Selection Of Target Sequences Research Articles

Comparative study on the target sequences of thaumatin-like protein (TLP) and internal transcribed spacer (ITS) in the detection of corn component in edible oil by ladder-shape melting temperature isothermal amplification (LMTIA)

Single copy thaumatin-like protein (TLP) genes and multiple copy internal transcribed spacer (ITS) genes from the nucleus of corn were selected as targets, designed and screened primers. The impact of target sequence selection on ladder-shape melting temperature isothermal amplification (LMTIA) detection of corn components in edible oil from aspects such as specificity, sensitivity, and stability were studied. The results showed that the LMTIA system targeting TLP has an optimal reaction temperature of 58 °C and can detect a minimum of 100 pg/μL of corn DNA, and the LMTIA system established with ITS as the target has higher sensitivity, with an optimal temperature of 59 °C, and can detect corn DNA as low as 1 pg/μL. Both systems have good specificity, stable reactions, and no false positive results were observed. Conclusion: ITS is more suitable as a target sequence for LMTIA detection of corn components in edible oil.

A bioinformatics framework for targeted gene expression assay design: Application to in vitro developmental neurotoxicity screening in a rat model

Brain development involves a series of intricately choreographed neuronal differentiation and maturation steps that are acutely vulnerable to interferences from chemical exposures. Many genes involved in neurodevelopmental processes show evolutionarily conserved expression patterns in mammals and may constitute useful indicators/biomarkers for the evaluation of potential developmental neurotoxicity. Based on these premises, this study developed a bioinformatics framework to guide the design of a gene expression-based in vitro developmental neurotoxicity assay targeting evolutionary conserved genes associated with neuronal differentiation and maturation in rat cerebellar granule cells (CGCs). Rat, mouse and human genes involved in neurodevelopment and presenting one-to-one orthology were selected and orthologous exons within these genes were identified. PCR primer sets were designed within these orthologous exons and their specificity was evaluated in silico. The performance and specificity of rat, mouse and human PCR primer sets were then confirmed experimentally. Finally, RT-qPCR analyses in CGCs exposed in vitro to well-known neurotoxicants (Chlorpyrifos and Chlorpyrifos oxon) uncovered perturbations of expression levels for most of the selected genes. This bioinformatics framework for gene and target sequence selection may facilitate the identification of transcriptional biomarkers for developmental neurotoxicity assays and the comparison of gene expression data across experimental models from different mammalian species.

In Silico Prediction and Selection of Target Sequences in the SARS-CoV-2 RNA Genome for an Antiviral Attack.

The SARS-CoV-2 pandemic has urged the development of protective vaccines and the search for specific antiviral drugs. The modern molecular biology tools provides alternative methods, such as CRISPR-Cas and RNA interference, that can be adapted as antiviral approaches, and contribute to this search. The unique CRISPR-Cas13d system, with the small crRNA guide molecule, mediates a sequence-specific attack on RNA, and can be developed as an anti-coronavirus strategy. We analyzed the SARS-CoV-2 genome to localize the hypothetically best crRNA-annealing sites of 23 nucleotides based on our extensive expertise with sequence-specific antiviral strategies. We considered target sites of which the sequence is well-conserved among SARS-CoV-2 isolates. As we should prepare for a potential future outbreak of related viruses, we screened for targets that are conserved between SARS-CoV-2 and SARS-CoV. To further broaden the search, we screened for targets that are conserved between SARS-CoV-2 and the more distantly related MERS-CoV, as well as the four other human coronaviruses (OC43, 229E, NL63, HKU1). Finally, we performed a search for pan-corona target sequences that are conserved among all these coronaviruses, including the new Omicron variant, that are able to replicate in humans. This survey may contribute to the design of effective, safe, and escape-proof antiviral strategies to prepare for future pandemics.

Design of Oligonucleotides for Allele-Specific Amplification Based on PCR and Isothermal Techniques.

Single-nucleotide variations have been associated to various genetic diseases, variations on drug efficiency, and differences in cancer prognostics. The detection of these changes in nucleic acid sequences from patient samples is particularly useful for accurate diagnosis, therapeutics, and disease management. A reliable allele-specific amplification is still an important challenge for molecular-based diagnostic technologies. In the last years, allele-specific primers have been designed for promoting the enrichment of certain variants, based on a higher stability of primer/template duplexes. Also, several methods are based on the addition of a blocking oligonucleotide that prevent the amplification of a specific variant, enabling that other DNA variants can be observed. In this context, genotyping methods based on isothermal amplification techniques are increasing, especially those assays aimed to be deployed at point-of-care applications. The correct selection of target sequences is crucial for reaching the required analytical performances, in terms of reaction time, amplification yield, and selectivity. The present chapter describes the design criteria for the selection of primers and blockers for relevant PCR approaches and novel isothermal strategies. Several successful examples are provided in order to highlight the main design restrictions and the potential to be extended to other applications.

Genomic variations in SARS-CoV-2 strains at the target sequences of nucleic acid amplification tests

IntroductionNucleic acid amplification is the main method used to detect infections of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the false-negative rate of nucleic acid tests cannot be ignored.Material and methodsHerein, we demonstrated genomic variations at the target sequences for the tests and the geographical distribution of the variations across countries by analyzing the whole-genome sequencing data of SARS-CoV-2 strains from the 2019 Novel Coronavirus Resource (2019nCoVR) database.ResultsAmong the 21 pairs of primer sequences in regions ORF1ab, S, E, and N, the total length of primer and probe target sequences was 938 bp, with 131 (13.97%) variant loci in 2415 (38.96%) isolates. Primer targets in the N region contained the most variations that were distributed among the most isolates, and the E region contained the fewest. Single nucleotide polymorphisms were the most frequent variation, with C to T transitions being detected in the most variant loci. G to A transitions and G to C transversions were the most common and had the highest isolate density. Genomic variations at the three mutation sites N: 28881, N: 28882, and N: 28883 were the most commonly detected, including in 608 SARS-CoV-2 strains from 33 countries, especially in the United Kingdom, Portugal, and Belgium.ConclusionsOur work comprehensively analyzed genomic variations at the target sequences of the nucleic acid amplification tests, offering evidence to optimize primer and probe target sequence selection, thereby improving the performance of the SARS-CoV-2 diagnostic test.

FOXO4 Transactivation Domain Interaction with Forkhead DNA Binding Domain and Effect on Selective DNA Recognition for Transcription Initiation

Forkhead box O4 (FOXO4) is a human transcription factor (TF) that participates in cell homeostasis. While the structure and DNA binding properties of the conserved forkhead domain (FHD) have been thoroughly investigated, how the transactivation domain (TAD) regulates the DNA binding properties of the protein remains elusive. Here, we investigated the role of TAD in modulating the DNA binding properties of FOXO4 using solution NMR. We found that TAD and FHD form an intramolecular complex mainly governed by hydrophobic interaction. Remarkably, TAD and DNA share the same surface of FHD for binding. While FHD did not differentiate binding to target and non-target DNA, the FHD-TAD complex showed different behaviors depending on the DNA sequence. In the presence of TAD, free and DNA-bound FHD exhibited a slow exchange with target DNA and a fast exchange with non-target DNA. The interaction of the two domains affected the kinetic function of FHD depending on the type of DNA. Based on these findings, we suggest a transcription initiation model by which TAD modulates FOXO4 recognition of its target promoter DNA sequences. This study describes the function of TAD in FOXO4 and provides a new kinetic perspective on target sequence selection by TFs.

Gapmer Antisense Oligonucleotides Containing 2',3'-Dideoxy-2'-fluoro-3'-C-hydroxymethyl-β-d-lyxofuranosyl Nucleotides Display Site-Specific RNase H Cleavage and Induce Gene Silencing.

Off-target effects remain a significant challenge in the therapeutic use of gapmer antisense oligonucleotides (AONs). Over the years various modifications have been synthesized and incorporated into AONs, however, precise control of RNase H-induced cleavage and target sequence selectivity has yet to be realized. Herein, the synthesis of the uracil and cytosine derivatives of a novel class of 2'-deoxy-2'-fluoro-3'-C-hydroxymethyl-β-d-lyxo-configured nucleotides has been accomplished and the target molecules have been incorporated into AONs. Experiments on exonuclease degradation showed improved nucleolytic stability relative to the unmodified control. Upon the introduction of one or two of the novel 2'-fluoro-3'-C-hydroxymethyl nucleotides as modifications in the gap region of a gapmer AON was associated with efficient RNase H-mediated cleavage of the RNA strand of the corresponding AON:RNA duplex. Notably, a tailored single cleavage event could be engineered depending on the positioning of a single modification. The effect of single mismatched base pairs was scanned along the full gap region demonstrating that the modification enables a remarkable specificity of RNase H cleavage. A cell-based model system was used to demonstrate the potential of gapmer AONs containing the novel modification to mediate gene silencing.

Multimode MicroRNA Sensing via Multiple Enzyme-Free Signal Amplification and Cation-Exchange Reaction.

The detection of biomarkers requires not only high sensitivity but also different signal reading methods depending on the actual situation. Herein, the luminescent properties of CdTe quantum dots (QDs) were exploited, where CdTe QDs were used as shared signal molecules. Combining multiple types of nucleic acid and chemical signal amplification techniques, and various signal detection techniques, a magnetic nanoparticle (NP) and filter-assisted separation multimode sensing strategy has been developed. In this work, miRNA-141 was selected as a representative target, which can trigger the catalyzed hairpin assembly and hybrid chain reaction enzyme-free nucleic acid signal amplification that generates long double-stranded DNA. Subsequently, the chemical amplification of silver NPs (Ag NPs) that release a large amount of Ag+ was introduced into the system. Finally, the cation-exchange reaction between CdTe QDs and Ag+ was utilized to quench the fluorescence (FL) of the CdTe QDs, releasing free Cd2+. The visual/FL/chemical vapor generation-atomic fluorescence spectrometry (CVG-AFS)/inductively coupled plasma mass spectrometry (ICP-MS) method could then be performed for the analysis of miRNA. After investigating its experimental performance, it has been found that 10 fM can be differentiated from the blank solution with the naked eye. In addition, FL/CVG-AFS/ICP-MS methods all displayed good analytical capability for target detection, and the limits of detection (LODs) are as low as fM, which show high target sequence selectivity. This platform was applied to investigate miRNA-141 expression in various cancer cells, which can accurately detect in the range of 100-100 000 MDA-MB-231 cells (breast cancer cell lines), with an LOD of 15 cells. Therefore, the multimode sensing strategy based on a single signal molecule and multiple signal amplification strategies is an applicable and versatile detection method of biomarkers; it can even achieve point-of-care testing, improving the accuracy and efficiency of medical diagnosis.

MorphoCatcher: a multiple-alignment based web tool for target selection and designing taxon-specific primers in the loop-mediated isothermal amplification method.

BackgroundAdvantages of loop-mediated isothermal amplification in molecular diagnostics allow to consider the method as a promising technology of nucleic acid detection in agriculture and medicine. A bioinformatics tool that provides rapid screening and selection of target nucleotide sequences with subsequent taxon-specific primer design toward polymorphic orthologous genes, not only unique or conserved common regions of genome, would contribute to the development of more specific and sensitive diagnostic assays. However, considering features of the original software for primer selection, also known as the PrimerExplorer (Eiken Chemical Co. LTD, Tokyo, Japan), the taxon-specific primer design using multiple sequence alignments of orthologs or even viral genomes with conservative architecture is still complicated.FindingsHere, MorphoCatcher is introduced as a fast and simple web plugin for PrimerExplorer with a clear interface. It enables an execution of multiple-alignment based search of taxon-specific mutations, visual screening and selection of target sequences, and easy-to-start specific primer design using the PrimerExplorer software. The combination of MorphoCatcher and PrimerExplorer allows to perform processing of the multiple alignments of orthologs for informative sliding-window plot analysis, which is used to identify the sequence regions with a high density of taxon-specific mutations and cover them by the primer ends for better specificity of amplification.ConclusionsWe hope that this new bioinformatics tool developed for target selection and taxon-specific primer design, called the MorphoCatcher, will gain more popularity of the loop-mediated isothermal amplification method for molecular diagnostics community. MorphoCatcher is a simple web plugin tool for the PrimerExplorer software which is freely available only for non-commercial and academic users at http://morphocatcher.ru.

Novel Microbial Modification Tools to Convert Lipids into Other Value-Added Products.

CRISPR-Cas9 Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated (Cas) is a microbial adaptive immune system that has revolutionized the field of molecular biology and genome engineering. The Type II CRISPR system consists of Cas9 nuclease of Streptococcus pyogenes and the RNA complex that guides Cas9 nuclease to a specific sequence of DNA in the genome. The CRISPR-Cas9 technology has reformed our ability to edit DNA and to regulate expression levels of genes of interest to high precision and accuracy. It is a powerful technology, which is used for genome engineering of a wide range of organisms for various applications. Here, we describe a method involving CRISPR-Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) mechanisms for biotechnological applications in yeast. The complete procedure of genome editing including target sequence selection, cloning gRNA with a target sequence, transformation, and verification of the desired mutation/deletion or insertion can be achieved within 2-3weeks in yeast.

CRISPR/Cas precision: do we need to worry about off-targeting in plants?

The CRISPR/Cas technology has recently become the tool of choice for targeted genome modification in plants and beyond. Although CRSIPR/Cas offers a rapid and facile way of introducing changes at genomic loci of interest, its application is associated with off-targeting, i.e. introduction of unintended mutations at off-target sites within the genome, which has been reported frequently in the mammalian field. Here we summarise the current knowledge on the precision of CRISPR/Cas in plant systems and provide a summary of state-of the-art strategies for avoiding off-target mutations, as well as unintended on-target changes, in plants. These include using natural (e.g. Cas12a) or engineered (e.g. SpCas9-HF) CRISPR/Cas nucleases characterised by higher precision, as compared to the commonly used wild type SpCas9. In addition, we discuss the usage of CRISPR/Cas nucleases in the form of ribonucleoproteins (RNPs) as an option for reducing off-targeting in plants. Finally, we conclude that the most important factor for reducing CRISPR/Cas off-targeting remains careful selection of target sequences, for which we provide an overview of available online software tools and experimental guidance.

Efficient production of reuterin from glycerol by Lactobacillus reuteri

Cpf1 has been harnessed as a tool for genome manipulation in various species because of its simplicity and high efficiency. Our recent study demonstrated that FnCpf1 could be utilized for human genome editing with notable advantages for target sequence selection due to the flexibility of the protospacer adjacent motif (PAM) sequence. Multiplex genome editing provides a powerful tool for targeting members of multigene families, dissecting gene networks, modeling multigenic disorders in vivo, and applying gene therapy. However, there are no reports at present that show FnCpf1-mediated multiplex genome editing via a single customized CRISPR RNA (crRNA) array. In the present study, we utilize a single customized crRNA array to simultaneously target multiple genes in human cells. In addition, we also demonstrate that a single customized crRNA array to target multiple sites in one gene could be achieved. Collectively, FnCpf1, a powerful genome-editing tool for multiple genomic targets, can be harnessed for effective manipulation of the human genome.

A Single Multiplex crRNA Array for FnCpf1-Mediated Human Genome Editing.

Cpf1 has been harnessed as a tool for genome manipulation in various species because of its simplicity and high efficiency. Our recent study demonstrated that FnCpf1 could be utilized for human genome editing with notable advantages for target sequence selection due to the flexibility of the protospacer adjacent motif (PAM) sequence. Multiplex genome editing provides a powerful tool for targeting members of multigene families, dissecting gene networks, modeling multigenic disorders invivo, and applying gene therapy. However, there are no reports at present that show FnCpf1-mediated multiplex genome editing via a single customized CRISPR RNA (crRNA) array. In the present study, we utilize a single customized crRNA array to simultaneously target multiple genes in human cells. In addition, we also demonstrate that a single customized crRNA array to target multiple sites in one gene could be achieved. Collectively, FnCpf1, a powerful genome-editing tool for multiple genomic targets, can be harnessed for effective manipulation of the human genome.

Engineering resistance to a resistance-breaking strain of Cucumber mosaic virus in plants utilizing viral dsRNA

Cucumber mosaic virus Ca-P1 strain, a P0 resistance-breaking virus, was isolated from the leaves of virus-infected Capsicum annuum ‘Manidda’ (a P0 resistant cultivar) in South Korea. Since CMV-Ca-P1 was first reported in 2006, this virus causing damage on pepper production has been constantly detected in South Korea. We constructed three CMV RNAi vectors based on the post-transcriptional gene silencing of defense system against virus infection. These independent vectors (hpCMV1, hpCMV2, and hpCMV2 + 1 RNAi vectors) were designed to produce dsRNAs containing each hpCMV1 (1270–1629 nt), hpCMV2 (1–300 nt), and hpCMV2 + 1 (fused hpCMV2 and hpCMV1) fragments, in RNA-1 (replicase gene) of CMV-Ca-P1, which were then confirmed by Agrobacterium-mediated transformation transient assay. Among these, dsRNAs expressed from the hpCMV2 + 1 vector showed resistance to both CMV-Ca-P1 and CMV-Fny (ordinary strain). To obtain high level of resistance to both CMV-Ca-P1 and CMV-Fny, transgenic Nicotiana benthamiana plants containing hpCMV2 + 1 vector were developed and conferred resistance to both CMV-Ca-P1 and CMV-Fny. This study contributes to the effective selection of target sequences that may inhibit CMV infection.

A bend, flip and trap mechanism for transposon integration.

Cut-and-paste DNA transposons of the mariner/Tc1 family are useful tools for genome engineering and are inserted specifically at TA target sites. A crystal structure of the mariner transposase Mos1 (derived from Drosophila mauritiana), in complex with transposon ends covalently joined to target DNA, portrays the transposition machinery after DNA integration. It reveals severe distortion of target DNA and flipping of the target adenines into extra-helical positions. Fluorescence experiments confirm dynamic base flipping in solution. Transposase residues W159, R186, F187 and K190 stabilise the target DNA distortions and are required for efficient transposon integration and transposition in vitro. Transposase recognises the flipped target adenines via base-specific interactions with backbone atoms, offering a molecular basis for TA target sequence selection. Our results will provide a template for re-designing mariner/Tc1 transposases with modified target specificities.

RNA-guided CRISPR-Cas technologies for genome-scale investigation of disease processes.

From its discovery as an adaptive bacterial and archaea immune system, the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has quickly been developed into a powerful and groundbreaking programmable nuclease technology for the global and precise editing of the genome in cells. This system allows for comprehensive unbiased functional studies and is already advancing the field by revealing genes that have previously unknown roles in disease processes. In this review, we examine and compare recently developed CRISPR-Cas platforms for global genome editing and examine the advancements these platforms have made in guide RNA design, guide RNA/Cas9 interaction, on-target specificity, and target sequence selection. We also explore some of the exciting therapeutic potentials of the CRISPR-Cas technology as well as some of the innovative new uses of this technology beyond genome editing.

Transforming growth factor-β gene silencing using adenovirus expressing TGF-β1 or TGF-β2 shRNA

Tumor cells secrete a variety of cytokines to outgrow and evade host immune surveillance. In this context, transforming growth factor-β1 (TGF-β1) is an extremely interesting cytokine because it has biphasic effects in cancer cells and normal cells. TGF-β1 acts as a growth inhibitor in normal cells, whereas it promotes tumor growth and progression in tumor cells. Overexpression of TGF-β1 in tumor cells also provides additional oncogenic activities by circumventing the host immune surveillance. Therefore, this study ultimately aimed to test the hypothesis that suppression of TGF-β1 in tumor cells by RNA interference can have antitumorigenic effects. However, we demonstrated here that the interrelation between TGF-β isotypes should be carefully considered for the antitumor effect in addition to the selection of target sequences with highest efficacy. The target sequences were proven to be highly specific and effective for suppressing both TGF-β1 mRNA and protein expression in cells after infection with an adenovirus expressing TGF-β1 short hairpin RNA (shRNA). A single base pair change in the shRNA sequence completely abrogated the suppressive effect on TGF-β1. Surprisingly, the suppression of TGF-β1 induced TGF-β3 upregulation, and the suppression of TGF-β2 induced another unexpected downregulation of both TGF-β1 and TGF-β3. Taken together, this information may prove useful when considering the design for a novel cancer immunogene therapy.

Thermodynamic basis of selectivity in guide‐target‐mismatched rna interference

Silencing in RNAi is strongly affected by guide-strand/target-mRNA mismatches. Target nucleation is thought to occur at positions 2-8 of the guide ("seed region"); successful hybridization in this region is the primary determinant of target-binding affinity and hence target cleavage. To define a molecular basis for the target sequence selectivity in RNAi, we studied all possible distinct single mismatches in seven positions of the seed region-a total of 21 substitutions. We report results from soft-core thermodynamic integration simulations to determine changes in targeting binding-free energies to Argonaute due to single mismatches in the guide strand, which arise during binding of an imperfectly matched target mRNA. In agreement with experiment, most mismatches impair target binding, consistent with a prominent role for binding affinity changes in RNAi sequence selectivity. Individual Argonaute residues located near the mismatched base pair are found to contribute significantly to binding affinity changes. We also use this methodology to analyze the mismatch-dependent free energy changes for dissociation of a DNA•RNA hybrid from Argonaute, as a model for the escape of miRNAs from the silencing pathway. Several mismatched sequences of the miRNA have increased affinity to Argonaute, implying that some mismatches may reduce the probability for escape. Furthermore, calculations of base-substitution-dependent free energy changes for binding ssDNA reveal mild sequence sensitivity as expected for guide strand binding to Argonaute. Our findings give a thermodynamic basis for RNAi target sequence selectivity and suggest that miRNA mismatches may increase silencing effectiveness and thus could be evolutionarily advantageous.

Selection of target sequences as well as sequence identity determine the outcome of RNAi approach for resistance against cotton leaf curl geminivirus complex

Cotton leaf curl disease is caused by a geminivirus complex that involves multiple distinct begomoviruses and a disease-specific DNA satellite, cotton leaf curl Multan betasatellite (CLCuMB), which is essential to induce disease symptoms. Here we have investigated the use of RNA interference (RNAi) for obtaining resistance against one of the viruses, Cotton leaf curl Multan virus (CLCuMV), associated with the disease. Three hairpin RNAi constructs were produced containing either complementary-sense genes essential for replication/pathogenicity or non-coding regulatory sequences of CLCuMV. In transient assays all three RNAi constructs significantly reduced the replication of the virus in inoculated tissues. However, only one of the constructs, that targeting the overlapping genes involved in virus replication and pathogenicity (the replication-associated protein (Rep), the transcriptional activator protein and the replication enhancer protein) was able to prevent systemic movement of the virus, although the other constructs significantly reduced the levels of virus in systemic tissues. In the presence of CLCuMB, however, a small number of plants co-inoculated with even the most efficient RNAi construct developed symptoms of virus infection, suggesting that the betasatellite may compromise resistance. Further analyses, using Rep gene sequences of distinct begomoviruses expressed from a PVX vector as the target, are consistent with the idea that the success of the RNAi approach depends on sequence identity to the target virus. The results show that selection of both the target sequence, as well as the levels of identity between the construct and target sequence, determine the outcome of RNAi-based resistance against geminivirus complexes.

Targeting specific genes for RNA interference is crucial to the development of strong resistance to Rice stripe virus

Rice stripe virus (RSV) has a serious negative effect on rice production in temperate regions of East Asia. Focusing on the putative importance of the selection of target sequences for RNA interference (RNAi), we analysed the effects of potential target sequences in each of the coding genes in the RSV genome, using transgenic rice plants that expressed a set of inverted-repeat (IR) constructs. The reactions of inoculated transgenic T(1) plants to RSV were divided subjectively into three classes, namely highly resistant, moderately resistant and lacking enhanced resistance to RSV, even though plants that harboured any constructs accumulated transgene-specific siRNAs prior to inoculation with RSV. Transgenic plants that harboured IR constructs specific for the gene for pC3, which encodes nucleocapsid protein, and for pC4, which encodes a viral movement protein, were immune to infection by RSV and were more resistant to infection than the natural resistant cultivars that have been used to control the disease in the field. By contrast, the IR construct specific for the gene for pC2, which encodes a glycoprotein of unknown function, and for p4, which encodes a major non-structural protein of unknown function, did not result in resistance. Our results indicate that not all RNAi constructs against viral RNAs are equally effective in preventing RSV infection and that it is important to identify the viral 'Achilles heel' for RNAi attack in the engineering of plants.