Target DNA Research Articles

Analysis of bovine beta-casein A1 and A2 allele frequency in Holstein-Friesian cows by Real-time PCR with fluorescent hybridization probes

A2 milk popularity is increasing across the world and novel molecular techniques have been evaluated to develop reliable methods. This study aimed to genotype Holstein-Friesian cows concerning their A1/A2 status using Real-time PCR assay with the specifically designed FRET hybridization probes. In this context, DNA samples were obtained from 310 Holstein-Friesian milk samples. Concerning the Real-time PCR assay, the melting temperature of each amplicon was analyzed and the melting data was converted to a derivative plot using the LightCycler 480 System. The sensor probe was designed to match the wild-type sequence in the target DNA. In the Real-time PCR assay, the melting peaks obtained in the Real-time PCR assay were highly decisive and consistent for each genotype regarding CCT→CAT alteration. The results indicated a remarkably high frequency of the A2 allele (68%) and a considerable frequency of heterozygous animals (0.41). Population genetic analysis showed intermediate levels of genetic variability and biodiversity. The A2-herd conversion process is a complex process consisting of genetic testing of both cows and calves, evaluating replacement rates, and the conversion of heterozygotes by using A2-genotyped bull semen. In this sense, the key point is a reliable and rapid genotyping method to produce A1-free milk. This study suggests that Real-time PCR assay with the specifically designed FRET hybridization probes is a preferable method for A2 genotyping, and may be useful for further studies and instructive for companies or breeders who aim to produce A2 milk.

Detection of Escherichia coli by capillary electrophoresis assisted by large volume sample stacking and nicking endonuclease signal amplification

Efficient, accurate and economical detection of pathogenic bacteria is crucial in ensuring food safety and preventing foodborne illnesses. In this study, a capillary electrophoresis coupled laser-induced fluorescence assay (CE-LIF) was developed for the detection of Escherichia coli (E. coli) by detecting its specific DNA. The CE-LIF was assisted by both online enrichment and offline amplification to improve the detection sensitivity of bacterial DNA. Here the online amplification was performed by large volume sample stacking (LVSS), while the offline amplification was nicking endonuclease signal amplification (NESA). Under the optimal experimental conditions, the detection limit of bacterial target DNA was 2.5 fM, and the conversion concentration of E. coli was 3 CFU · mL−1. The method had been applied to the detection of commercially available skim milk samples with good results, which proved that it could be used as an effective tool for food and environmental bacteria monitoring.

Noncanonical binding of transcription factors: time to revisit specificity?

Transcription factors (TFs) are one of the most studied classes of DNA-binding proteins that have a direct functional impact on gene transcription and thus, on human physiology and disease. The mechanisms that TFs use for recognizing target DNA binding sites have been studied for nearly five decades, yet they remain poorly understood. It is classically assumed that a TF recognizes a specific sequence pattern, or motif, as its binding sites. However, recent studies are consistently finding examples of noncanonical binding, that is, TFs binding at sites that do not resemble their sequence motifs. Here we review the current literature on four major types of noncanonical TF binding, namely binding based on DNA shape readout, at Guanine-quadruplex structures, at repeat sequences, and bispecific binding. These examples point to a critical need for studies to unify our current observations, many of which are at odds with the "one TF, one motif" view, into a more comprehensive definition of the DNA-binding specificity of TFs.

Visual detection of Fusarium temperatum by using CRISPR-Cas12a empowered LAMP assay coupled with AuNPs-based colorimetric reaction

Fusarium temperatum (F. temperatum) causes maize stalk rot disease, reducing grain yield and producing multiple harmful mycotoxins that threaten food safety and quality. However, current detection methods are limited by the requirement of well-trained personnel, complicated operations and expensive instruments. To address this challenge, gold nanoparticles (AuNPs)-based colorimetric assay using loop-mediated isothermal amplification (LAMP) coupled with CRISPR-Cas12a (Au-CRISPR) was tested for F. temperatum detection. The assay utilizes DNA-modified AuNPs as a colorimetric probe and the results can be readily observed by the naked eye. After optimization, the method can specifically detect as low as 100 copies/μL of target DNA or 10−8 ng/μL of extracted DNA from F. temperatum. The detection platform allows high-throughput testing by integrating microtiter plates and microplate reader. Furthermore, a portable smartphone-based device was developed to realize point of care (POC) detection of F. temperatum in resource-limited settings. This simple, inexpensive and sensitive detection platform has great potential for the applications in field detection.

Conditional probability as found in nature: Facilitated diffusion

Search processes are ubiquitous in physical and biological phenomena, often involving the random motion of molecules. In particular, transcription factors (TFs) are proteins that regulate gene expression and need to find their DNA targets quickly—which is difficult to achieve with random motion alone. Nature came up with a remarkable solution known as facilitated diffusion, combining 1D diffusion along the DNA and “excursions” of diffusion in 3D that help the TF to quickly arrive at distant parts of the DNA. In this paper, we show that this process can be analyzed naturally using the concept of conditional probability, providing an alternative intuition to the effectiveness of this mechanism.

Asymmetric dimerization in a transcription factor superfamily is promoted by allosteric interactions with DNA.

Transcription factors, such as nuclear receptors achieve precise transcriptional regulation by means of a tight and reciprocal communication with DNA, where cooperativity gained by receptor dimerization is added to binding site sequence specificity to expand the range of DNA target gene sequences. To unravel the evolutionary steps in the emergence of DNA selection by steroid receptors (SRs) from monomeric to dimeric palindromic binding sites, we carried out crystallographic, biophysical and phylogenetic studies, focusing on the estrogen-related receptors (ERRs, NR3B) that represent closest relatives of SRs. Our results, showing the structure of the ERR DNA-binding domain bound to a palindromic response element (RE), unveil the molecular mechanisms of ERR dimerization which are imprinted in the protein itself with DNA acting as an allosteric driver by allowing the formation of a novel extended asymmetric dimerization region (KR-box). Phylogenetic analyses suggest that this dimerization asymmetry is an ancestral feature necessary for establishing a strong overall dimerization interface, which was progressively modified in other SRs in the course of evolution.

Roles of DNA Target in Cancer Cell-Selective Cytotoxicity by Dicopper Complexes with DNA Target/Ligand Conjugates.

The DNA target/ligand conjugates (HLX, X = Pn and Mn, n = 1-3) were synthesized where various lengths of -CONH(CH2CH2O)nCH2CH2NHCO- linkers with a 9-phenanthrenyl (P) or methyl (M) terminal as DNA targets replace the methyl group of 2,6-di(amide-tether cyclen)-p-cresol ligand (HL). DNA binding, DNA cleavage, cellular uptake, and cytotoxicity of [Cu2(μ-OH)(LX)](ClO4)2 (1X) are examined and compared with those of [Cu2(μ-OH)(L)](ClO4)2 (1) to clarify roles of DNA targets. Upon reaction of 1X with H2O2, μ-1,1-O2H complexes are formed for DNA cleavage. 1P1, 1P2, and 1P3 are 22-, 11-, 3-fold more active for conversion of Form II to III in the cleavage of supercoiled plasmid DNA with H2O2 than 1, where the short P-linker may fix a dicopper moiety within a small number of base pairs to facilitate DNA double-strand breaks (dsb). This enhances the proapoptotic activity of 1P1, 1P2, and 1P3, which are 30-, 12-, and 9.9-fold cytotoxic against HeLa cells than 1. DNA dsb and cytotoxicity are 44% correlated in 1P1-3 but 5% in 1M1-3, suggesting specific DNA binding of P-linkers and nonspecific binding of M-linkers in biological cells. 1P1-3 exert cancer cell-selective cytotoxicity against lung and pancreas cancer and normal cells where the short P-linker enhances the selectivity, but 1M1-3 do not. Intracellular visualization, apoptosis assay, and caspase activity assay clarify mitochondrial apoptosis caused by 1P1-3. The highest cancer cell selectivity of 1P1 may be enabled by the short P-linker promoting dsb of mitochondrial DNA with H2O2 increased by mitochondrial dysfunction in cancer cells.

A label-free electrochemical biosensor for the detection of alpha-thalassemia 1 (SEA deletion) carriers using screen-printed carbon electrodes

A label-free electrochemical DNA biosensor based on electrochemical impedance spectroscopy (EIS) biosensor has been extensively developed for diagnosing human genetic diseases. However, its application has been limited to simulated target DNA. The aim of this study was to develop an EIS biosensor that can identify carriers of alpha (α)-thalassemia 1 with Southeast Asia (SEA) deletion. The biosensor was coupled with screen-printed carbon electrodes (SPCEs) and Hoechst 33,258 dye. To determine the optimal conditions and cutoff criteria, we evaluated forty samples with known DNA genotypes. Our findings suggested that the cutoff value for identifying α-thalassemia 1 (SEA deletion) carriers was more than 26.84 kΩ of negative imaginary impedance (-Z″) and 48.83 kΩ of charge transfer resistance (Rct). The sensitivity of the developed EIS biosensor was comparable to that of conventional gel electrophoresis, with no cross-reactivity observed in α-thalassemia 1 (THAI deletion), α-thalassemia 2 (3.7 deletion), α-thalassemia 2 (4.2 deletion), and beta (β)-thalassemia carriers. The diagnostic potential of the developed EIS biosensor was evaluated using 81 clinical blood samples, demonstrating 100% sensitivity, 88.2% specificity, 83.3% positive predictive value (PPV), 100% negative predictive value (NPV) and 92.6% accuracy for Rct measurement. The developed EIS biosensor presents an attractive screening method for the detecting α-thalassemia 1 (SEA deletion) carriers due to its simplicity, cost-effectiveness, and 45-fold reduction in turnaround time compared to conventional gel electrophoresis. Consequently, the identification of α-thalassemia 1 (SEA deletion) carrier through this approach represents the most effective strategy for the management and prevention of the most severe form of thalassemia.

Evolution of molecular switches for regulation of transgene expression by clinically licensed gluconate.

Synthetic biology holds great promise to improve the safety and efficacy of future gene and engineered cell therapies by providing new means of endogenous or exogenous control of the embedded therapeutic programs. Here, we focused on gluconate as a clinically licensed small-molecule inducer and engineered gluconate-sensitive molecular switches to regulate transgene expression in human cell cultures and in mice. Several switch designs were assembled based on the gluconate-responsive transcriptional repressor GntR from Escherichia coli. Initially we assembled OFF- and ON-type switches by rewiring the native gluconate-dependent binding of GntR to target DNA sequences in mammalian cells. Then, we utilized the ability of GntR to dimerize in the presence of gluconate to activate gene expression from a split transcriptional activator. By means of random mutagenesis of GntR combined with phenotypic screening, we identified variants that significantly enhanced the functionality of the genetic devices, enabling the construction of robust two-input logic gates. We also demonstrated the potential utility of the synthetic switch in two in vivo settings, one employing implantation of alginate-encapsulated engineered cells and the other involving modification of host cells by DNA delivery. Then, as proof-of-concept, the gluconate-actuated genetic switch was connected to insulin secretion, and the components encoding gluconate-induced insulin production were introduced intotype-1 diabetic miceas naked DNA via hydrodynamic tail vein injection. Normoglycemia was restored, thereby showcasing the suitability of oral gluconate to regulate in situ production of a therapeutic protein.

AcrIIC4 inhibits type II-C Cas9 by preventing R-loop formation

Anti-CRISPR (Acr) proteins are encoded by phages and other mobile genetic elements and inhibit host CRISPR-Cas immunity using versatile strategies. AcrIIC4 is a broad-spectrum Acr that inhibits the type II-C CRISPR-Cas9 system in several species by an unknown mechanism. Here, we determined a series of structures of Haemophilus parainfluenzae Cas9 (HpaCas9)-sgRNA in complex with AcrIIC4 and/or target DNA, as well as the crystal structure of AcrIIC4 alone. We found that AcrIIC4 resides in the crevice between the REC1 and REC2 domains of HpaCas9, where its extensive interactions restrict the mobility of the REC2 domain and prevent the unwinding of target double-stranded (ds) DNA at the PAM-distal end. Therefore, the full-length guide RNA:target DNA heteroduplex fails to form in the presence of AcrIIC4, preventing Cas9 nuclease activation. Altogether, our structural and biochemical studies illuminate a unique Acr mechanism that allows DNA binding to the Cas9 effector complex but blocks its cleavage by preventing R-loop formation, a key step supporting DNA cleavage by Cas9.

Lipid-, Inorganic-, Polymer-, and DNA-Based Nanocarriers for Delivery of the CRISPR/Cas9 system.

The clustered regularly interspaced short palindromic repeat (CRISPR)/associated protein 9 (CRISPR/Cas9) system has been widely explored for the precise manipulation of target DNA and has enabled efficient genomic editing in cells. Recently, CRISPR/Cas9 has shown promising potential in biomedical applications, including disease treatment, transcriptional regulation and genome-wide screening. Despite these exciting achievements, efficient and controlled delivery of the CRISPR/Cas9 system has remained a critical obstacle to its further application. Herein, we elaborate on the three delivery forms of the CRISPR/Cas9 system, and discuss the composition, advantages and limitations of these forms. Then we provide a comprehensive overview of the carriers of the system, and focus on the nonviral nanocarriers in chemical methods that facilitate efficient and controlled delivery of the CRISPR/Cas9 system. Finally, we discuss the challenges and prospects of the delivery methods of the CRISPR/Cas9 system in depth, and propose strategies to address the intracellular and extracellular barriers to delivery in clinical applications.

Synthesis and Biological Activity of 2-Benzo[b]thienyl and 2-Bithienyl Amidino-Substituted Benzothiazole and Benzimidazole Derivatives.

Novel benzo[b]thienyl- and 2,2'-bithienyl-derived benzothiazoles and benzimidazoles were synthesized to study their antiproliferative and antitrypanosomal activities in vitro. Specifically, we assessed the impact that amidine group substitutions and the type of thiophene backbone have on biological activity. In general, the benzothiazole derivatives were more active than their benzimidazole analogs as both antiproliferative and antitrypanosomal agents. The 2,2'-bithienyl-substituted benzothiazoles with unsubstituted and 2-imidazolinyl amidine showed the most potent antitrypanosomal activity, and the greatest selectivity was observed for the benzimidazole derivatives bearing isopropyl, unsubstituted and 2-imidazolinyl amidine. The 2,2'-bithiophene derivatives showed most selective antiproliferative activity. Whereas the all 2,2'-bithienyl-substituted benzothiazoles were selectively active against lung carcinoma, the benzimidazoles were selective against cervical carcinoma cells. The compounds with an unsubstituted amidine group also produced strong antiproliferative effects. The more pronounced antiproliferative activity of the benzothiazole derivatives was attributed to different cytotoxicity mechanisms. Cell cycle analysis, and DNA binding experiments provide evidence that the benzimidazoles target DNA, whereas the benzothiazoles have a different cellular target because they are localized in the cytoplasm and do not interact with DNA.

Streamlined and on-demand preparation of mRNA products on a universal integrated platform

Vaccines are used to protect human beings from various diseases. mRNA vaccines simplify the development process and reduce the production cost of conventional vaccines, making it possible to respond rapidly to acute and severe diseases, such as coronavirus disease 2019. In this study, a universal integrated platform for the streamlined and on-demand preparation of mRNA products directly from DNA templates was established. Target DNA templates were amplified in vitro by a polymerase chain reaction module and transcribed into mRNA sequences, which were magnetically purified and encapsulated in lipid nanoparticles. As an initial example, enhanced green fluorescent protein (eGFP) was used to test the platform. The expression capacity and efficiency of the products were evaluated by transfecting them into HEK-293T cells. The batch production rate was estimated to be 200–300 μg of eGFP mRNA in 8 h. Furthermore, an mRNA vaccine encoding the receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein was produced by this platform. The proposed integrated platform shows advantages for the universal and on-demand preparation of mRNA products, offering the potential to facilitate broad access to mRNA technology and enable the development of mRNA products, including the rapid supply of new mRNA-based vaccines in pandemic situations and personalized mRNA-based therapies for oncology and chronic infectious diseases, such as viral hepatitis and acquired immune deficiency syndrome.

Novel hybridization indicator for DNA sequences related to the hepatitis B virus: Electrochemical and in-silico approach

In the present study, esculetin, as a novel electroactive indicator, was used to produce an electrochemical DNA hybridization biosensor for Hepatitis B Virus (HBV) detection. At first, alkanothiol-modified DNA probe (from Hepatitis B) was self-assembled on the surface of a glassy carbon electrode modified by the nanocomposite of polyaniline and Au nanoparticles (PANI-Au). Hybridization conditions were enhanced, including the immobilization of the probe strands, the hybridization process and the indicator accumulation. For evaluating the DNA hybridization, the differential pulse voltammetric response of the accumulated esculetin was used as an electroactive indicator on different electrode surfaces. According to the obtained results, the voltammetric responses of esculetin accumulated on the probes interacted with non-complementary, mismatch and complementary targets were significantly different. Therefore, in optimum conditions, electrochemical signals of esculetin were linear in the concentration range of 0.01–3.00 × 10−6 M of the complementary strands, and a detection limit of 3.80 × 10−9 M was obtained for the target. Subsequently, the modified electrode was utilized for the detection of the HBV in spiked serum and plasma samples. The results obtained from electrochemical technique were confirmed by computational molecular simulation using MOE software. Moreover, the simulation outcomes provided valuable information about quantum chemical properties including the HOMO–LUMO maps and chemical reactivity for esculetin-the DNA target components.

Coupling 2-Aminopurine with DNA Copper Nanoparticles as a Rapid and Enzyme-Free System for Operating DNA Contrary Logic Pairs

Exploring affordable and efficient platform for innovative DNA computing is of great significance. Herein, by coupling 2-aminopurine (2AP) with DNA copper nanoparticles (CuNPs) as two universal opposite outputs, we, for the first time, fabricated a rapid and enzyme-free system for operating DNA contrary logic pairs (D-CLPs). Notably, derived from the rapid and concomitant response of both fluorescent probes, different D-CLPs can be achieved via a “double-results-half-efforts” manner in less than 20 min with low-cost. Moreover, based on the same system, the smart ratiometric analysis of target DNA was realized by employing the high reliability and accuracy of D-CLPs, providing a robust and typical paradigm for the exploration of smart nucleic acid sensors.

A disposable electrochemiluminescence biosensor sensitized with multipedal DNA walker for in situ quenching detection of DNA methylation

Herein, a disposable electrochemiluminescence (ECL) biosensor sensitized with target-triggered multi-DNA release and multipedal DNA walker was designed for in situ quenching detection of methylated target DNA. In this work, zirconium(IV)-based metal organic frameworks encapsulated with Ru(bpy)32+ emitter (UiO-66-NH2@Ru) were attached onto indium tin oxide (ITO) electrode as sensing substrate. With the assistance of magnetic bead separation, one methylated DNA released three kinds of DNAs, achieving a one-to-multiple signal transduction and amplification. Then, these multi-DNA strands hybridized with swing arm immobilized on ITO surface as DNA walking strands. The further hybridization between multi-DNA strands and DNA tracks resulted in the formation of cleavage sites, which were digested by a restriction endonuclease, causing the cleavage of DNA tracks into two short fragments. Subsequently, the dissociated DNA walking strands explored adjacent DNA tracks, and the multipedal walking procedure was conducted. Relying on the in situ quenching of H2O2 for ECL emission of Ru(bpy)32+ system, this method achieved the quantification of methylated target DNA. Moreover, taking the advantages of inexpensiveness, high walking kinetics, great amplification efficiency, good reproducibility and practicability, this disposable biosensor held great potential for analyzing DNA methylation levels in resource-limited environments.

Rapid and highly sensitive LAMP-CRISPR/Cas12a-based identification of bovine mastitis milk samples contaminated by Escherichia coli

Mastitis is a prevalent disease affecting dairy cows, leading to significant economic losses in the dairy industry. Conventional diagnostic methods such as microbiology and PCR are expensive and time-consuming, emphasizing the need for alternative diagnostic approaches. The field of novel diagnostics is expanding rapidly due to the application of a modern molecular detection methods based on the CRISPR/Cas system. This system functions by targeting specific genetic sequences of the pathogen, including Escherichia coli, and detects the presence of the pathogen by employing a CRISPR RNA that complements the pathogen's genetic sequence and a Cas12a enzyme that cleaves the particular DNA sequence. In this paper, we present a novel pathogen detection technology that combines the loop-mediated isothermal amplification (LAMP) reaction and Cas12a collateral activity. We have successfully developed a rapid and precise method for identifying E.coli genomic DNA using the LAMP-Cas12a technology, which exhibits high analytical sensitivity within an hour, detecting as low as 1.3 × 101 copies of target DNA. This technology also has the ability to differentiate E. coli from other prevalent mastitis pathogens such as Staphylococcus aureus and Streptococcus agalactiae. Furthermore, we employed a novel MbCas12a nuclease, which demonstrated excellent diagnostic performance in our study when identifying E.coli isolates isolated from bovine mastitis milk samples. The development of such new methods has the potential to expand agricultural tools for use in point-of-care (POC) diagnostics.

Specific oligonucleotide-modified QDs as a powerful platform for SARS Cov-2 virus detection by FRET-based biosensors

FRET-based nano-bioconjugate sensors (nano-biosensors) could be examined in coronavirus disease (COVID-19) diagnosis. In this work, a nano-biosensor for SARS Cov-2 virus detection was established based on fluorescence resonance energy transfer (FRET) between oligonucleotide-modified CdTe/ZnS quantum dots (QDs) as a donor and Cy3 as an acceptor. UV–visible spectrophotometry, FT-IR spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and atomic force microscopy (AFM) were used to characterize the chemical and physical structures of the nano-biosensor. Under optimized conditions, the detection limit of complementary target DNA (ctDNA) was 2.19 × 10−9 µM. The linear equation and regression were y = 181.8x + 1 and R2 = 0.9762. In addition, capture DNA/QDs ratios were modified under different conditions. Finally, under optimal conditions, the feasibility of the current nano-biosensor evaluated for monitoring SARS Cov-2 in real samples.

CRISPR-Cas Systems: Programmable Nuclease Revolutionizing the Molecular Diagnosis.

CRISPR-Cas system has evolved as a highly preferred genetic engineering tool to perform target gene manipulation via alteration of the guide RNA (gRNA) sequence. The ability to recognize and cleave a specific target with high precision has led to its applicability in multiple frontiers pertaining to human health and medicine. From basic research focused on understanding the molecular basis of disease to translational approach leading to early and precise disease diagnosis as well as developing effective therapeutics, the CRISPR-Cas system has proved to be a quite versatile tool. The coupling of CRISPR-Cas mediated cleavage with isothermal amplification (ISA) of target DNA, followed by a read-out using fluorescent or colorimetric reporters appears quite promising in providing a solution to the urgent need for nucleic acid-based point-of-care diagnostic. Hence, it has been recognized as a highly sophisticated molecular diagnostic tool for the detection of disease-specific biomarkers not limited to nucleic acids-based detection but also of non-nucleic acid targets such as proteins, exosomes, and other small molecules. In this review, we have presented salient features and principles of class 2 type II, V, and VI CRISPR-Cas systems represented by Cas9, Cas12, and Cas13 endonucleases which are frequently used in molecular diagnosis. The article then highlights different medical diagnostic applications of CRISPR-Cas systems focusing on the diagnosis of SARS-CoV-2, Dengue, Mycobacterium tuberculosis, and Listeria monocytogenes. Lastly, we discuss existing obstacles and potential future pathways concerning this subject in a concise manner.

Dual Signal Amplification by Urease Catalysis and Silver Nanoparticles for Ultrasensitive Colorimetric Detection of Nucleic Acids.

Signal amplification techniques are highly desirable for the analysis of low-level targets that are closely related with diseases and the monitoring of important biological processes. However, it is still challenging to achieve this goal in a facile and economical way. Herein, we developed a novel dual signal amplification strategy by combining urease catalysis with the release of Ag+ from silver nanoparticles (AgNPs). This strategy was used for quantifying a DNA sequence (HIV-1) related with human immunodeficiency virus (HIV). DNA target HIV-1 hybridizes with the capture DNA probe on magnetic beads and the reporter DNA probe on AgNPs, forming a sandwich complex. The captured AgNPs are then transformed into numerous Ag+ ions that inactivate numerous ureases. Without catalytic production of ammonia from urea, the substrate solution shows a low pH 5.8 that will increase otherwise. The pH change is monitored by a pH indicator (phenol red), which allows for colorimetric detection. The proposed approach is sensitive, easy to use, economic, and universal, exhibiting a low detection limit of 9.7 fM (i.e., 1.94 attomoles) and a dynamic linear range of 4 orders for HIV-1 sequence detection.