Designing siRNA that distinguish between genes that differ by a single nucleotide
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE5291 | GEO | 2006/08/04
SECONDARY ACCESSION(S): PRJNA96413
REPOSITORIES: GEO
Similar Datasets
Project description:Small interfering RNAs (siRNAs), the guides that direct RNA interference (RNAi), provide a powerful tool to reduce the expression of a single gene in human cells. Ideally, dominant, gain-of-function human diseases could be treated using siRNAs that specifically silence the mutant disease allele, while leaving expression of the wild-type allele unperturbed. Previous reports suggest that siRNAs can be designed with single nucleotide specificity, but no rational basis for the design of siRNAs with single nucleotide discrimination has been proposed. We systematically identified siRNAs that discriminate between the wild-type and mutant alleles of two disease genes: the human Cu, Zn superoxide dismutase (SOD1) gene, which contributes to the progression of hereditary amyotrophic lateral sclerosis through the gain of a toxic property, and the huntingtin (HTT) gene, which causes Huntington disease when its CAG-repeat region expands beyond approximately 35 repeats. Using cell-free RNAi reactions in Drosophila embryo lysate and reporter assays and microarray analysis of off-target effects in cultured human cells, we identified positions within an siRNA that are most sensitive to mismatches. We also show that purine:purine mismatches imbue an siRNA with greater discriminatory power than other types of base mismatches. siRNAs in which either a G:U wobble or a mismatch is located in the "seed" sequence, the specialized siRNA guide region responsible for target binding, displayed lower levels of selectivity than those in which the mismatch was located 3' to the seed; this region of an siRNA is critical for target cleavage but not siRNA binding. Our data suggest that siRNAs can be designed to discriminate between the wild-type and mutant alleles of many genes that differ by just a single nucleotide.
| S-EPMC1560399 | biostudies-literature
Project description:The use of DNA microarrays to identify nucleotide variation is almost 20 years old. A variety of improvements in probe design and experimental conditions have brought this technology to the point that single-nucleotide differences can be efficiently detected in unmixed samples, although developing reliable methods for detection of mixed sequences (e.g. heterozygotes) remains challenging. Surprisingly, a comprehensive study of the probe design parameters and experimental conditions that optimize discrimination of single nucleotide polymorphisms (SNPs) has yet to be reported, so the limits of this technology remain uncertain. By targeting 24,549 SNPs that differ between two Saccharomyces cerevisiae strains, we studied the effect of SNPs on hybridization efficiency to DNA microarray probes of different lengths under different hybridization conditions. We found that the critical parameter for optimization of sequence discrimination is the relationship between probe melting temperature (Tm) and the temperature at which the hybridization reaction is performed. This relationship can be exploited through the design of microarrays containing probes of equal Tm by varying the length of probes. We demonstrate that using such a microarray we detect >90% homozygous SNPs and >80% heterozygous SNPs using the SNPScanner algorithm. The optimized design and experimental parameters determined in this study should guide DNA microarray designs for applications that require sequence discrimination such as mutation detection, genotyping of unmixed and mixed samples and allele-specific gene expression. Moreover, designing microarray probes with optimized sensitivity to mismatches should increase the accuracy of standard microarray applications such as copy number variation detection and gene expression analysis. Keyword(s): MutationDetection_CGH Comparison of polymorphic vs non-polymorphic genomes when hybridrized to short probe microarrays
2010-05-18 | E-GEOD-19319 | biostudies-arrayexpress
Project description:The use of DNA microarrays to identify nucleotide variation is almost 20 years old. A variety of improvements in probe design and experimental conditions have brought this technology to the point that single-nucleotide differences can be efficiently detected in unmixed samples, although developing reliable methods for detection of mixed sequences (e.g. heterozygotes) remains challenging. Surprisingly, a comprehensive study of the probe design parameters and experimental conditions that optimize discrimination of single nucleotide polymorphisms (SNPs) has yet to be reported, so the limits of this technology remain uncertain. By targeting 24,549 SNPs that differ between two Saccharomyces cerevisiae strains, we studied the effect of SNPs on hybridization efficiency to DNA microarray probes of different lengths under different hybridization conditions. We found that the critical parameter for optimization of sequence discrimination is the relationship between probe melting temperature (Tm) and the temperature at which the hybridization reaction is performed. This relationship can be exploited through the design of microarrays containing probes of equal Tm by varying the length of probes. We demonstrate that using such a microarray we detect >90% homozygous SNPs and >80% heterozygous SNPs using the SNPScanner algorithm. The optimized design and experimental parameters determined in this study should guide DNA microarray designs for applications that require sequence discrimination such as mutation detection, genotyping of unmixed and mixed samples and allele-specific gene expression. Moreover, designing microarray probes with optimized sensitivity to mismatches should increase the accuracy of standard microarray applications such as copy number variation detection and gene expression analysis. Keyword(s): MutationDetection_CGH
2009-12-10 | GSE19319 | GEO
Project description:Members of the ribonuclease (RNase) III family regulate gene expression by processing double-stranded (ds) RNA. The founding member of the family, Escherichia coli (Ec) RNase III, is the most comprehensively studied and its E38A mutant (EcE38A) is an economical reagent for the preparation of small interfering (si) RNA cocktails. Previously, it was shown that EcRNase III recognizes dsRNA with little specificity and that EcE38A mainly produces 23-nucleotide (nt) siRNAs. To characterize substrate specificity and product size, we performed in vitro cleavage of dsRNAs by bacterial RNase IIIs and delineated the cleavage products by next generation sequencing. Surprisingly, we found that RNase III cleaves dsRNA at preferred sites and most siRNAs produced by EcE38A are 22 nt long. We eliminated the sequence specificity of EcE38A through the introduction of additional mutations, thereby creating a reagent that is ideally suited for producing heterogeneous siRNA cocktails to be used in gene silencing studies.
2019-05-26 | GSE120052 | GEO
Project description:Interventions: Endoscopic examinations of mucosal membrane using three-dimensional optical coherence tomography probe.
Primary outcome(s): Discrimination ability of mucosal membrane structure. Discrimination ability of cancer tissue.
Study Design: Single arm Non-randomized
| 2618337 | ecrin-mdr-crc
Project description:We tested the ability of trans siRNAs to establish a silent allele of endogenous ade6+ or an ade6+ transgene inserted within euchromatic loci. We found that a silent ade6-OFF epi-allele that grows red on low adenine medium could be established in several mutant backgrounds (mlo3∆, leo1∆) in the presence of ectopic siRNAs. Furthermore, we found that the silent ade6-OFF epi-allele could be meiotically inherited to wildtype progeny lacking the establishing mutation or ectopic siRNAs. Here, we sequenced genome-wide small RNAs and found widespread generation of secondary siRNAs at genes surrounding the ade6-OFF locus in cells expressing the ectopic siRNAs source cen::ade6. In wildtype meiotic progeny lacking the driver cen::ade6 siRNAs, we observed establishment of a local siRNA producing locus at genes adjacent to the endogenous ade6-OFF or at a kanMX-ade6-OFF transgene inserted within euchromatic loci. In contrast, spreading of secondary siRNAs was limited or nonexistent in ade6-ON cells. Finally, we found that spreading of secondary siRNAs at the endogenous ade6 locus was limited when an ade6 hairpin construct served as the ectopic siRNA driver locus instead of cen::ade6.
2018-04-26 | GSE111858 | GEO
Project description:Allele-specific circular chromosome conformation capture sequencing (4C-seq) using the single nucleotide polymorphism rs2836411 as a bait was performed in two cell types: human umibilical vein endothelial cells (HUVECs) and human aortic smooth muscle cells (HASMCs).
2019-10-11 | GSE137288 | GEO
Project description:Establishing the functional roles of genetic variants remains a significant challenge in the post-genomic era. Here, we present a method, allele-specific alternative mRNA processing (ASARP), to identify genetically influenced mRNA processing events using transcriptome sequencing (RNA-Seq) data. The method examines RNA-Seq data at both single nucleotide and whole-gene/isoform levels to identify allele-specific expression (ASE) and existence of allele-specific regulation of mRNA processing. We applied the methods to data obtained from the human glioblastoma cell line U87MG and primary breast cancer tissues and found that 26M-bM-^@M-^S45% of all genes with sufficient read coverage demonstrated ASE, with significant overlap between the two cell types. Our methods predicted potential mechanisms underlying ASE due to regulations affecting either whole-gene-level expression or alternative mRNA processing, including alternative splicing, alternative polyadenylation and alternative transcriptional initiation. Allele-specific alternative splicing and alternative polyadenylation may explain ASE in hundreds of genes in each cell type. Reporter studies following these predictions identified the causal single nucleotide variants (SNVs) for several allele-specific alternative splicing events. Finally, many genes identified in our study were also reported as disease/phenotype-associated genes in genome-wide association studies. Future applications of our approach may provide ample insights for a better understanding of the genetic basis of gene regulation underlying phenotypic diversity and disease mechanisms. Examine allele-specific gene expression and alternative RNA processing in U87MG cell line
2012-03-29 | E-GEOD-29738 | biostudies-arrayexpress
Project description:Our fine-mapping of 76 non-MHC loci associated with risk for rheumatoid arthritis (RA, 11,475 cases, 15,870 controls) has recently identified rs117701653, a non-coding single nucleotide polymorphism (SNP) in the CTLA4/CD28/ICOS locus, as the variant most likely to modulate RA risk within this region, with the minor allele (C) showing disease protection compared to the major allele (A). Notably, this SNP exhibits allele-specific protein binding, further supporting its regulatory nature, including greater binding of proteins from Jurkat T cell nuclear extract to the protective allele (C) than to the risk allele (A) by electrophoretic mobility shift assay (EMSA). To identify this protein or protein complex, we applied an efficient DNA pulldown technique, flanking restriction enhanced pulldown (FREP), using nuclear extract from Jurkat T cells, bait DNA corresponding to the (C) allele of rs117701653, competitor DNA fragment, and irrelevant DNA sequence as a negative control. Mass spectrometry analysis of peptides released from FREP identified 43 proteins. Our strongest candidate was structural maintenance of chromosomes flexible hinge domain-containing protein 1 (SMCHD1), which exhibited specific binding to the (C) allele of rs117701653. We confirmed the allelic affinity of SMCHD1 using multiple orthogonal approaches, including FREP and western blotting, EMSA with anti-SMCHD1 antibodies, and CHIP-qPCR with CRISPR-modified Jurkat clones bearing different genotype at rs117701653. In this study, we identified SMCHD1, a chromatin regulator that binds allelically to the rs117701653 allele (C) associated with protection against RA risk.
2024-05-23 | PXD048977 | Pride
Project description:Genome-wide association studies implicate multiple loci in risk for systemic lupus erythematosus (SLE), but few contain exonic variants, rendering systematic identification of non-coding variants essential to decoding SLE genetics. We utilized SNP-seq and bioinformatic enrichment to interrogate 2180 single-nucleotide polymorphisms (SNPs) from 87 SLE risk loci for potential binding of transcription factors and related proteins from B cells. 52 SNPs that passed initial screening were tested by electrophoretic mobility shift (EMSA) and luciferase reporter assays. To identify binding of transcription factors and/or other nuclear proteins in an allele-determined manner, we employed pulldown using nuclear extract from Daudi cells and silver staining in SNPs that had exhibited allele-specific differential binding by EMSA. Each pulldown product for each allele of the five high-probability SNPs (rs2297550 C/G, rs13213604 C/G, rs276461 T/C, rs9907955 C/T, rs7302634 T/C) was evaluated by mass spectrometry (MS) to identify binding nuclear proteins, yielding a set of candidate proteins for each.
2024-08-02 | PXD048367 | Pride