Showing papers on "Small hairpin RNA published in 2001"
TL;DR: In this article, the authors examined design rules for efficient gene silencing, in terms of both the proportion of independent transgenic plants showing silencing and the degree of silencing.
Abstract: Post-transcriptional silencing of plant genes using anti-sense or co-suppression constructs usually results in only a modest proportion of silenced individuals. Recent work has demonstrated the potential for constructs encoding self-complementary 'hairpin' RNA (hpRNA) to efficiently silence genes. In this study we examine design rules for efficient gene silencing, in terms of both the proportion of independent transgenic plants showing silencing, and the degree of silencing. Using hpRNA constructs containing sense/anti-sense arms ranging from 98 to 853 nt gave efficient silencing in a wide range of plant species, and inclusion of an intron in these constructs had a consistently enhancing effect. Intron-containing constructs (ihpRNA) generally gave 90-100% of independent transgenic plants showing silencing. The degree of silencing with these constructs was much greater than that obtained using either co-suppression or anti-sense constructs. We have made a generic vector, pHANNIBAL, that allows a simple, single PCR product from a gene of interest to be easily converted into a highly effective ihpRNA silencing construct. We have also created a high-throughput vector, pHELLSGATE, that should facilitate the cloning of gene libraries or large numbers of defined genes, such as those in EST collections, using an in vitro recombinase system. This system may facilitate the large-scale determination and discovery of plant gene functions in the same way as RNAi is being used to examine gene function in Caenorhabditis elegans.
1,642 citations
Journal Article•
TL;DR: A generic vector is made that allows a simple, single PCR product from a gene of interest to be easily converted into a highly effective ihpRNA silencing construct, and a high-throughput vector that should facilitate the cloning of gene libraries or large numbers of defined genes, such as those in EST collections, using an in vitro recombinase system.
Abstract: Post-transcriptional silencing of plant genes using anti-sense or co-suppression constructs usually results in only a modest proportion of silenced individuals. Recent work has demonstrated the potential for constructs encoding self-complementary 'hairpin' RNA (hpRNA) to efficiently silence genes. In this study we examine design rules for efficient gene silencing, in terms of both the proportion of independent transgenic plants showing silencing, and the degree of silencing. Using hpRNA constructs containing sense/anti-sense arms ranging from 98 to 853 nt gave efficient silencing in a wide range of plant species, and inclusion of an intron in these constructs had a consistently enhancing effect. Intron-containing constructs (ihpRNA) generally gave 90-100% of independent transgenic plants showing silencing. The degree of silencing with these constructs was much greater than that obtained using either co-suppression or anti-sense constructs. We have made a generic vector, pHANNIBAL, that allows a simple, single PCR product from a gene of interest to be easily converted into a highly effective ihpRNA silencing construct. We have also created a high-throughput vector, pHELLSGATE, that should facilitate the cloning of gene libraries or large numbers of defined genes, such as those in EST collections, using an in vitro recombinase system. This system may facilitate the large-scale determination and discovery of plant gene functions in the same way as RNAi is being used to examine gene function in Caenorhabditis elegans.
1,446 citations
TL;DR: Genetic studies have expanded the biology of RNAi to cosuppression, transposon silencing, and the first hints of relationships to regulation of translation and development, as well as expanding the possible roles of RNA-dependent RNA polymerase (RdRp) in RNAi.
Abstract: In the few years since the discovery of RNA interference (RNAi; Fire et al. 1998), it has become clear that this process is ancient. RNAi, the oldest and most ubiquitous antiviral system, appeared before the divergence of plants and animals. Because aspects of RNAi, known as cosuppression, also control the expression of transposable elements and repetitive sequences (Ketting et al. 1999; Tabara et al. 1999), the interplay of RNAi and transposon activities have almost certainly shaped the structure of the genome of most organisms. Surprisingly, we are only now beginning to explore the molecular processes responsible for RNAi and to appreciate the breadth of its function in biology. Practical applications of this knowledge have allowed rapid surveys of gene functions (see Fraser et al. 2000 and Gönczey et al. 2000 for RNAi analysis of genes on chromosome I and III of Caenorhabditis elegans) and will possibly result in new therapeutic interventions. Genetic studies have expanded the biology of RNAi to cosuppression, transposon silencing, and the first hints of relationships to regulation of translation and development. The possible roles of RNA-dependent RNA polymerase (RdRp) in RNAi have been expanded. Many experiments indicate that dsRNA directs gene-specific methylation of DNA and, thus, regulation at the stage of transcription in plants. Cosuppression may involve regulation by polycomb complexes at the level of transcription in C. elegans and Drosophila. This article will review these topics and primarily summarize advances in the study of RNAi over the past year.
774 citations
Patent•
16 Mar 2001
TL;DR: In this paper, a hairpin RNA was used to attenuate gene expression in a cell, especially in a mammalian cell, using gene-targeted double stranded RNA (dsRNA).
Abstract: The present invention provides methods for attenuating gene expression in a cell, especially in a mammalian cell, using gene-targeted double stranded RNA (dsRNA), such as a hairpin RNA. The dsRNA contains a nucleotide sequence that hybridizes under physiologic conditions of the cell to the nucleotide sequence of at least a portion of the gene to be inhibited (the “target” gene).
599 citations
TL;DR: The discovery of RNAi has changed the understanding of how cells guard their genomes, led to the development of new strategies for blocking gene function, and may yet yield RNA-based drugs to treat human disease.
Abstract: The term RNA interference (RNAi) describes the use of double-stranded RNA to target specific mRNAs for degradation, thereby silencing their expression. RNAi is one manifestation of a broad class of RNA silencing phenomena that are found in plants, animals and fungi. The discovery of RNAi has changed our understanding of how cells guard their genomes, led to the development of new strategies for blocking gene function, and may yet yield RNA-based drugs to treat human disease.
447 citations
TL;DR: This minireview will highlight recent advances in understanding the molecular mechanism of RNAi and its biological function and identify components of the RNAi machinery required for posttranscriptional silencing by cosuppression.
Abstract: The term aRNA interferenceo (RNAi) was coined after the groundbreaking discovery that injection of double-stranded RNA (dsRNA) into the nematode Caenorhabditis elegans leads to specific silencing of genes highly homologous in sequence to the delivered dsRNA. The RNAi phenotype is either identical to the genetic null mutant or resembles an allelic series of mutants. The dsRNA can also be delivered by feeding bacteria that express dsRNA from recombinant plasmids to the worm or by soaking the worm in a solution containing the dsRNA. 3] In rapid sequence, RNAi was observed in other animals including mice, 5] and therefore this process possibly exists also in humans. RNAi appears to be related to the posttranscriptional gene silencing (PTGS) mechanism of cosuppression in plants and quelling in fungi. Cosuppression is the ability of some transgenes to silence both themselves and homologous chromosomal loci simultaneously. The initiator molecule for cosuppression is believed to be aberrant RNA, possibly dsRNA, and some components of the RNAi machinery are required for posttranscriptional silencing by cosuppression. 8, 13] The natural function of RNAi and cosuppression is thought to be protection of the genome against invasion by mobile genetic elements such as transposons and viruses, which produce aberrant RNA or dsRNA in the host cell when they become active. Thus, specific mRNA degradation is thought to prevent transposon and virus replication. This minireview will highlight recent advances in understanding the molecular mechanism of RNAi and its biological function. The reader is also referred to a number of excellent reviews that have appeared recently (see refs. [18 ± 24]).
426 citations
TL;DR: Results of the experiments indicated that hairpin dsRNA was just as effective as ds RNA in promoting the destruction of targeted mRNA, the EGFP marker could be expressed from the construct, and the distance of the SV40 intron from the inverted repeat was critical for the transcribed RNA to function in RNAi.
146 citations
TL;DR: A modified RNA interference method for generating gene knock-outs in Drosophila melanogaster using the sequence of the yellow (y) locus to construct an inverted repeat that will form a double-stranded hairpin structure (y-IR) that is under the control of the upstream activating sequence (UAS) of the yeast transcriptional activator GAL4.
Abstract: We have developed a modified RNA interference (RNAi) method for generating gene knock-outs in Drosophila melanogaster. We used the sequence of the yellow (y) locus to construct an inverted repeat that will form a double-stranded hairpin structure (y-IR) that is under the control of the upstream activating sequence (UAS) of the yeast transcriptional activator GAL4. Hairpins are extremely difficult to manipulate in Escherichia coli, so our method makes use of a heterologous 330 bp spacer encoding sequences from green fluorescent protein to facilitate the cloning steps. When the UAS–y–IR hairpin is expressed under the control of different promoter–GAL4 fusions, a high frequency of y pigment phenocopies is obtained in adults. Consequently this method for producing gene knock-outs has several advantages over previous methods in that it is applicable to any gene within the fly genome, greatly facilitates cloning of the hairpin, can be used if required with GAL4 drivers to avoid lethality or to induce RNAi in a specific developmental stage and/or tissue, is useful for generating knock-outs of adult phenotypes as reported here and, finally, the system can be manipulated to investigate the trans-acting factors that are involved in the RNAi mechanism.
144 citations
Patent•
07 Nov 2001
TL;DR: In this article, a process is provided to deliver small interfering RNA to cells in vivo for the purpose of inhibiting gene expression in that cell, which is performed on post-embryonic animals.
Abstract: A process is provided to deliver small interfering RNA to cells in vivo for the purpose of inhibiting gene expression in that cell. The small interfering RNA is less than 50 base-pairs in length. This process is practiced on post-embryonic animals. Inhibition is sequence-specific and relies on sequence identity of the small interfering RNA and the target nucleic acid molecule.
117 citations
TL;DR: It is proposed that the natural strand-transfer reaction is enhanced by interaction of the anti-TAR ssDNA hairpin with the TAR hairpin in the 3'R acceptor, which can also fold a hairpin structure, that is critical for strand transfer.
Abstract: Two obligatory DNA strand transfers take place during reverse transcription of a retroviral RNA genome. The first strand transfer is facilitated by terminal repeat (R) elements in the viral genome. This strand-transfer reaction depends on base pairing between the cDNA of the 5'R and the 3'R. There is accumulating evidence that retroviral R regions contain features other than sequence complementarity that stimulate this critical nucleic acid hybridization step. The R region of the human immunodeficiency virus type 1 (HIV-1) is relatively extended (97 nt) and encodes two well-conserved stem-loop structures, the TAR and poly(A) hairpins. The role of these motifs was studied in an in vitro strand-transfer assay with two separate templates, the 5'R donor and the 3'R acceptor, and mutants thereof. The results indicate that the upper part of the TAR hairpin structure in the 5'R donor is critical for efficient strand transfer. This seems to pose a paradox, as the 5'R template is degraded by RNase H before strand transfer occurs. We propose that it is not the RNA hairpin motif in the 5'R donor, but rather the antisense motif in the ssDNA copy, which can also fold a hairpin structure, that is critical for strand transfer. Mutation of the loop sequence in the TAR hairpin of the donor RNA, which is copied in the loop of the cDNA hairpin, reduces the transfer efficiency more than fivefold. It is proposed that the natural strand-transfer reaction is enhanced by interaction of the anti-TAR ssDNA hairpin with the TAR hairpin in the 3'R acceptor. Base pairing can occur between the complementary loops ("loop-loop kissing"), and strand transfer is completed by the subsequent formation of an extended RNA-cDNA duplex.
103 citations
TL;DR: The results suggest that the binding of DbpA to RNA requires sequence-specific interactions with hairpin 92 as well as nonspecific interactions with the RNA extension, which is a critical recognition element for DBPA.
Abstract: The Escherichia coli DEAD protein DbpA is an RNA-specific ATPase that is activated by a 153-nt fragment within domain V of 23S rRNA. A series of RNA subfragments and sequence changes were used to identify the recognition elements of this RNA-protein interaction. Reducing the size of the fully active 153-nt RNA yields compromised substrates in which both RNA and ATP binding are weakened considerably without affecting the maximal rate of ATP hydrolysis. All RNAs that stimulate ATPase activity contain hairpin 92 of 23S rRNA, which is known to interact with the 3' end of tRNAs in the ribosomal A-site. RNAs with base mutations within this hairpin fail to activate ATP hydrolysis, suggesting that it is a critical recognition element for DbpA. Although the isolated hairpin fails to activate DbpA, RNAs with an extension of approximately 15 nt on either the 5' or 3' side of hairpin 92 elicit full ATPase activity. These results suggest that the binding of DbpA to RNA requires sequence-specific interactions with hairpin 92 as well as nonspecific interactions with the RNA extension. A model relating the RNA binding and ATPase activities of DbpA is presented.
TL;DR: It is unequivocally demonstrated that the DIS hairpin is the main determinant for HIV-2 RNA dimerization, supported by phylogenetic analysis of leader RNA sequences from different viral isolates, indicating that RNA genomeDimerization occurs by a similar mechanism for all members of the human and simian immunodeficiency viruses.
TL;DR: A quantitative study of the effects of RNAi in cultured plant cells that are uniform and divide synchronously for functional analysis of genes of interest and constructed dsRNA expression plasmids for a luciferase gene under the control of the cauliflower mosaic virus 35S promoter by simply connecting sense and antisense sequences in a head-to-head manner.
Abstract: Suppression by double-stranded RNA (dsRNA) of the expression of a target gene is known as RNA interference (RNAi). No quantitative analysis of the effects of RNAi on the expression of specific genes in cultured plant cells has been reported. However, as it is possible to produce populations of cultured plant cells that are uniform and divide synchronously for functional analysis of genes of interest, we performed a quantitative study of the effects of RNAi in such cells. We constructed dsRNA expression plasmids for a luciferase gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter by simply connecting sense and antisense sequences in a head-to-head manner. An RNAi effect was observed 24 hours after the introduction of dsRNA expression plasmids into tobacco BY-2 cells by electroporation. The simple system for suppression of specific genes in plant cells should be useful in attempts to elucidate the roles of individual genes in plant cells.
TL;DR: In vitro analysis with a partially purified TCV RdRp preparation demonstrated that the motif1-hairpin in its minus-sense orientation was able to stimulate RNA synthesis from the satC hairpin promoter by almost twofold, supporting the hypothesis that the design is primarily involved in enhancing plus-strand synthesis.
TL;DR: The results suggest that endonuclease activity is absolutely dependent on the presence of a 3′ hairpin loop structure, the first direct evidence for RNA secondary structure within the 3′ arm being required for a specific stage, i.e., end onuclease cleavage, in the influenza virus replicative cycle.
Abstract: Previous studies have shown that the 5′ arm of the influenza A virus virion RNA promoter requires a hairpin loop structure for efficient endonuclease activity of influenza virus RNA polymerase, an activity that is required for the cap-snatching activity of primers from host pre-mRNA. Here we examine whether a hairpin loop is also required in the 3′ arm of the viral RNA promoter. We study point mutations at each nucleotide position (1 to 12) within the 3′ arm of the promoter as well as complementary “rescue” mutations which restored base pairing in the stem of a potential hairpin loop. Our results suggest that endonuclease activity is absolutely dependent on the presence of a 3′ hairpin loop structure. This is the first direct evidence for RNA secondary structure within the 3′ arm being required for a specific stage, i.e., endonuclease cleavage, in the influenza virus replicative cycle.
Patent•
10 May 2001
TL;DR: In this article, the authors proposed methods to produce RNA viral sequences, recombinant RNA viruses, mutants of RNA viruses and RNA virus-derived vectors in cell culture and in vitro using non-viable, replication defective, helper vaccinia recombinants.
Abstract: The invention provides methods to produce RNA viral sequences, recombinant RNA viruses, mutants of RNA viruses and RNA virus-derived vectors in cell culture and in vitro using non-viable, replication defective, helper vaccinia recombinants. These methods allow generation of RNA virus sequences and viral particles in cell culture and in vitro independent of their natural replication pathways, bypassing the limitation of any cellular barriers. The invention also provides novel RNA viral sequences and viral particles using these methods.
Patent•
30 Mar 2001TL;DR: In this article, a Drosophila in vitro system was used to demonstrate that dsRNA is processed to RNA segments 21-23 nucleotides (nt) in length.
Abstract: The present invention relates to a Drosophila in vitro system which was used to demonstrate that dsRNA is processed to RNA segments 21-23 nucleotides (nt) in length. Furthermore, when these 21-23 nt fragments are purified and added back to Drosophila extracts, they mediate RNA interference in the absence of long dsRNA. Thus, these 21-23 nt fragments are the sequence-specific mediators of RNA degradation. A molecular signal, which may be their specific length, must be present in these 21-23 nt fragments to recruit cellular factors involved in RNAi. This present invention encompasses these 21-23 nt fragments and their use for specifically inactivating gene function.; The use of these fragments (or chemically synthesized oligonucleotides of the same or similar nature) enables the targeting of specific mRNAs for degradation in mammalian cells, where the use of long dsRNAs to elicit RNAi is usually not practical, presumably because of the deleterious effects of the interferon response. This specific targeting of a particular gene function is useful in functional genomic and therapeutic applications.
TL;DR: It is concluded that HpRz prepared in vitro possesses specific catalytic activity, indicating that it is possible for H pRz to intracellularly inhibit the replication of HBV.
Abstract: To study the preparation and cleavage of hairpin ribozyme (HpRz) directed against the transcript of HBV core regionin vitro, HRz gene designed by computer targeting the transcript of HBV core gene was cloned into the vector p1. 5 between 5′-cis-Rz and 3′-cis-Rz.32P-labeled HpRz transcript proved whether the vector fit for the preparation of hairpin ribozyme.32P-labeled pKC transcript containing HBV core region as targets-RNA was transcribed by using T7 RNA polymerase and purified by PAGE. Cold HpRz transcript was incubated with32P-labeled target-RNAs under different conditions and radioautographed after denaturing polyacrylamide gel electrophoresis. The results showed that HpRz had the ability of cleavage at 37°C and 12 mmol/L MgCl2 and the design of ribozyme was correct. It is concluded that HpRz preparedin vitro possesses specific catalytic activity, indicating that it is possible for HpRz to intracellularly inhibit the replication of HBV. It may be developed into a nucleic acid drug in the treatment of hepatitis B in the future.
TL;DR: Specific inhibition of gene expression by exogenous homologous double-stranded RNA in invertebrates and in the early development of vertebrates is termed RNA interference and RNA interference was also studied in cells cotransfected with plasmids directing synthesis of sense and antisense RNAs.
Abstract: Specific inhibition of gene expression by exogenous homologous double-stranded RNA (dsRNA) in invertebrates and in the early development of vertebrates is termed RNA interference. Cultured cells were cotransfected with reporter plasmids and dsRNA. The inhibitory effect on reporter gene expression depended on the extent of homology between dsRNA and the target gene. RNA interference was also studied in cells cotransfected with plasmids directing synthesis of sense and antisense RNAs. Production of antisense RNA only slightly inhibited expression of the reporter gene. Simultaneous expression of both sense and antisense RNAs caused by cotransfection by corresponding plasmids did not inhibit expression of the reporter construct.
Patent•
15 May 2001TL;DR: In this article, an expression system was proposed to reproduce conditions allowing the exertion of the function of a viral protein along the inherent life cycle of the virus and, at the same time, evaluate with the use of a reporter gene without forming any infective virus.
Abstract: It is intended to establish an expression system whereby conditions allowing the exertion of the function of a viral protein can be reproduced along the inherent life cycle of the virus and, at the same time, the function of the viral protein can be evaluated with the use of a reporter gene without forming any infective virus. Thus, an assay system which contains a vector containing a DNA encoding an RNA-dependent RNA polymerase originating in RNA and a reporter gene expressed in the antisense direction is provided.
Patent•
29 Nov 2001
TL;DR: Application of invention ensures target-gene silencing, mediated by target-specific RNA interference, in medicine and in pharmaceutical industry.
Abstract: FIELD: medicine, pharmaceutics. SUBSTANCE: invention relates to field of molecular biology and biotechnology and can be used in medicine and in pharmaceutical industry. RNA molecule, capable of target-specific RNA interference, represents double-stranded RNA molecule, 23 nucleotides long, which has 3'-overhang from 1-5 nucleotides. It is obtained by joining two RNA strands and used for obtaining pharmaceutical composition. When introduced into multicellular eukaryotic organism or in a cell of multicellular eukaryotic organism, said RNA molecule ensures promotion of target-specific RNA interference and leads to reduction of target-gene expression level or to target-gene knockout. Method of promoting target-specific RNA interference by means of said RNA molecule is applied for determination or modulation of gene function. Cell, containing endogenic target nucleic acid, RNA molecule, capable of target-specific RNA interference, and exogenic target nucleic acid, is used in analytical procedures. EFFECT: application of invention ensures target-gene silencing, mediated by target-specific RNA interference. 40 cl, 23 dwg, 3 ex
Journal Article•
TL;DR: Adenine deamination in double-stranded RNA by specific ADENine deaminases efficiently blocks RNA interference in vitro as mentioned in this paper, but not in the presence of interference.
Abstract: Adenine deamination in double-stranded RNA by specific adenine deaminases efficiently blocks RNA interference in vitro