Single-stranded Nucleotides Research Articles

An overview of aptamer: Design strategy, prominent applications, and potential challenge in plants

Aptamers, serving as highly efficient molecular recognition and biotechnology tools, have garnered increasing interest in the realm of plant science in recent years. Aptamers are synthetic single-stranded short nucleotides or peptides, that bind targets with high specificity and affinity, triggering precise biological responses. As an alternative to antibodies, aptamers present promising avenues for advancement in biological researches. Aptamers function in a range of fields, encompassing cell signaling, drug development, biosensor technology, as well as botany, agricultural and forestry sciences. In this review, we introduce classifications and screening methods of aptamers, as well as aptamer-based technologies, highlighting their significant contributions to recent advancements. With their powerful functionality and ability to bind targets with high specificity and affinity, aptamers offer promising opportunities for breakthroughs in plant research.

SOD2 in platelets: with age comes responsibility

SOD2 in platelets: with age comes responsibility

Nucleotide Sequence Assessment of Four ORFs of Citrus Tristeza Virus: Evidence of Recombination

Citrus Tristeza Virus (CTV), usually occurs in nature as a mixture of genotypes. Six naturally infected citrus (Citrus sinensis) trees grafted on sour orange rootstock were collected from three citrus growing governorates in Egypt (Sharqia, Qalyubia and Garbia). In this study, RT-PCR, Single-Strand Conformation Polymorphism (SSCP) and nucleotide sequence analysis were used for four independent CTV genomic regions (p65, p18, p20, and p23) to detect and assess the sequence and genetic variabilities among CTV Egyptian isolates. RTPCR products (650 bp) for the CTV p23 gene obtained from the selected isolates were used for the SSCP analysis and DNA sequencing. SSCP patterns of p23 gene for individual isolates yielded different complex haplotype patterns. Nucleotide sequence analysis of p23 region amplified from six isolates under study revealed that p23 shared high nucleotide identity 98.7% with T36 isolate from USA, Florida. Phylogenetic analysis of p23 gene indicated a close evolutionary relationship between all examined isolates and Qaha isolate (T36 isolate group), suggesting that they may have originated from closely related ancestors. Nucleotide sequence analysis of the three genes located on CTV 3′-coterminal overhang, p18, p20 and p65, amplified from isolate A3, Sharqia governorate, revealed that the p18, p65, and p20 genes were related to the T3-KB isolate from South Africa with 99%–100% sequence homology. Phylogenetic relationship analysis for p65, p18 and p20 ORFs clustered the current A3 isolate with T3 genotype group. The recombination analysis identified three of six isolates from Sharqia, and Garbia as potential recombinant for p23 gene. The isolates T36 and T3 were identified as major donors for recombination events in isolate A3. Our results concluded that p23 ORF likely to be as a hotspot region for recombination and originated through recombination event. The current study indicated that recombination is an important factor for the origin of CTV strains in Egypt.

Determination of RNA Structure with In Vitro SHAPE Experiments.

The structure of an RNA molecule is often critical for its correct functioning, post-transcriptional regulation, and/or translation. Predicting RNA secondary structures with in silico tools is relatively straightforward with the large array of software and webservers available. However, for long RNAs and RNA at high temperatures, in silico predictions are less accurate and require experimental validation. To this end, a variety of structural probing reagents are commonly used, both for in vitro and in vivo mapping of RNA structure. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) experiments make use of a nonbase-specific modifying reagent, acylating all conformationally flexible (mainly single-stranded or unpaired) nucleotides and have been employed both for in vitro and in vivo modification of RNA. Here, we describe a protocol for an in vitro SHAPE experiment, starting from in vitro transcribed RNA using a T7 polymerase system. This RNA is folded and subsequently modified in vitro with the SHAPE-reagent N-methyl isatoic anhydride (NMIA). Primer extension employing a radioactive 32P-labeled primer that binds the RNA downstream of the structure of interest will generate cDNA until the reverse transcriptase enzyme is halted by the introduced SHAPE modifications. Denaturing acrylamide gel electrophoresis of the pool of 32P-labeled cDNAs and the corresponding sequencing ladders, followed by autoradiography, will expose these stops in reverse transcription (RT) and will therefore enable to identify single-stranded nucleotides in the RNA of interest. These RT stops and NMIA-modification efficiencies can be quantified with ImageJ software and can be used to validate or increase the accuracy of RNA secondary structure predictions.

DNA Aptamers Block the Receptor Binding Domain at the Spike Protein of SARS-CoV-2.

DNA aptamers are versatile molecular species obtained by the folding of short single-stranded nucleotide sequences, with highly specific recognition capabilities against proteins. Here we test the ability of DNA aptamers to interact with the spike (S-)protein of the SARS-CoV-2 viral capsid. The S-protein, a trimer made up of several subdomains, develops the crucial function of recognizing the ACE2 receptors on the surface of human cells, and subsequent fusioning of the virus membrane with the host cell membrane. In order to achieve this, the S1 domain of one protomer switches between a closed conformation, in which the binding site is inaccessible to the cell receptors, and an open conformation, in which ACE2 can bind, thereby initiating the entry process of the viral genetic material in the host cell. Here we show, by means of state-of-the-art molecular simulations, that small DNA aptamers experimentally identified can recognize the S-protein of SARS-CoV-2, and characterize the details of the binding process. We find that their interaction with different subdomains of the S-protein can effectively block, or at least considerably slow down the opening process of the S1 domain, thereby significantly reducing the probability of virus-cell binding. We provide evidence that, as a consequence, binding of the human ACE2 receptor may be crucially affected under such conditions. Given the facility and low cost of fabrication of specific aptamers, the present findings could open the way to both an innovative viral screening technique with sub-nanomolar sensitivity, and to an effective and low impact curative strategy.

Integrated analysis of miRNA-mRNA interaction in pediatric dilated cardiomyopathy.

BackgroundmicroRNAs (miRNAs) are short single stranded nucleotides that can regulate gene expression. Although we previously evaluated expression of miRNAs in pediatric dilated cardiomyopathy (DCM) by miRNA array, pathway prediction based on changes in mRNA expression has not been previous analyzed in this population. The current study aimed to determine regulation of miRNA expression by miRNA-Seq and, through mRNA-Seq, analyze their putative target genes and altered pathways in pediatric DCM hearts.MethodsmiRNA expression was determined by miRNA-Seq [n=10 non-failing (NF), n=20 DCM]. Expression of a subset of miRNAs was evaluated in adult DCM patients (n=11 NF, n=13 DCM). miRNA-mRNA prediction analysis was performed using mRNA-Seq data (n=7 NF, n=7 DCM) from matched samples.ResultsExpression of 393 miRNAs was significantly different (p<0.05) in pediatric DCM patients compared to NF controls. TargetScan-based miRNA-mRNA analysis revealed 808 significantly inversely expressed genes. Functional analysis suggests up-regulated pathways related regulation of stem cell differentiation and cardiac muscle contraction, and down-regulated pathways related to regulation of protein phosphorylation, signal transduction and cell communication.ConclusionOur results demonstrated a unique age-dependent regulation of miRNAs and their putative target genes which may contribute to distinctive phenotypic characteristics of DCM in children.

Recent Trends in Design and Development of Nanomaterial-based Aptasensors

The past decade has witnessed extensive research in the field of biosensors, where nanoaptasensors achieved widespread interest. Aptamer, the single-stranded DNA or RNA nucleotide, is extensively employed as a bioreceptor due to its stability, ease in a modification that ensures convenient immobilization strategies, reducing the cost of sensor manufacturing. The detection limit and sensitivity were notably improved when modified with nanomaterials such as metal nanoparticles, carbon nanomaterials, graphene, quantum dots, and other nanocomposites. This paper introduces various design strategies for the fabrication of sensors utilizing aptamers and nanomaterials in ¬developing signal-readout mechanism, focusing mainly on the latest research in food, biomedical and environmental applications. Aptasensors that utilize various signal recognition methods such as electrochemistry, colorimetry, luminescence, and fluorescence are highlighted. This review offers a wide range of outlook for future developments of aptamer bioreceptors employing the unique physicochemical nanomaterials as efficient transducers and amplifiers. Furthermore, this review will also provide an insight into aptamer-aided biosensors' technology over the last three to five years of work.

Systematic analysis of factors that improve homologous direct repair (HDR) efficiency in CRISPR/Cas9 technique.

The CRISPR/Cas9 bacterial system has proven to be an powerful tool for genetic manipulation in several organisms, but the efficiency of sequence replacement by homologous direct repair (HDR) is substantially lower than random indel creation. Many studies focused on improving HDR efficiency using double sgRNA, cell synchronization cycle, and the delivery of single-stranded oligo DNA nucleotides (ssODN) with a rational design. In this study, we evaluate these three methods’ synergistic effects to improve HDR efficiency. For our tests, we have chosen the TNFα gene (NM_000594) for its crucial role in various biological processes and diseases. For the first time, our results showed how the use of two sgRNA with asymmetric donor design and triple transfection events dramatically increase the HDR efficiency from an undetectable HDR event to 39% of HDR efficiency and provide a new strategy to facilitate CRISPR/Cas9-mediated human genome editing. Besides, we demonstrated that the TNFα locus could be edited with CRISPR/Cas9 methodology, an opportunity to safely correct, in the future, the specific mutations of each patient.

The Possibility of Nucleic Acids to Act as Anti-Viral Therapeutic Agents—A Review

For just about 30 years, researchers have considered the likelihood to utilize nucleic acids as antiviral therapeutics. In principle, small single-stranded nucleotide sequence (oligonucleotide) could hybridize to a particular gene or messenger RNA and diminish transcription or translation, respectively, in this manner decreasing the amount of protein that is synthesized. Until now, an incredible number of antisense oligonucleotides, double-stranded oligonucleotides, aptamers, ribozymes, deoxyribozymes, interfering RNAs, chimeric RNA-DNA molecules, antibody genes has been created artificially and applied effectively for comprehension and manipulating biological processes and in clinical preliminaries to treat a variety of diseases. Their versatility and potency make them similarly fit candidates for fighting viral infections. However, troubles with their efficiency, off-target effects, toxicity, delivery, and stability halted the development of nucleic acid-based therapeutics that can be utilized in the clinic. The potential for nucleic acid therapeutic agents is significant and is quite recently beginning to be realized. In this review, we have summarized some of the recent advancements made in the area of nucleic acid based therapeutics and focused on the methods of their delivery and associated challenges.

CRISPR/Cas9-mediated correction of mutated copper transporter ATP7B.

Wilson's disease (WD) is a monogenetic liver disease that is based on a mutation of the ATP7B gene and leads to a functional deterioration in copper (Cu) excretion in the liver. The excess Cu accumulates in various organs such as the liver and brain. WD patients show clinical heterogeneity, which can range from acute or chronic liver failure to neurological symptoms. The course of the disease can be improved by a life-long treatment with zinc or chelators such as D-penicillamine in a majority of patients, but serious side effects have been observed in a significant portion of patients, e.g. neurological deterioration and nephrotoxicity, so that a liver transplant would be inevitable. An alternative therapy option would be the genetic correction of the ATP7B gene. The novel gene therapy method CRISPR/Cas9, which has recently been used in the clinic, may represent a suitable therapeutic opportunity. In this study, we first initiated an artificial ATP7B point mutation in a human cell line using CRISPR/Cas9 gene editing, and corrected this mutation by the additional use of single-stranded oligo DNA nucleotides (ssODNs), simulating a gene correction of a WD point mutation in vitro. By the addition of 0.5 mM of Cu three days after lipofection, a high yield of CRISPR/Cas9-mediated ATP7B repaired cell clones was achieved (60%). Moreover, the repair efficiency was enhanced using ssODNs that incorporated three blocking mutations. The repaired cell clones showed a high resistance to Cu after exposure to increasing Cu concentrations. Our findings indicate that CRISPR/Cas9-mediated correction of ATP7B point mutations is feasible and may have the potential to be transferred to the clinic.

Label-Free Electrochemical Detection of DNA Hybridization: A Method for COVID-19 Diagnosis.

This paper presents label-free electrochemical transduction as a suitable scheme for COVID-19-specific viral RNA/c-DNA detection, with an aim to facilitate point of care diagnosis. In lieu of this, we discuss the proposed electrochemical biosensing scheme, based on electrodeposited gold nanoparticles as the transducing elements. Specific to this approach, here, the protocols associated with the immobilization of the single-stranded probe nucleotide on to the biosensor, have also been laid out. This paper also discusses the methods of electrochemical analysis, to be used for data acquisition and subsequent calibration, in relation to target analyte detection. Towards facilitating portable diagnosis, development of miniaturized sensors and their integration with readout units have also been discussed.

Development of a Novel Lateral Flow Biosensor Combined With Aptamer-Based Isolation: Application for Rapid Detection of Grouper Nervous Necrosis Virus.

Nervous necrosis virus (NNV) has infected more than 50 fish species worldwide, and has caused serious economic losses in the aquaculture industries. However, there is no effective antiviral therapy. The development of a rapid and accurate point-of-care diagnostic method for the prevention and control of NNV infection is urgently required. Commonly used methods for NNV detection include the cell culture-based assay, antibody-based assay and polymerase chain reaction (PCR)-based assay. However, these methods have disadvantages as they are time-consuming and complex. In the present study, we developed a simple and sensitive aptamer-based lateral flow biosensor (LFB) method for the rapid detection of red-spotted grouper nervous necrosis virus (RGNNV). An aptamer is a single-stranded nucleotide, which can specifically bind to the target and has many advantages. Based on a previously selected aptamer, which specifically bound to the coat protein of RGNNV (RGNNV-CP), two modified aptamers were used in this study. One aptamer was used for magnetic bead enrichment and the other was used for isothermal strand displacement amplification (SDA). After amplification, the product was further tested by the LFB, and the detection results were observed by the naked eye within 5 min with high specificity and sensitivity. The LFB method could detect RGNNV-CP protein as low as 5 ng/mL or 5 × 103 RGNNV-infected GB (grouper brain) cells. Overall, it is the first application of a LFB combined with aptamer in the rapid diagnosis of virus from aquatic animals, which provides a new option for virus detection in aquaculture.

Quantitative detection of neurotransmitter using aptamer: From diagnosis to therapeutics

Neurotransmitters, the small molecule chemical messenger responsible for nervous system regulation and can control joy, fear, depression, insomnia, craving for carbohydrates, drugs, and alcohols. Variation in neurotransmitter levels is a characteristic manifestation of several neurological diseases. Accurate diagnosis of these diseases caused due to an imbalance in neurotransmitter level followed by impaired transmission of signals between neurons and other body parts remains a great challenge for the clinicians. Recent evidences reveal, artificial single-stranded nucleotides called 'aptamer' are widely used as biosensors, antibody substitutes, diagnostic agents, and for targeted therapy. These aptamers are superior candidate both for early detection and diagnosis of many neurological disorders caused due to suboptimal level of neurotransmitters. Presently, noninvasive neurotransmitter detection by aptamer has been found to be an easy, fast, and cost-effective choice. In addition, increased specificity, stability, affinity, and reproducibility of aptamers, high throughput screening of aptamer-based sensing platforms have been observed. Moreover, clinical applicability of aptamer has also proved to be efficacious, though still at a preliminary stage. Herein, we review salient features of aptamerbased sensing technology used for neurotransmitter detection particularly their chemical modifications, selection, assay development, immobilization, therapeutic efficiency, and stability for early diagnosis of diseases caused due to neurotransmitter imbalance.

DNA repair gene expression is associated with differential prognosis between HPV16 and HPV18 positive cervical cancer patients following radiation therapy

Cervical cancers are almost always induced by HPV infections, of which HPV16 and HPV18 are predominant. Cancers associated with these strains are induced through DNA repair factors and have a differential response to radiation therapy. Hence this study focuses on finding DNA repair gene expression differences in HPV16 and HPV18 positive cervical cancers after radiation therapy. A higher number of somatic mutations were observed in HPV16 positive cervical tumours for patients that were disease free when compared to those who recurred/progressed. Moreover, hierarchal clustering of RNAseq data from The Cancer Genome Atlas was conducted to identify groups of DNA repair genes associated with a differential prognosis for cervical cancer following postoperative radiation therapy. TP53BP1, MCM9 (at higher than mean levels), POLR2F and SIRT6 (at lower than mean levels), were associated with an increase in patients experiencing cervical cancer recurrence/progression following postoperative radiation therapy when HPV18 positive, but not HPV16 positive. The expression patterns of these genes provide an explanation for the higher rate of postoperative radiation therapy resistance associated with HPV18 positive cervical cancer patients. Therefore, HPV18 positive cervical tumours may be more likely retain a greater non-homologous end joining and homologous recombination pathway activity, which could dampen the effect of postoperative radiation therapy. Moreover, greater susceptibility to postoperative radiation therapy could be caused by the reliance of cervical cancer cells upon the single-strand annealing and nucleotide excision pathways for repair of DNA damage.

Structural Basis of 7SK RNA 5′-Gamma-Phosphate Methylation and Retention by MePCE

7SK non-coding RNA is an essential regulator of eukaryotic transcription elongation through dynamic RNP assemblies that sequester or release the positive transcription elongation factor b (P-TEFb). All 7SK RNPs include a core comprising 7SK RNA, methylphosphate capping enzyme (MePCE), and La-related protein 7 (Larp7). In addition to its non-enzymatic role as a core component of 7SK RNPs, MePCE catalyzes the formation of a gamma-phosphate monomethylation cap, unique to 7SK and U6 in humans. How MePCE can specifically recognize and modify its RNA substrate and remain bound to 7SK to form the core 7SK RNP has not been previously known. We report 2.0 and 2.1 Å X-ray crystal structures of human MePCE methyltransferase domain bound to S-adenosylhomocysteine (SAH) and uncapped or capped 7SK substrates, respectively. We find that 7SK recognition is achieved by an extensive network of protein contacts to a 5' hairpin-single-stranded RNA region, involving the triphosphate group, two terminal basepairs and six single-stranded nucleotides, thus explaining MePCE specificity for 7SK and U6. The large size of the RNA binding site required for recognition and catalysis is unique among RNA methyltransferases studied to date. The structures reveal SAH and product RNA in a near-transition state geometry, where the cofactor SAH is buried inside the active site by the RNA-interacting residues. Unexpectedly, binding experiments show that MePCE has higher affinity for capped vs uncapped 7SK, with kinetic data supporting a slow product release model. Taken together, these data describe the features of an unusual RNA capping enzyme, and reveal the molecular mechanism of methyl transfer and 7SK retention by MePCE for subsequent assembly of the core 7SK RNP.

Changes in Electrophysiological Features of Rats’ Working Atrial Myocardium after Its Lipotransfection with MicroRNAs miR-1-3p, miR-153-3p, or miR-133a-3p

MicroRNA is a small, single-stranded nucleotide sequence, which can regulate gene expression at the posttranscriptional level. To date, it is known that some microRNAs play an important role in physiology and pathophysiology of the cardio-vascular system. The effects of lipotransfection with microRNAs, such as miR-1-3p, miR-153-3p, or miR-133-3p, on the electrophysiological indices of the myocardium tissue were studied here for the first time. With the use of the standard microelectrode technique, action potentials (AP) in isolated perfused samples of the rat atrial tissue were recorded after their treatment with a transfection mixture of a liposome forming reagent and miR-1-3p, miR-153-3p, or miR-133-3p. It is demonstrated that the treatment of the myocardial tissue with a transfection agent results in the prolongation of the repolarization phase of AP. It was found that miR-1-3p and miR-153-3p did not affect the pattern of AP within 6 h after the treatment of tissue samples. However, miR133a-3p evoked a statistically significant increase in the AP duration under 90% repolarization. The duration was maximum 4 h after the transfection. Search and analysis of possible targets for miR133a-3p using bioinformatic methods were performed and it was assumed that this microRNA can interact with mRNA of some protein phosphatases. Suppression of protein phosphatases’ expression in cardiomyocytes may underlie the increased AP duration during the application of miR133a-3p found in the present study due to the effect on the proteins of calcium turnover.

Mapping the RNA Chaperone Activity of the T. brucei Editosome Using SHAPE Chemical Probing.

Mitochondrial pre-mRNAs in African trypanosomes adopt intricately folded, highly stable 2D and 3D structures. The RNA molecules are substrates of a U-nucleotide-specific insertion/deletion-type RNA editing reaction, which is catalyzed by a 0.8MDa protein complex known as the editosome. RNA binding to the editosome is followed by a chaperone-mediated RNA remodeling reaction. The reaction increases the dynamic of specifically U-nucleotides to lower their base-pairing probability and as a consequence generates a simplified RNA folding landscape that is critical for the progression of the editing reaction cycle. Here we describe a chemical mapping method to quantitatively monitor the chaperone-driven structural changes of pre-edited mRNAs upon editosome binding. The method is known as selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE). SHAPE is based on the differential electrophilic modification of ribose 2'-hydroxyl groups in structurally constraint (double-stranded) versus structurally unconstrained (single-stranded) nucleotides. Electrophilic anhydrides such as 1-methyl-7-nitroisatoic anhydride are used as probing reagents, and the ribose 2'-modified nucleotides are mapped as abortive cDNA synthesis products. As a result, SHAPE allows the identification of all single-stranded and base-paired regions in a given RNA, and the data are used to compute experimentally derived RNA 2D structures. A side-by-side comparison of the RNA 2D folds in the pre- and post-chaperone states finally maps the chaperone-induced dynamic of the different pre-mRNAs with single-nucleotide resolution.

IcSHAPE-pipe: A comprehensive toolkit for icSHAPE data analysis and evaluation.

RNA molecules have the intrinsic ability to fold into complex structures that are important in regulating many biological processes, including transcription, translation, processing and degradation. However, our knowledge for RNA structures remains very limited. Previously, we developed icSHAPE, a high-throughput method to probe single-stranded RNA nucleotide in cells. To recover the structural profile of an RNA or a transcript by icSHAPE, it is essential to accurately calculate an icSHAPE reactivity as the RNA structure score for each base. Here, we present icSHAPE-pipe, a comprehensive toolkit for the analysis of RNA structure sequencing data obtained from icSHAPE experiments. Compared to the original icSHAPE data processing protocol, icSHAPE-pipe calculates RNA structural information with higher accuracy and achieves higher coverage of the transcriptome. In addition, icSHAPE-pipe can perform quality control, and generate reports on sequencing data and the statistics of results. In sum, icSHAPE-pipe provides a convenient workflow for researchers to analyze RNA structural data from icSHAPE sequencing experiments.

In Vitro Synthesis and RNA Structure Probing of CUG Triplet Repeat RNA.

Aberrant RNA structure plays a central role in the molecular mechanisms guided by repeat RNAs in diseases like myotonic dystrophy (DM), C9orf72-linked amyotrophic lateral sclerosis (ALS) and fragile X tremor/ataxia syndrome (FXTAS). Much knowledge remains to be gained about how these repeat-expanded transcripts are actually folded, especially regarding the properties specific to very long and interrupted repeats. RNA structure can be interrogated by chemical as well as enzymatic probes. These probes usually bind or cleave single-stranded nucleotides, which can subsequently be detected using reverse transcriptase primer extension. In this chapter, we describe methodology for in vitro synthesis and structure probing of expanded CUG repeat RNAs associated with DM type 1 and approaches for the associated data analysis.

DNA translocation mechanism of the MCM complex and implications for replication initiation

The DNA translocation activity of the minichromosome maintenance (MCM) complex powers DNA strand separation of the replication forks of eukaryotes and archaea. Here we illustrate an atomic level mechanism for this activity with a crystal structure of an archaeal MCM hexamer bound to single-stranded DNA and nucleotide cofactors. Sequence conservation indicates this rotary mechanism is fully possible for all eukaryotes and archaea. The structure definitively demonstrates the ring orients during translocation with the N-terminal domain leading, indicating that the translocation activity could also provide the physical basis of replication initiation where a double-hexamer idly encircling double-stranded DNA transforms to single-hexamers that encircle only one strand. In this mechanism, each strand binds to the N-terminal tier of one hexamer and the AAA+ tier of the other hexamer such that one ring pulls on the other, aligning equivalent interfaces to enable each hexamer to pull its translocation strand outside of the opposing hexamer.