascade. The 8-loci ensemble stands as the optimal marker for MSI-H identification in CRC. ACVR2A, a central element within this group, deters CRC progression, while its suppression amplifies PI3K/AKT/mTOR signaling and angiogenesis under hypoxic stress.
Topics: Colorectal Neoplasms; Humans; Microsatellite Instability; Activin Receptors, Type II; Disease Progression; Animals; Cell Movement; Mice; Cell Line, Tumor; Cell Proliferation; Biomarkers, Tumor; Signal Transduction; Male; High-Throughput Nucleotide Sequencing; Female; Proto-Oncogene Proteins c-akt
PubMed: 38898042
DOI: 10.1038/s41598-024-62753-1