Complementary Target Sequences Research Articles

Profiling the eukaryotic soil microbiome with differential primers and an antifungal peptide nucleic acid probe (PNA): Implications for diversity assessment

The analysis of 18S rRNA gene amplicons is an important tool to characterize the diversity of the eukaryotic soil microbiome. Here we analyzed two primer sets (TAReuk, EKeuk) and the impact of a newly designed antifungal peptide nucleic acid to enhance the detection of protists in silico and with soil DNA extracted from croplands and a forest. The in silico analyses showed for TAReuk pronounced specificities for protist SAR supergroup and Metazoa, while EKeuk was particularly specific for Ascomycota and Basidiomycota. In silico, the PNA matched with the majority of Ascomycota (81.3 %) and Basidiomycota (65.4 %), but with <6 % of protists. The contrasting primer specificities were confirmed with soil DNA, but the proportion of protist amplicons was similar. In contrast to in silico, effects of the PNA were not as clear with soil DNA, even though it completely inhibited the amplification of the targeted fungal sequences. PNA effects were more pronounced with TAReuk, and results with cropland and forest soil DNA were not consistent, e.g., for cropland, PNA decreased the relative abundance of fungi but for forest it was the opposite, possibly because of different fungal diversity. The divergence between PNA in silico-predictions and results with soil DNA are likely an outcome of primer binding to <100 % complementary target sequences and a still limited DNA sequence databases for soil microbial eukaryotes. With TAReuk, the presence of PNA enhanced the detection of Conosa and, thus, could be a useful tool to study this group in the future.

MicroRNA-29-mediated cross-talk between metabolic organs in the pathogenesis of diabetes mellitus and its complications: A narrative review.

Diabetes mellitus (DM) is a heterogeneous group of disorders characterized by hyperglycemia. Microribonucleic acids (microRNAs) are noncoding RNA molecules synthesized in the nucleus, modified, and exported to the extracellular environment to bind to their complementary target sequences. It regulates protein synthesis in the targeted cells by inhibiting translation or triggering the degradation of the target messenger. MicroRNA-29 is one of noncoding RNA that can be secreted by adipose tissue, hepatocytes, islet cells, and brain cells. The expression level of the microRNA-29 family in several metabolic organs is regulated by body weight, blood concentrations of inflammatory mediators, serum glucose levels, and smoking habits. Several experimental studies have demonstrated the effect of microRNA-29 on the expression of target genes involved in glucose metabolism, insulin synthesis and secretion, islet cell survival, and proliferation. These findings shed new light on the role of microRNA-29 in the pathogenesis of diabetes and its complications, which plays a vital role in developing appropriate therapies. Different molecular pathways have been proposed to explain how microRNA-29 promotes the development of diabetes and its complications. However, to the best of our knowledge, no published review article has summarized the molecular mechanism of microRNA-29-mediated initiation of DM and its complications. Therefore, this narrative review aims to summarize the role of microRNA-29-mediated cross-talk between metabolic organs in the pathogenesis of diabetes and its complications.

A new method for the robust expression and single-step purification of dCas9 for CRISPR interference/activation (CRISPRi/a) applications

CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated enzyme 9) is known for its simplicity, versatility, and scalability in genome editing applications. In vitro Cas9, when complexed with sgRNA, binds and cleaves the complementary target sequences with almost perfect precision. The enzyme is exploited for various applications in understanding and changing gene function. dCas9 (deactivated or dead Cas9) is a double mutated version of Cas9 that bears mutations in the nuclease domains of the enzyme and thus cannot cleave the target DNA. dCas9 is equally advantageous since it can alter gene expression using various transcriptional activators CRISPRa and repressors CRISPRi. Additionally, dCas9 can bind to the desired target gene without cleaving it, making it a unique reagent to study the kinetics and stability of RNA-protein-DNA interactions required to design more efficient and specific gene-editing nucleases. An appreciable quantity of pure and homogeneous protein is needed to characterise dCas9 for its structural and functional understanding. This study used an N-terminal acidic tag to express the dCas9 in an E. coli-bacterial host. A simple single-step protocol for robust and efficient production of dCas9 has been described. The study and methods are distinctive as the purification is performed in a single step using inexpensive multi-modal hydroxyapatite chromatography. The purified protein can be used in different in vitro and in vivo studies.

Polydopamine functionalized dendritic fibrous silica nanoparticles as a generic platform for nucleic acid-based biosensing

Accurate and rapid detection of nucleic acid sequences is of utmost importance in various fields, including disease monitoring, clinical treatment, gene analysis and drug discovery. In this study, we developed a "turn-on" fluorescence biosensor that enables simple and highly efficient detection of nucleic acid biomarkers. Our approach involves the utilization of 6-carboxyfluorescein modified single-stranded DNA (FAM-ssDNA) as molecular recognition element, along with polydopamine-functionalized dendritic fibrous nanosilica (DFNS). FAM-ssDNA serves as both specific molecular recognition element for the target analyte and reporter capable of transducing a detectable signal through Watson–Crick base pairing. The polydopamine-functionalized DFNS (DFNS@DA) exhibits strong binding to FAM-ssDNA via polyvalent metal mediated coordination leading to effective quenching by fluorescence resonance energy transfer. In the presence of a complementary target sequence, FAM-ssDNA forms hybridized structure and detaches from DFNS@DA, which causes an increased fluorescence emission. The analytical system based on FAM-ssDNA and DFNS@DA demonstrates exceptional sensitivity, selectivity, and rapid response for the detection of nucleic acid sequences, leveraging the high adsorption and quenching properties of DFNS@DA. For the first proof of concept, we demonstrated the successful detection of microRNA (miR-21) in cancer cells using the FAM-ssDNA/DFNS@DA system. Our results highlight the promising capabilities of DFNS@DA and nucleic acid-based biosensors, offering a generic and cost-effective solution for the detection of nucleic acid-related biomarkers.Graphical abstract

DNA-Targeted Complexes of Tc and Re for Biomedical Applications.

Due to their favorable chemical features, Re and Tc complexes have been widely used for the development of new therapeutic agents and imaging probes to solve problems of biomedical relevance. This review provides an update of the most relevant research efforts towards the development of novel cancer theranostic agents using Re and Tc-based compounds interacting with specific DNA structures. This includes a variety of homometallic complexes, namely those containing M(CO)3 (M=Re, Tc) moieties, that exhibit different modes of interaction with DNA, such as covalent binding, intercalation, groove binding or G-quadruplex DNA binding. Additionally, heterometallic complexes, designed to potentiate synergistic effects of different metal centers to improve DNA-targeting, cytotoxicity and fluorescence properties, are also reviewed. Particular attention is also given to 99m Tc- and 188 Re-labeled oligonucleotides that have been widely explored to develop imaging and therapeutic radiopharmaceuticals through the in vivo hybridization with a specific complementary DNA or RNA target sequence to provide useful molecular tools in precision medicine for cancer diagnosis and treatment. Finally, the need for further improvement of DNA-targeted Re and Tc-based compounds as potential therapeutic and diagnostic agents is highlighted, and future directions are discussed.

A label-free sensor based on a carbon nanotube-graphene platform for the detection of non-Hodgkin lymphoma genes

Early diagnosis of non-Hodgkin lymphoma (NHL) is crucial for effective management. We report an ultrasensitive electrochemical genosensor using multi-walled carbon nanotube (MWCNT)-graphene nanocomposite for detection of lymphoma biomarker gene. MWCNTs were dispersed over graphene nanoplatelets to form a conductive nanocomposite platform. The high surface area and π-π stacking ability enabled efficient immobilization of lymphoma probe DNA. Detailed materials characterization was performed using microscopy and spectroscopy techniques. The sensor was fabricated by drop-casting probe-conjugated nanocomposite suspension onto a glassy carbon electrode. The genosensor exhibited a wide dynamic range of 1 fM to 1 nM with a sensitivity of 0.895 kΩ per decade of target concentration. The sensor showed excellent selectivity for fully complementary target sequence over even single base mismatch. Hybridization detection by electrochemical impedance spectroscopy was further validated using differential pulse voltammetry which showed drastic reduction in redox peak current after incubating with target. The nanocomposite genosensor demonstrated good reproducibility with relative standard deviation of 3.72 % among five independently prepared electrodes. Clinical applicability was tested by spiking lymphoma gene biomarker in 10 % serum and recoveries of 93–99 % were obtained. The proposed MWCNT-graphene nanocomposite genosensor provides a simple, specific and ultrasensitive platform for point-of-care diagnosis of NHL.

Beyond Loading: Functions of Plant ARGONAUTE Proteins.

ARGONAUTE (AGO) proteins are key components of the RNA-induced silencing complex (RISC) that mediates gene silencing in eukaryotes. Small-RNA (sRNA) cargoes are selectively loaded into different members of the AGO protein family and then target complementary sequences to in-duce transcriptional repression, mRNA cleavage, or translation inhibition. Previous reviews have mainly focused on the traditional roles of AGOs in specific biological processes or on the molecular mechanisms of sRNA sorting. In this review, we summarize the biological significance of canonical sRNA loading, including the balance among distinct sRNA pathways, cross-regulation of different RISC activities during plant development and defense, and, especially, the emerging roles of AGOs in sRNA movement. We also discuss recent advances in novel non-canonical functions of plant AGOs. Perspectives for future functional studies of this evolutionarily conserved eukaryotic protein family will facilitate a more comprehensive understanding of the multi-faceted AGO proteins.

The Evolution of Meganucleases Genome Engineering Tools: an overview

Human beings today have been able to pursue goals with non-random changes in the genome of living organisms. This is the process of genetic engineering or genome editing, which involves replacing, inserting, or deleting an arbitrary genomic sequence using an artificial restriction enzyme capable of cleaving a specific part of the genome. The ability to manipulate and modify genes and functional studies as well as awareness of the molecular basis of diseases and the development of new and targeted therapies with these techniques have been created. To date, different techniques have now been developed with the ability to edit targeted genomics including Zinc-finger nucleases (ZFNs), Transcription activator-like effector nucleases (TALEN) and Clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR associated protein 9 (Cas9) (CRISPR/Cas9). The fusing of a zinc-finger DNA-binding domain to a DNA-cleavage domain produces artificial restriction enzymes known as Zinc-finger nucleases (ZFNs). Zinc finger domains can be designed to target a specific DNA sequence which causes ZFNs to target unique sequences in complex genomes. TALENs are made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain to cut DNA strands at specific locations. CRISPR/Cas9 is a bacterial immune system against viruses in which the Cas9 nucleus connects with a single-strand guide to a complementary target sequence and makes changes. In this study, we will review the evolution and various aspects of each one over time.

Graphene-Oxide and Ionic Liquid Modified Electrodes for Electrochemical Sensing of Breast Cancer 1 Gene.

Graphene-oxide and ionic liquid composite-modified pencil graphite electrodes (GO-IL-PGEs) were developed and used as a sensing platform for breast cancer 1 (BRCA1) gene detection. The characterization of GO-IL modified electrodes was executed by scanning electron microscopy (SEM), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). The nucleic-acid hybridization was monitored by a differential pulse voltammetry (DPV) technique by directly measuring the guanine oxidation signal without using any indicator. The effects of the IL concentration, the probe concentration, and the hybridization time were optimized to the biosensor response. The limit of detection (LOD) was calculated in the concentration range of 2–10 μg/mL for the BRCA1 gene and found to be 1.48 µg/mL. The sensitivity of the sensor was calculated as 1.49 µA mL/µg cm2. The developed biosensor can effectively discriminate the complementary target sequence in comparison to a three-base-mismatched sequence or the non-complementary one.

ZIKV Infection and miRNA Network in Pathogenesis and Immune Response.

Over the years, viral infections have caused severe illness in humans. Zika Virus (ZIKV) is a flavivirus transmitted by mosquito vectors that leads to notable neurological impairment, whose most dramatic impact is the Congenital ZIKV Syndrome (CZS). ZIKV targets neuronal precursor cells leading to apoptosis and further impairment of neuronal development, causing microcephaly, lissencephaly, ventriculomegaly, and calcifications. Several regulators of biological processes are involved in CZS development, and in this context, microRNAs (miRNAs) seem to have a fundamental role. miRNAs are important regulators of protein translation, as they form the RISC silencing complex and interact with complementary mRNA target sequences to further post-transcriptional repression. In this context, little is known about their participation in the pathogenesis of viral infections. In this review, we discuss how miRNAs could relate to ZIKV and other flavivirus infections.

Current widely-used web-based tools for CRISPR nucleases, base editors, and prime editors

CRISPR-Cas nucleases, base editors (BEs), and prime editors (PEs) are efficient genome editing tools used widely in diverse research fields, including biology, biotechnology, and medicine. While the genome editing mechanism is different for each tool, the target specificity is conferred in common by binding of guide RNAs (gRNAs) and their complementary target sequences. However, gRNAs can bind to off-target sequences with a few mismatches, provoking off-target editing, and the editing activities/outcomes vary depending on the gRNAs. Therefore, selection of gRNAs as well as analysis of the outcomes is crucial to improve the editing strategies. In this review, we introduce various programs currently used in selection of gRNAs and analysis of results for each genome editing tool and briefly describe the purpose and features of each program, which will be informative to researchers when planning genome editing.

Self-assembled miRNA-switch nanoparticles target denuded regions and prevent restenosis

Cardiovascular disease is the leading cause of death and disability worldwide. Effective delivery of cell-selective therapies that target atherosclerotic plaques and neointimal growth while sparing the endothelium remains the Achilles heel of percutaneous interventions. The current study utilizes synthetic microRNA switch therapy that self-assembles to form a compacted, nuclease-resistant nanoparticle <200nM in size when mixed with cationic amphipathic cell-penetrating peptide (p5RHH). These nanoparticles possess intrinsic endosomolytic activity that requires endosomal acidification. When administered in a femoral artery wire injury mouse model invivo, the mRNA-p5RHH nanoparticles deliver their payload specifically to the regions of endothelial denudation and not to the lungs, liver, kidney, or spleen. Moreover, repeated administration of nanoparticles containing a microRNA switch, consisting of synthetically modified mRNA encoding for the cyclin-dependent kinase inhibitor p27Kip1 that contains one complementary target sequence of the endothelial cell-specific miR-126 at its 5' UTR, drastically reduced neointima formation after wire injury and allowed for vessel reendothelialization. This cell-selective nanotherapy is a valuable tool that has the potential to advance the fight against neointimal hyperplasia and atherosclerosis.

Response surface methodology optimized electrochemical DNA biosensor based on HAPNPTs/PPY/MWCNTs nanocomposite for detecting Mycobacterium tuberculosis

An important issue in the prognosis of tuberculosis (TB) is a short period between correct diagnosis and start the suitable antibiotic therapy. So, a rapid and valid method for detection of Mycobacterium tuberculosis (M. tb) complex is considered as a necessity. Herein, a rapid, low-cost, and PCR-free DNA biosensor was developed based on multi-walled carbon nanotubes (MWCNTs), polypyrrole (PPy), and hydroxyapatite nanoparticles (HAPNPs) for highly sensitive and specific recognition of M.tb. The biosensor consisted of M.tb ssDNA probe covalently attached to the HANPs/PPy/MWCNTs/GCE surface that hybridized to a complementary target sequence to form a duplex DNA. The M.tb target recognition was based on the oxidation signal of the electroactive Methylene Blue (MB) on the surface of the modified GCE using differential pulse voltammetry (DPV) method. It is worth to mention that for the first time Plackett-Burman (PB) screening design and response surface method (RSM) based on central composite design (CCD) was applied as a powerful and an efficient approach to find optimal conditions for maximum M.tb biosensor performance leading to simplicity and rapidity of operation. The proposed DNA biosensor exhibits a wide detection range from 0.25 to 200.0 nM with a low detection limit of 0.141 nM. The performance of designed biosensor for clinical diagnosis and practical applications was revealed through hybridization between DNA probe-modified GCE and extracted DNA from sputum clinical samples.

Will CRISPR-Cas9 Have Cards to Play Against Cancer? An Update on its Applications.

Genome editing employs targeted nucleases as powerful tools to precisely alter the genome of target cells and regulate functional genes. Various strategies have been risen so far as the molecular scissors-mediated genome editing that includes zinc finger nuclease, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats—CRISPR-related protein 9. These tools allow researchers to understand the basics of manipulating the genome, create animal models to study human diseases, understand host–pathogen interactions and design disease targets. Targeted genome modification utilizing RNA-guided nucleases are of recent curiosity, as it is a fast and effective strategy that enables the researchers to manipulate the gene of interest, carry out functional studies, understand the molecular basis of the disease and design targeted therapies. CRISPR-Cas9, a bacterial defense system employed against viruses, consists of a single-strand RNA-guided Cas9 nuclease connected to the corresponding complementary target sequence. This powerful and versatile tool has gained tremendous attention among the researchers, owing to its ability to correct genetic disorders. To help illustrate the potential of this gene editor in unexplored corners of oncology, we describe the history of CRISPR-Cas9, its rapid progression in cancer research as well as future perspectives.

The role of miRNA in kidney development

MicroRNAs (miRNAs) are endogenous small non-coding RNAs (19-25 nucleotides) that negatively regulate gene expression at the post transcriptional level by binding to complementary target sequences in the target mRNA. miRNAs play an important role in a wide range of biological processes, including organ development. Recent studies have shown that some miRNAs are highly expressed in the kidney and are closely related to kidney development and diseases, suggesting that miRNAs are important regulators in kidney physiology and pathology. This review will focus on the research progress of miRNA in kidney development, and discuss the role of miRNAs in the occurrence and development of renal dysplasia, which will provide a reference for the diagnosis and research of diseases related to kidney development.

Establishing a Field-Effect Transistor Sensor for the Detection of Mutations in the Tumour Protein 53 Gene (TP53)-An Electrochemical Optimisation Approach.

We present a low-cost, sensitive and specific DNA field-effect transistor sensor for the rapid detection of a common mutation to the tumour protein 53 gene (TP53). The sensor consists of a commercially available, low-cost, field-effect transistor attached in series to a gold electrode sensing pad for DNA hybridisation. The sensor has been predominantly optimised electrochemically, particularly with respect to open-circuit potentiometry as a route towards understanding potential (voltage) changes upon DNA hybridisation using a transistor. The developed sensor responds sensitively to TP53 mutant DNA as low as 100 nM concentration. The sensor responds linearly as a function of DNA target concentration and is able to differentiate between complementary and noncomplementary DNA target sequences.

Argonaute bypasses cellular obstacles without hindrance during target search

Argonaute (Ago) proteins are key players in both gene regulation (eukaryotes) and host defense (prokaryotes). Acting on single-stranded nucleic-acid substrates, Ago relies on base pairing between a small nucleic-acid guide and its complementary target sequences for specificity. To efficiently scan nucleic-acid chains for targets, Ago diffuses laterally along the substrate and must bypass secondary structures as well as protein barriers. Using single-molecule FRET in conjunction with kinetic modelling, we reveal that target scanning is mediated through loose protein-nucleic acid interactions, allowing Ago to slide short distances over secondary structures, as well as to bypass protein barriers via intersegmental transfer. Our combined single-molecule experiment and kinetic modelling approach may serve as a platform to dissect search processes and study the effect of sequence on search kinetics for other nucleic acid-guided proteins.

Novel electrochemical genosensor for Zika virus based on a poly-(3-amino-4-hydroxybenzoic acid)-modified pencil carbon graphite electrode

The Zika virus has recently emerged as a major worldwide threat because it induces microencephaly and other brain damage in babies born to infected mothers. Epidemiological monitoring of infection has been hampered by the lack of reliable tests capable of distinguishing between Zika and other Flavivirus infections, especially the dengue virus. The present work describes the electrochemical modification of pencil carbon graphite electrodes with a material derived from 3-amino-4-hydroxybenzoic acid. Solutions were prepared in acid medium and the material formed showed insulating behavior. Optical and electrical data suggested that carboxyl groups did not participate in the reaction mechanism. FT-IR spectra indicated that these groups were retained, enabling the proposal of a mechanism based on the data obtained. The material formed was used for immobilization of the ssDNAzv aminated oligonucleotide. Detection of the hybridization was based on the Δip resulting from the higher electrostatic repulsion of [Fe(CN)6]3−, caused by the increase of phosphates by addition of the complementary target sequence. The system showed a good linear correlation coefficient (r² = 0.997), with satisfactory selectivity and reproducibility, a low limit of detection (25.4 pM), and precision (RSD) of 3.1%, using real samples of human serum enriched with total extract of Zika virus RNA. Tests of the system using the total RNA extracted from dengue virus types 2 and 3 cultures showed little difference in the observed current, hence confirming the specificity of the system. These findings demonstrated the potential of the proposed biosensor.

Cetyltrimethyl ammonium bromide modified gold nanostructure supported by chitosan as a novel scaffold for immobilization of DNA and ultra-sensitive bioassay of Legionella pneumophila

Legionella pneumophila is a causative agent of Pontiac fever and Legionaries' disease and its identification. Also L. pnumophila is one of the threatening pathogens that has many challenges for its rapid and specific detection. The present study is aimed to designing an appropriate DNA based bioassay to identification of L. pneumophila. To achieve ideal biosensor, gold nanoparticles supported by chitosan and cetyltrimethyl ammonium bromide (CTAB) was electrodeposited on the surface of gold electrode. Cyclic voltammetry technique was used to coating gold nanostructure. Also Field emission scanning electron microscope (FE-SEM) and energy dispersive spectroscopy (EDX) were applied for morphological and elementary analysis investigation. The prepared substrate was used for immobilization of pDNA and hybridization by complementary target sequence (cDNA). Therefore, an innovative method to the sensitive detection of target DNA based was proposed. Used probe sequence was. Obtained results demonstrated that in the optimum conditions, linear range was 1 μM to 1 fM and low limit of quantification (LLOQ) was 1 f-molar. Engineered DNA biosensor not only shows good sensitivity and selectivity but also displays rapid performance in detection of L. pneumophila. Findings of this study indicate that for designing an ideal biosensor for this pathogen, we can use the wide range of nanomaterials to increase the sensitivity and accuracy of the future biosensors.

An innovative nucleic acid based biosensor toward detection of Legionella pneumophila using DNA immobilization and hybridization: A novel genosensor

Legionella pneumophila is a causative agent of Pontiac fever and Legionaries' disease. L. pneumophila is a gram-negative, fastidious and hard to grow bacterium. Since severe growth of this pathogen, molecular diagnostics based on DNA can be very important. Mip gene is one of the strongest constructed in Legionella pneumophila that targeted in several molecular detection methods. Also, L. pneumophila is one of the threatening pathogens that has many challenges for its rapid and specific detection. This study is an attempt to identify Legionella pneumophila specific genes using a DNA-based bioassay. In this work, gold nanostructures supported by cysteamine (Cys A/AuNPs) were deposited on the surface of gold electrode to engineer an innovative DNA based biosensor. The Cys A/AuNPs interface provides a large surface area for the effective immobilization of ssDNA, as well as it ascertains the bioactivity and stability of immobilized of pDNA. Field emission scanning electron microscope (FE-SEM), and EDS photoelectron spectroscopies were used to monitor the sensor fabrication. Cyclic voltammetry (CV) and square wave voltammetry (SWV) were used for electrochemical characterization of DNA immobilization and hybridization by cDNA as complementary target sequence. Obtained results showed good sensitivity, selectivity and stability. Finally, target DNA was quantified at a linear range from 1 μM to 1 ZM and low limit of quantification (LLOQ) was 1 Zepto-molar. The results of this study indicate that nucleic acid based bioassay in the near future can be a good alternative to the detection of L. pneumophila.