-
Bioorganic & Medicinal Chemistry Letters Oct 2023Mitoxantrone (MX) is a robust chemotherapeutic with well-characterized applications in treating certain leukemias and advanced breast and prostate cancers. The canonical...
Mitoxantrone (MX) is a robust chemotherapeutic with well-characterized applications in treating certain leukemias and advanced breast and prostate cancers. The canonical mechanism of action associated with MX is its ability to intercalate DNA and inhibit topoisomerase II, giving it the designation of a topoisomerase II poison. Years after FDA approval, investigations have unveiled novel protein-binding partners, such as methyl-CpG-binding domain protein (MBD2), PIM1 serine/threonine kinase, RAD52, and others that may contribute to the therapeutic profile of MX. Moreover, recent proteomic studies have revealed MX's ability to modulate protein expression, illuminating the complex cellular interactions of MX. Although mechanistically relevant, the differential expression across the proteome does not address the direct interaction with potential binding partners. Identification and characterization of these MX-binding cellular partners will provide the molecular basis for the alternate mechanisms that influence MX's cytotoxicity. Here, we describe the design and synthesis of a MX-biotin probe (MXP) and negative control (MXP-NC) that can be used to define MX's cellular targets and expand our understanding of the proteome-wide profile for MX. In proof of concept studies, we used MXP to successfully isolate a recently identified protein-binding partner of MX, RAD52, in a cell lysate pulldown with streptavidin beads and western blotting.
Topics: Humans; Male; DNA Topoisomerases, Type II; DNA-Binding Proteins; Mitoxantrone; Prostatic Neoplasms; Proteome; Proteomics; Molecular Probes; Breast Neoplasms; Female
PubMed: 37669721
DOI: 10.1016/j.bmcl.2023.129465 -
Analytica Chimica Acta Jul 2023Electrochemical DNA sensors can be operated in either static or flow-based detection schemes. In static schemes, manual washing steps are still necessary, resulting in a...
Electrochemical DNA sensors can be operated in either static or flow-based detection schemes. In static schemes, manual washing steps are still necessary, resulting in a tedious and time-consuming process. In contrast, in flow-based electrochemical sensors, the current response is collected when the solution flows through the electrode continuously. However, the drawback of such a flow system is the low sensitivity due to the limited time for the interaction between the capturing element and the target. Herein, we propose a novel electrochemical capillary-driven microfluidic DNA sensor to combine the advantages of static and flow-based electrochemical detection systems into a single device by incorporating burst valve technology. The microfluidic device with a two-electrode configuration was applied for the simultaneous detection of two different DNA markers, human immunodeficiency virus-1 (HIV-1) and hepatitis C virus (HCV) cDNA, via the specific interaction between pyrrolidinyl peptide nucleic acids (PNA) probes and the DNA target. The integrated system, while requiring a small sample volume (7 μL for each sample loading port) and less analysis time, achieved good performance in terms of the limits of detection (LOD) (3SD/slope) and quantification (LOQ) (10SD/slope) at 1.45 nM and 4.79 nM for HIV and 1.20 nM and 3.96 nM for HCV, respectively. The simultaneous detection of HIV-1 and HCV cDNA prepared from human blood samples showed results that are in complete agreement with the RT‒PCR assay. The results qualify this platform as a promising alternative for the analysis of either HIV-1/HCV or coinfection that can be easily adapted for other clinically important nucleic acid-based markers.
Topics: Humans; Hepacivirus; Microfluidics; HIV-1; DNA, Complementary; Coinfection; DNA; Hepatitis C; HIV Infections
PubMed: 37230584
DOI: 10.1016/j.aca.2023.341257 -
ACS Applied Materials & Interfaces Oct 2023Signal amplification methods based on DNA molecular interactions are promising tools for detecting various biomarkers in low abundance. The entropy-driven circuit (EDC),...
Signal amplification methods based on DNA molecular interactions are promising tools for detecting various biomarkers in low abundance. The entropy-driven circuit (EDC), as an enzyme-free signal amplification method, has been used in detecting and imaging a variety of biomarkers. The localization strategy can effectively increase the local concentration of the DNA reaction modules to improve the signal amplification effect. However, the localization strategy may also amplify the leak reaction of the EDC, and effective signal amplification can be limited by the unclear structure-function relationship. Herein, we utilized locked nucleic acid (LNA) modification to enhance the stability of the localized entropy-driven circuit (LEDC), which suppressed a 94.6% leak signal. The coarse-grained model molecular simulation was used to guide the structure design of the LEDC, and the influence of critical factors such as the localized distance and spacer length was analyzed at the molecular level to obtain the best reaction performance. The sensitivities of miR-21 and miR-141 detected by a simulation-guided optimal LEDC probe were 17.45 and 65 pM, 1345 and 521 times higher than free-EDC, respectively. The LEDC was further employed for the fluorescence imaging of miRNA in cancer cells, showing excellent specificity and sensitivity. This work utilizes LNA and molecular simulations to comprehensively improve the performance of a localized DNA signal amplification circuit, providing an advanced DNA probe design strategy for biosensing and imaging as well as valuable information for the designers of DNA-based probes.
Topics: Entropy; DNA; DNA Probes; MicroRNAs; Biomarkers; Nucleic Acid Amplification Techniques; Biosensing Techniques
PubMed: 37773989
DOI: 10.1021/acsami.3c11189 -
Methods in Molecular Biology (Clifton,... 2024Circular ssDNA viruses are ubiquitous and can be found in both prokaryotes and eukaryotes. To understand the interaction of ssDNA viruses with their hosts, it is...
Circular ssDNA viruses are ubiquitous and can be found in both prokaryotes and eukaryotes. To understand the interaction of ssDNA viruses with their hosts, it is important to characterize the dynamics of viral sense (VS) and complementary-sense (CS) viral strands during the infection process. Here, we present a simple and rapid protocol that allows sensitive and accurate determination of the VS and CS strands generated during viral infection.The method consists of a two-step qPCR in which the first step uses a strand-specific (CS or VS) labeled primer and T4 DNA polymerase that lacks strand displacement activity and makes a single copy per VS or CS strand. Next, the T4 DNA polymerase and unincorporated oligonucleotides are removed by a silica membrane spin column. Finally, the purified VS or CS strands are quantified by qPCR in a second step in which amplification uses a tag primer and a specific primer. Absolute quantification of VS and CS strands is obtained by extrapolating the Cq data to a standard curve of ssDNA, which can be generated by phagemid expression. Quantification of VS and CS strands of two geminiviruses in infections of Solanum lycopersicum (tomato) and Nicotiana benthamiana plants using this method is shown.
Topics: DNA, Complementary; DNA, Single-Stranded; Geminiviridae; Solanum lycopersicum; Virion; DNA-Directed DNA Polymerase
PubMed: 37987901
DOI: 10.1007/978-1-0716-3485-1_8 -
Spectrochimica Acta. Part A, Molecular... Feb 2024For early diagnosis of disease, ultrasensitive mircoRNA-21 detection has considerable potential. In this paper, an ultra-sensitive fluorescence detection method for...
For early diagnosis of disease, ultrasensitive mircoRNA-21 detection has considerable potential. In this paper, an ultra-sensitive fluorescence detection method for microRNA was developed by atom transfer radical polymerization (ATRP). This ATRP reaction was first initiated by using flavin mononucleotide (FADH). The DNA probe 1 modified with amino group was fixed on the magnetic nanoparticle FeO, and microRNA-21 was added to form the probe 1-microRNA-21. Another carboxy-modified DNA 2 forms a sandwich structure with the bound microRNA-21. Two terminally modified DNA types are used as microRNA probes, using complementary base pairing to form a stable super-sandwich structure between the DNA probe and the microRNA. Under optimal conditions, microRNA was detected in PBS buffer with a detection limit of 0.19 fM. And even in 10% of human serum, microRNA-21 can be detected with a detection limit of 47.8 fM. Results show that this method has high selectivity, efficiency and stability, which broad application prospect in microRNA ultra-sensitive detection.
Topics: Humans; Polymerization; Limit of Detection; DNA; MicroRNAs; DNA Probes; Biosensing Techniques; Electrochemical Techniques
PubMed: 37871544
DOI: 10.1016/j.saa.2023.123548 -
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 -
Biosensors & Bioelectronics Dec 2023Herein, a novel multifunctional photoelectrochemical (PEC) biosensor based on AgInS (AIS)/ZnS quantum dots (QDs) sensitized-WSe nanoflowers and DNA nanostructure signal...
Herein, a novel multifunctional photoelectrochemical (PEC) biosensor based on AgInS (AIS)/ZnS quantum dots (QDs) sensitized-WSe nanoflowers and DNA nanostructure signal probe was designed to achieve ultra-sensitive "On-Off" detection of human tumor necrosis factor α (TNF-α) and methylase Dam MTase (MTase). AIS/ZnS QDs as an excellent photosensitive material was found to match WSe in energy level for the first time, and the photocurrent signal after sensitization was 65 times that of WSe nanoflowers and 17.9 times that of AIS/ZnS QDs. Moreover, abundant AIS/ZnS QDs were loaded on the TiO nanoparticles with good conductivity by DNA to fabricate a multifunctional probe, which can not only amplify signal but also specifically recognize target. When target TNF-α was present, the AIS/ZnS QDs signal probe was attached to the WSe nanoflowers-modified electrode through binding to aptamer, and the amplified PEC signal was generated for "on" assay of TNF-α. Furthermore, Dam MTase as second target induced methylation of hairpin H, so it is cleaved by the endonuclease DpnI, resulting in the shedding of AIS/ZnS QDs signal probe for signal "off" detection of MTase. This work opened a new photosensitized probe and developed a promising PEC biosensor for dual-targets assay. By programming the DNA nanostructure, the biosensor can detect versatile targets in a simple and sensitive method, which has good practical application value in human serum.
Topics: Humans; Tumor Necrosis Factor-alpha; Electrochemical Techniques; Quantum Dots; Biosensing Techniques; Nanostructures; DNA; DNA Probes
PubMed: 37748401
DOI: 10.1016/j.bios.2023.115704 -
Journal of Visualized Experiments : JoVE Feb 2024AQRNA-seq provides a direct linear relationship between sequencing read counts and small RNA copy numbers in a biological sample, thus enabling accurate quantification...
AQRNA-seq provides a direct linear relationship between sequencing read counts and small RNA copy numbers in a biological sample, thus enabling accurate quantification of the pool of small RNAs. The AQRNA-seq library preparation procedure described here involves the use of custom-designed sequencing linkers and a step for reducing methylation RNA modifications that block reverse transcription processivity, which results in an increased yield of full-length cDNAs. In addition, a detailed implementation of the accompanying bioinformatics pipeline is presented. This demonstration of AQRNA-seq was conducted through a quantitative analysis of the 45 tRNAs in Mycobacterium bovis BCG harvested on 5 selected days across a 20-day time course of nutrient deprivation and 6 days of resuscitation. Ongoing efforts to improve the efficiency and rigor of AQRNA-seq will also be discussed here. This includes exploring methods to obviate gel purification for mitigating primer dimer issues after PCR amplification and to increase the proportion of full-length reads to enable more accurate read mapping. Future enhancements to AQRNA-seq will be focused on facilitating automation and high-throughput implementation of this technology for quantifying all small RNA species in cell and tissue samples from diverse organisms.
Topics: High-Throughput Nucleotide Sequencing; RNA; RNA, Transfer; Gene Library; DNA, Complementary; Sequence Analysis, RNA
PubMed: 38372376
DOI: 10.3791/66335 -
RNA (New York, N.Y.) Jul 2023RNA sequencing has spurred a significant number of research areas in recent years. Most protocols rely on synthesizing a more stable complementary DNA (cDNA) copy of the... (Review)
Review
RNA sequencing has spurred a significant number of research areas in recent years. Most protocols rely on synthesizing a more stable complementary DNA (cDNA) copy of the RNA molecule during the reverse transcription reaction. The resulting cDNA pool is often wrongfully assumed to be quantitatively and molecularly similar to the original RNA input. Sadly, biases and artifacts confound the resulting cDNA mixture. These issues are often overlooked or ignored in the literature by those that rely on the reverse transcription process. In this review, we confront the reader with intra- and intersample biases and artifacts caused by the reverse transcription reaction during RNA sequencing experiments. To fight the reader's despair, we also provide solutions to most issues and inform on good RNA sequencing practices. We hope the reader can use this review to their advantage, thereby contributing to scientifically sound RNA studies.
Topics: Reverse Transcription; DNA, Complementary; Artifacts; RNA; Sequence Analysis, RNA; Bias
PubMed: 36990512
DOI: 10.1261/rna.079623.123 -
Journal of the American Chemical Society Mar 2024Regulatory modules for controlling the kinetics of toehold-mediated strand displacement (TMSD) play critical roles in designing dynamic and dissipative DNA chemical...
Regulatory modules for controlling the kinetics of toehold-mediated strand displacement (TMSD) play critical roles in designing dynamic and dissipative DNA chemical reaction networks (CRNs) but are hardwired into sequence designs. Herein, we introduce antitoehold (At), a plug-and-play module for reversible and continuous tuning of TMSD kinetics by temporarily occupying the toehold domain via a metastable duplex and base stacking. We demonstrate that kinetic control can be readily activated or deactivated in real time for any TMSD by simply adding At or anti-At. Continuous tuning of TMSD kinetics can also be achieved by altering the concentration of At. Moreover, the simple addition of At could readily reprogram existing TMSDs into a pulse-generation DNA CRN with continuous tunability. Our At approach also offers a new way for engineering continuously tunable DNA hybridization probes, which may find practical uses for discriminating clinically important mutations. Because of the simplicity, we anticipate that At will find wide applications for engineering DNA CRNs with diverse dynamic and dissipative behaviors, and DNA hybridization probes with tunable affinity and selectivity.
Topics: DNA; Nucleic Acid Hybridization; Kinetics; DNA Probes
PubMed: 38411013
DOI: 10.1021/jacs.3c09242