Single-stranded Fragments Research Articles

Icerya purchasi Maskell (Hemiptera: Monophlebidae) Control Using Low Carbon Footprint Oligonucleotide Insecticides.

Climate change creates favourable conditions for the growth of insect populations. Today, the world is seeing an increase in the number of insect pest infestations associated with a long-term increase in the average temperature of climatic systems. For example, local invasions of Icerya purchasi Maskell, a citrus pest recognized worldwide, have increased in size and number in recent years. Controlling this pest is complicated because not all chemical insecticides are effective, and their use is undesirable since citrus fruit is used for food and chemical agents cumulatively harm human health. In this article, we demonstrated for the first time the successful use of a short single-stranded fragment of the 28S ribosomal RNA gene called "oligoICER-11" to control cottony cushion scale, and we propose the use of green oligonucleotide insecticides with a low carbon footprint for large-scale implementation in agriculture and forestry. Using the contact oligonucleotide insecticide oligoICER-11 at a concentration of 100 ng/μL on I. purchasi larvae resulted in a mortality of 70.55 ± 0.77% within 10 days. Thus, climate change is driving the need in both agriculture and forestry for oligonucleotide insecticides (DNA insecticides, olinscides): safe, effective, affordable insecticides with a low carbon footprint and long operational life.

Synthesis of the scFv fragment of anti-Frizzled-7 antibody and evaluation of its effects on triple-negative breast cancer in vitro study.

Among the most promising antibody formats in terms of inhibiting carcinogenesis are single-stranded variable fragments, whose targeted binding to the Fzd7 receptor has been proven effective at suppressing tumorigenesis. In this study, we investigated the effectiveness of an anti-Fzd7 antibody fragment against both tumor growth and metastasis of breast cancer cells. To develop anti-Fzd7 antibodies, bioinformatics approaches were used and the antibodies were expressed recombinantly in E. coli BL21 (DE3). The expression of anti-Fzd7 fragments was verified by Western blotting. Analysis of the antibody's binding capacity to Fzd7 was conducted by flow cytometry. Cell death and apoptosis were assessed by MTT and Annexin V/PI assays. The transwell migration and invasion assays, as well as the scratch method, were used to evaluate cell motility and invasiveness. The anti-Fzd7 antibody was expressed successfully as a single band of 31kDa. It could bind to 21.5% of MDA-MB-231 cells, as opposed to only 0.54% of SKBR-3 cells as negative control. According to MTT assay, induced apoptosis was 73.7% in MDA-MB-231 cells, compared with 29.5% in SKBR-3 cells. Also, the antibody exerted a significant inhibitory effect of 76% and 58% on migration and invasion of MDA-MB-231 cells, respectively. The recombinantly developed anti-Fzd7 scFv of this study could exhibit significant antiproliferative and antimigratory properties, along with a high apoptosis-inducing potential, making it suitable for the immunotherapy of triple negative breast cancer.

Distinct SARS-CoV-2 RNA fragments activate Toll-like receptors 7 and 8 and induce cytokine release from human macrophages and microglia.

The pandemic coronavirus disease 19 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is marked by thromboembolic events and an inflammatory response throughout the body, including the brain. Employing the machine learning approach BrainDead we systematically screened for SARS-CoV-2 genome-derived single-stranded (ss) RNA fragments with high potential to activate the viral RNA-sensing innate immune receptors Toll-like receptor (TLR)7 and/or TLR8. Analyzing HEK TLR7/8 reporter cells we tested such RNA fragments with respect to their potential to induce activation of human TLR7 and TLR8 and to activate human macrophages, as well as iPSC-derived human microglia, the resident immune cells in the brain. We experimentally validated several sequence-specific RNA fragment candidates out of the SARS-CoV-2 RNA fragments predicted in silico as activators of human TLR7 and TLR8. Moreover, these SARS-CoV-2 ssRNAs induced cytokine release from human macrophages and iPSC-derived human microglia in a sequence- and species-specific fashion. Our findings determine TLR7 and TLR8 as key sensors of SARS-CoV-2-derived ssRNAs and may deepen our understanding of the mechanisms how this virus triggers, but also modulates an inflammatory response through innate immune signaling.

Application of microfluidic chip electrophoresis for high-throughput nucleic acid fluorescence fragment analysis assays.

Nucleic acid fragment analysis via separation and detection are routine operations in molecular biology. However, analysis of small single-stranded nucleic acid fragments (<100nt) is challenging and mainly limited to labor-intensive polyacrylamide gel electrophoresis or high-cost capillary electrophoresis methods. Here we report an alternative method, a microfluidic chip electrophoresis system that provides a size resolution of 5nt and a detection time of one minute per sample of fluorescence-labeled DNA/RNA fragments. The feasibility of this system was evaluated by quantifying CRISPR-Cas9 cleavage efficiency and the detection resolution was evaluated by analyzing ssDNA/RNA adenylation and phosphorylation. We employed this system to study the RNA capping efficiency and double-stranded DNA unwinding efficiency in isothermal amplification as two examples for assay design and evaluation. The microfluidic chip electrophoresis system provides a rapid, sensitive, and high-throughput fluorescence fragment analysis (FFA), and can be applied for enzyme characterization, reaction optimization, and product quality control in various molecular biology processes.

Resolving a guanine-quadruplex structure in the SARS-CoV-2 genome through circular dichroism and multiscale molecular modeling.

The genome of SARS-CoV-2 coronavirus is made up of a single-stranded RNA fragment that can assume a specific secondary structure, whose stability can influence the virus's ability to reproduce. Recent studies have identified putative guanine quadruplex sequences in SARS-CoV-2 genome fragments that are involved in coding for both structural and non-structural proteins. In this contribution, we focus on a specific G-rich sequence referred to as RG-2, which codes for the non-structural protein 10 (Nsp10) and assumes a guanine-quadruplex (G4) arrangement. We provide the secondary structure of RG-2 G4 at atomistic resolution by molecular modeling and simulation, validated by the superposition of experimental and calculated electronic circular dichroism spectra. Through both experimental and simulation approaches, we have demonstrated that pyridostatin (PDS), a widely recognized G4 binder, can bind to and stabilize RG-2 G4 more strongly than RG-1, another G4 forming sequence that was previously proposed as a potential target for antiviral drug candidates. Overall, this study highlights RG-2 as a valuable target to inhibit the translation and replication of SARS-CoV-2, paving the way towards original therapeutic approaches against emerging RNA viruses.

A Mad7 System for Genetic Engineering of Filamentous Fungi.

The introduction of CRISPR technologies has revolutionized strain engineering in filamentous fungi. However, its use in commercial applications has been hampered by concerns over intellectual property (IP) ownership, and there is a need for implementing Cas nucleases that are not limited by complex IP constraints. One promising candidate in this context is the Mad7 enzyme, and we here present a versatile Mad7-CRISPR vector-set that can be efficiently used for the genetic engineering of four different Aspergillus species: Aspergillus nidulans,&amp;nbsp;A. niger, A. oryzae and A. campestris, the latter being a species that has never previously been genetically engineered. We successfully used Mad7 to introduce unspecific as well as specific template-directed mutations including gene disruptions, gene insertions and gene deletions. Moreover, we demonstrate that both single-stranded oligonucleotides and PCR fragments equipped with short and long targeting sequences can be used for efficient marker-free gene editing. Importantly, our CRISPR/Mad7 system was functional in both non-homologous end-joining (NHEJ) proficient and deficient strains. Therefore, the newly implemented CRISPR/Mad7 was efficient to promote gene deletions and integrations using different types of DNA repair in four different Aspergillus species, resulting in the expansion of CRISPR toolboxes in fungal cell factories.

Detection of micro-RNA by a combination of nucleic acid sequence-based amplification and a novel chemiluminescent pyrophosphate assay.

Micro-RNA has attracted much attention as a biomarker for disease progression and malignancy. A compact, simple, rapid, and highly sensitive method is required to perform simple genetic analyses, such as point-of-care testing (POCT), at the clinic or bedside. Nucleic acid sequence-based amplification (NASBA) is a specific amplification method for a single-stranded RNA fragment that is useful for the highly sensitive detection of miRNAs. In this work, we developed a novel miRNA analytical system for POCT by combining the NASBA and chemiluminescence methods. Because the NASBA reaction is conducted at a constant temperature (41°C) and detection by chemiluminescence reaction does not require a light source, these methods could be combined to amplify 100 ng/assay miRNA. This combined miRNA detection method could be useful for the future development of compact POCT systems.

Short single-stranded DNAs with putative non-canonical structures comprise a new class of plasma cell-free DNA

BackgroundCell-free DNA (cfDNA), which is extracellular DNA present in the circulating plasma and other body fluids, is currently investigated as a minimally invasive, highly informative biomarker. While nucleosome-sized cfDNA fragments have been investigated intensively, shorter DNA fragments in the plasma have not been studied due to several technical limitations.ResultsWe aimed to investigate the existence of shorter cfDNA fragments in the blood. Using an improved cfDNA purification protocol and a 3′-end-labeling method, we found DNA fragments of approximately 50 nucleotides in length in the human plasma, present at a molar concentration comparable to that of nucleosome-sized fragments. Unfortunately, these short fragments cannot be recovered by widely used cfDNA isolation methods. In addition, they are composed of single-stranded DNA (ssDNA), thus escaping detection in previous studies. Therefore, we established a library-preparation protocol based on our unique ssDNA ligation technique and applied it to the isolated cfDNA. Deep sequencing of these libraries revealed that the short fragments are derived from hundreds of thousands of genomic sites in open chromatin regions and enriched with transcription factor-binding sites. Remarkably, antisense strands of putative G-quadruplex motifs occupy as much as one-third of the peaks by these short fragments.ConclusionsWe propose a new class of plasma cfDNA composed of short single-stranded fragments that potentially form non-canonical DNA structures.

Delivery of miR-26a Using an Exosomes-Based Nanosystem Inhibited Proliferation of Hepatocellular Carcinoma.

Background: MicroRNA (abbreviated miRNA)-based treatment holds great promise for application as clinical antitumor therapy, but good carriers for delivery of the miRNA drug are lacking. Exosomes secreted by mesenchymal stem cells (MSCs) have proved to be safe, and exogenously modified exosomes may potentially represent an excellent drug delivery vehicle.Methods: In this study, we designed a delivery nano system using single-stranded variable fragment (scFv)-modified exosomes derived from human cord blood MSCs. Genetic engineering technology was used to obtain anti-Glypican 3 (GPC3) scFv-modified exosomes, which were then loaded with miR-26a mimics through electroporation.Results: Results of electron microscopy and dynamic light scattering indicated that the diameter of the drug-carrying exosomes was about 160 nm. Furthermore, anti-GPC3 scFv-modified exosomes effectively delivered miR-26a to GPC3-positive hepatocellular carcinoma cells, thereby inhibiting cell proliferation and migration by regulating the expression of downstream target genes of miR-26a. The exosomes-based nano system displayed favorable anti-tumor effect in vivo with no obvious side effects.Conclusion: Our data provided a new perspective for the use of exosome delivery systems for miRNA-based antitumor therapy.

Entanglements of structure elements revealed in RNA 3D models.

Computational methods to predict RNA 3D structure have more and more practical applications in molecular biology and medicine. Therefore, it is crucial to intensify efforts to improve the accuracy and quality of predicted three-dimensional structures. A significant role in this is played by the RNA-Puzzles initiative that collects, evaluates, and shares RNAs built computationally within currently nearly 30 challenges. RNA-Puzzles datasets, subjected to multi-criteria analysis, allow revealing the strengths and weaknesses of computer prediction methods. Here, we study the issue of entangled RNA fragments in the predicted RNA 3D structure models. By entanglement, we mean an arrangement of two structural elements such that one of them passes through the other. We propose the classification of entanglements driven by their topology and components. It distinguishes two general classes, interlaces and lassos, and subclasses characterized by element types—loops, dinucleotide steps, open single-stranded fragments—and puncture multiplicity. Our computational pipeline for entanglement detection, applied for 1,017 non-redundant models from RNA-Puzzles, has shown the frequency of different entanglements and allowed identifying 138 structures with intersected assemblies.

Predicting aptamer sequences that interact with target proteins using an aptamer-protein interaction classifier and a Monte Carlo tree search approach.

Oligonucleotide-based aptamers, which have a three-dimensional structure with a single-stranded fragment, feature various characteristics with respect to size, toxicity, and permeability. Accordingly, aptamers are advantageous in terms of diagnosis and treatment and are materials that can be produced through relatively simple experiments. Systematic evolution of ligands by exponential enrichment (SELEX) is one of the most widely used experimental methods for generating aptamers; however, it is highly expensive and time-consuming. To reduce the related costs, recent studies have used in silico approaches, such as aptamer-protein interaction (API) classifiers that use sequence patterns to determine the binding affinity between RNA aptamers and proteins. Some of these methods generate candidate RNA aptamer sequences that bind to a target protein, but they are limited to producing candidates of a specific size. In this study, we present a machine learning approach for selecting candidate sequences of various sizes that have a high binding affinity for a specific sequence of a target protein. We applied the Monte Carlo tree search (MCTS) algorithm for generating the candidate sequences using a score function based on an API classifier. The tree structure that we designed with MCTS enables nucleotide sequence sampling, and the obtained sequences are potential aptamer candidates. We performed a quality assessment using the scores of docking simulations. Our validation datasets revealed that our model showed similar or better docking scores in ZDOCK docking simulations than the known aptamers. We expect that our method, which is size-independent and easy to use, can provide insights into searching for an appropriate aptamer sequence for a target protein during the simulation step of SELEX.

Antisense Oligonucleotide Therapy for Ophthalmic Conditions

ABSTRACT Antisense oligonucleotides (AON) are synthetic single-stranded fragments of nucleic acids that bind to a specific complementary messenger RNA (mRNA) sequence and change the final gene product. AON were initially approved for treating cytomegalovirus retinitis and have shown promise in treating Mendelian systemic disease. AON are currently being investigated as a treatment modality for many ophthalmic diseases, including inherited retinal disorders (IRD), inflammatory response and wound healing after glaucoma surgery, and macular degeneration. They provide a possible solution to gene therapy for IRD that are not candidates for adeno-associated virus (AAV) delivery. This chapter outlines the historical background of AON and reviews clinical applications and ongoing clinical trials.

Short single‐stranded DNA with putative non‐canonical structures comprises a novel class of plasma cell‐free DNA

Cell-free DNA (cfDNA) in human blood is currently being investigated as a minimally invasive, highly informative biomarker. Besides carrying genetic variations, cfDNA can also provide useful epigenetic information through its methylation and fragmentation pattern. Previous cfDNA studies have focused on nucleosome-sized fragments, leaving shorter ones neglected. Hence, we intended to investigate whether shorter cfDNA fragments exist in the blood or not. Using an improved cfDNA purification protocol and a 3ʹ-end labeling method, we found that human plasma contains DNA fragments of approximately 50 nucleotides, present at a molar concentration largely comparable to that of the nucleosome-sized fragments. These short fragments cannot be recovered by using popular cfDNA isolation methods, and they are composed of single-stranded DNA (ssDNA), thus escaping detection in previous studies. We established a library preparation protocol based on a ssDNA ligation technique unique developed by us and applied it to cfDNA isolated with the improved purification protocol. Deep sequencing of these libraries revealed that the short fragments are derived from hundreds of thousands of genomic sites in open chromatin regions and enriched with transcription factor-binding sites. Remarkably, antisense strands of putative G-quadruplex motifs occupy as much as one-third of these short fragments. Hence, we propose a novel class of plasma cfDNA composed of short single-stranded fragments that potentially form non-canonical DNA structures.

The Impact of Interferon-γ (IFN-γ) and IFN-γ-Inducible Protein 10 (IP-10) Genes’ Polymorphism on Risk of Hepatitis C Virus–Related Liver Cirrhosis

ABSTRACT Background Today there is increasing evidence concerning the association between individual genetic polymorphisms within proinflammatory cytokines and chronic hepatitis C (CHC) severity. It has been demonstrated that polymorphisms in some genes may significantly predict HCV infected patients’ susceptibility to developing liver cirrhosis or their responsiveness to the treatment. Aim We investigated the influence of single nucleotide polymorphisms (SNPs) in Interferon (IFN-γ) and Interferon Gamma-Inducible Protein 10 (IP-10) genes on cirrhosis risk in HCV-infected patients and their association with response to various direct-acting antiviral drugs (DAAs). Methods IFN-γ (+874T/A, +2109A/G) and IP-10 (−135G/A, −1447A/G) genotypes were determined in 175 CHC Egyptian HCV patients (69 liver cirrhotic and 106 non-cirrhotic patients) using either single-stranded polymorphism polymerase chain reaction (SSP-PCR) or Restriction fragment length-PCR (RFLP-PCR) methods. Results IFN-γ + 874 TA, IP-10 − 135AA, and IP-10 − 1447AA and IP-10 − 1447GG genotypes are increased in patients developing liver cirrhosis compared to non-cirrhotic ones. Although, no statistical significance in their distribution was demonstrated, indicating the lack of association between these SNPs and liver cirrhosis susceptibility in HCV-infected patients. Haplotypes analysis between different loci on all selected genes showed a significant increase in AGGA and TAGA and a significant decrease in TGGA haplotypes in cirrhotic patients. Genotype frequencies at loci −135 and −1447 of IP-10 appeared to be in complete Linkage disequilibrium (LD) (D’ = 0.999, r2 = 0.689). Conclusion Our data support the concept that IFN-γ and IP-10 gene polymorphisms are not predictors of disease progression among Egyptian patients with HCV infection.

Separation of RNA according to Size: Electrophoresis of RNA through Denaturing Urea Polyacrylamide Gels.

Thin (0.4-1.5 mm) polyacrylamide-urea gels provide high resolution of RNAs up to 1000 nt in size and are capable of resolving single-stranded fragments of RNA that differ in length by as little as 1 nt. The polyacrylamide gel is cast between two glass plates that are separated by two thin Teflon or nylon spacers. A so-called shark's tooth comb or, less frequently, a standard slotted comb forms the sample wells into which the RNA samples are loaded before electrophoresis. In contrast to electrophoresis using agarose gels, which occurs while the gel is horizontal, polyacrylamide gels are run while in the vertical position. Gels are also typically run at 45°C-55°C, which is the melting temperature of RNA, and in the presence of 6-8 m urea. The gel recipe and protocol presented here for 8 m urea/TBE polyacrylamide gels can be used for a variety of applications including mapping RNA with nuclease S1, ribonuclease protection assay, or analysis of RNA by primer extension.

Discovery of GS-9688 (Selgantolimod) as a Potent and Selective Oral Toll-Like Receptor 8 Agonist for the Treatment of Chronic Hepatitis B.

Toll-like receptor 8 (TLR8) recognizes pathogen-derived single-stranded RNA fragments to trigger innate and adaptive immune responses. Chronic hepatitis B (CHB) is associated with a dysfunctional immune response, and therefore a selective TLR8 agonist may be an effective treatment option. Structure-based optimization of a dual TLR7/8 agonist led to the identification of the selective TLR8 clinical candidate (R)-2-((2-amino-7-fluoropyrido[3,2-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (GS-9688, (R)-7). Potent TLR8 agonism (IL-12p40 EC50 = 220 nM) and >100-fold TLR7 selectivity (IFN-α EC50 > 50 μM) was observed in human peripheral blood mononuclear cells (PBMCs). The TLR8-ectodomain:(R)-7 complex confirmed TLR8 binding and a direct ligand interaction with TLR8 residue Asp545. Oral (R)-7 had good absorption and high first pass clearance in preclinical species. A reduction in viral markers was observed in HBV-infected primary human hepatocytes treated with media from PBMCs stimulated with (R)-7, supporting the clinical development of (R)-7 for the treatment of CHB.

A multiplex guide RNA expression system and its efficacy for plant genome engineering

BackgroundThe Streptococcus pyogenes CRISPR system is composed of a Cas9 endonuclease (SpCas9) and a single-stranded guide RNA (gRNA) harboring a target-specific sequence. Theoretically, SpCas9 proteins could cleave as many targeted loci as gRNAs bind in a genome.ResultsWe introduce a PCR-free multiple gRNA cloning system for editing plant genomes. This method consists of two steps: (1) cloning the annealed products of two single-stranded oligonucleotide fragments harboring a complimentary target-binding sequence on each strand between tRNA and gRNA scaffold sequences in a pGRNA vector; and (2) assembling tRNA-gRNA units from several pGRNA vectors with a plant binary vector containing a SpCas9 expression cassette using the Golden Gate assembly method. We validated the editing efficiency and patterns of the multiplex gRNA expression system in wild tobacco (Nicotiana attenuata) protoplasts and in transformed plants by performing targeted deep sequencing. Two proximal cleavages by SpCas9-gRNA largely increased the editing efficiency and induced large deletions between two cleavage sites.ConclusionsThis multiplex gRNA expression system enables high-throughput production of a single binary vector and increases the efficiency of plant genome editing.

Practical Guidance in Genome-Wide RNA:DNA Triple Helix Prediction.

Long noncoding RNAs (lncRNAs) play a key role in many cellular processes including chromatin regulation. To modify chromatin, lncRNAs often interact with DNA in a sequence-specific manner forming RNA:DNA triple helices. Computational tools for triple helix search do not always provide genome-wide predictions of sufficient quality. Here, we used four human lncRNAs (MEG3, DACOR1, TERC and HOTAIR) and their experimentally determined binding regions for evaluating triplex parameters that provide the highest prediction accuracy. Additionally, we combined triplex prediction with the lncRNA secondary structure and demonstrated that considering only single-stranded fragments of lncRNA can further improve DNA-RNA triplexes prediction.

Switchable DNA tweezer and G-quadruplex nanostructures for ultrasensitive voltammetric determination of the K-ras gene fragment.

Voltammetric detection of the K-ras gene fragment was accomplished through the combined application of (a) a switchable DNA nanostructure, (b) the use of hairpin probe and exonuclease III (Exo III)-assisted signal amplification, (c) a split G-quadruplex, and (d) by exploiting the redox activity of DNAzyme. Three assistant oligonucleotides were designed to construct a DNA tweezer on a gold electrode. It is in "open state" in the absence of K-ras DNA. Then, a hairpin probe was introduced, whose stem-loop structure can be opened through hybridization with the K-ras DNA. Exo III is added which hydrolyzes the complementary region of the hairpin sequence to release a single-stranded rest fragment. The ssDNA hybridizes with the DNA tweezer on the electrode which thereby is switched to the "closed state". This leads to the formation of G-quadruplex due to the shortened distance of the split G-quadruplex-forming sequences in the tweezer. The voltammetric signal of the G-quadruplex-hemin complex, with a peak near -0.3V vs. Ag/AgCl, is used as the signal output. Under the optimal conditions, the current response in differential pulse voltammetry (DPV) increases linearly with the concentration of K-ras DNA in the range of 0.01-1000 pM, and the detection limit is 2.4 fM. The assay can clearly discriminate K-ras DNA from a single-base mutation. The method has excellent selectivity and was applied to the determination of K-ras DNA in (spiked) serum samples. Graphical abstractSchematic representation ofa method for the determination of the K-ras gene fragment through a combination of switchable DNA tweezer, split G-quadruplex, and exonuclease III (ExoIII)-assisted target recycling signal amplification.

Sources of erroneous sequences and artifact chimeric reads in next generation sequencing of genomic DNA from formalin-fixed paraffin-embedded samples.

Tissues used in pathology laboratories are typically stored in the form of formalin-fixed, paraffin-embedded (FFPE) samples. One important consideration in repurposing FFPE material for next generation sequencing (NGS) analysis is the sequencing artifacts that can arise from the significant damage to nucleic acids due to treatment with formalin, storage at room temperature and extraction. One such class of artifacts consists of chimeric reads that appear to be derived from non-contiguous portions of the genome. Here, we show that a major proportion of such chimeric reads align to both the ‘Watson’ and ‘Crick’ strands of the reference genome. We refer to these as strand-split artifact reads (SSARs). This study provides a conceptual framework for the mechanistic basis of the genesis of SSARs and other chimeric artifacts along with supporting experimental evidence, which have led to approaches to reduce the levels of such artifacts. We demonstrate that one of these approaches, involving S1 nuclease-mediated removal of single-stranded fragments and overhangs, also reduces sequence bias, base error rates, and false positive detection of copy number and single nucleotide variants. Finally, we describe an analytical approach for quantifying SSARs from NGS data.