RNA interference (RNAi) is the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene. The mediators of sequence-specific messenger RNA degradation are 21- and 22-nucleotide small interfering RNAs (siRNAs) generated by ribonuclease III cleavage from longer dsRNAs. Here we show that 21-nucleotide siRNA duplexes specifically suppress expression of endogenous and heterologous genes in different mammalian cell lines, including human embryonic kidney (293) and HeLa cells. Therefore, 21-nucleotide siRNA duplexes provide a new tool for studying gene function in mammalian cells and may eventually be used as gene-specific therapeutics.

Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells

I read this book the same weekend that the Packers took on the Rams, and the experience of the latter event, obviously, colored my judgment. Although I abhor anything that smacks of being a handbook (like, \"How to Earn a Merit Badge in Neurosurgery\") because too many volumes in biomedical science already evince a boyscout-like approach, I must confess that parts of this volume are fast, scholarly, and significant, with certain reservations. I like parts of this well-illustrated book because Dr. Sj6strand, without so stating, develops certain subjects on technique in relation to the acquisition of judgment and sophistication. And this is important! So, given that the author (like all of us) is somewhat deficient in some areas, and biased in others, the book is still valuable if the uninitiated reader swallows it in a general fashion, realizing full well that what will be required from the reader is a modulation to fit his vision, propreception, adaptation and response, and the kind of problem he is undertaking. A major deficiency of this book is revealed by comparison of its use of physics and of chemistry to provide understanding and background for the application of high resolution electron microscopy to problems in biology. Since the volume is keyed to high resolution electron microscopy, which is a sophisticated form of structural analysis, but really morphology in a modern guise, the physical and mechanical background of The instrument and its ancillary tools are simply and well presented. The potential use of chemical or cytochemical information as it relates to biological fine structure , however, is quite deficient. I wonder when even sophisticated morphol-ogists will consider fixation a reaction and not a technique; only then will the fundamentals become self-evident and predictable and this sine qua flon will become less mystical. Staining reactions (the most inadequate chapter) ought to be something more than a technique to selectively enhance contrast of morphological elements; it ought to give the structural addresses of some of the chemical residents of cell components. Is it pertinent that auto-radiography gets singled out for more complete coverage than other significant aspects of cytochemistry by a high resolution microscopist, when it has a built-in minimal error of 1,000 A in standard practice? I don't mean to blind-side (in strict football terminology) Dr. Sj6strand's efforts for what is \"routinely used in our laboratory\"; what is done is usually well done. It's just that …

Methods in Enzymology.

Short interfering RNAs (siRNAs) are double-stranded RNAs of ’21‐25 nucleotides that have been shown to function as key intermediaries in triggering sequence-specific RNA degradation during posttranscriptional gene silencing in plants and RNA interference in invertebrates siRNAs have a characteristic structure, with 5*-phosphatey3*-hydroxyl ends and a 2-base 3* overhang on each strand of the duplex In this study, we present data that synthetic siRNAs can induce gene-specific inhibition of expression in Caenorhabditis elegans and in cell lines from humans and mice In each case, the interference by siRNAs was superior to the inhibition of gene expression mediated by single-stranded antisense oligonucleotides The siRNAs seem to avoid the well documented nonspecific effects triggered by longer double-stranded RNAs in mammalian cells These observations may open a path toward the use of siRNAs as a reverse genetic and therapeutic tool in mammalian cells

/pdf/specific-inhibition-of-gene-expression-by-small-double-4ot4xfxzyk.pdf

Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems.

Double-stranded RNA (dsRNA) induces sequence-specific post-transcriptional gene silencing in many organisms by a process known as RNA interference (RNAi). Using a Drosophila in vitro system, we demonstrate that 19-23 nt short RNA fragments are the sequence-specific mediators of RNAi. The short interfering RNAs (siRNAs) are generated by an RNase III-like processing reaction from long dsRNA. Chemically synthesized siRNA duplexes with overhanging 3′ ends mediate efficient target RNA cleavage in the lysate, and the cleavage site is located near the center of the region spanned by the guiding siRNA. Furthermore, we provide evidence that the direction of dsRNA processing determines whether sense or antisense target RNA can be cleaved by the produced siRNP complex.

Rna interference mediating small rna molecules

RNA interference (RNAi) is the process of sequence-specific post-transcriptional gene silencing triggered by double-stranded RNAs. In attempts to identify RNAi triggers that effectively function at lower concentrations, we found that synthetic RNA duplexes 25–30 nucleotides in length can be up to 100-fold more potent than corresponding conventional 21-mer small interfering RNAs (siRNAs). Some sites that are refractory to silencing by 21-mer siRNAs can be effectively targeted by 27-mer duplexes, with silencing lasting up to 10 d. Notably, the 27-mers do not induce interferon or activate protein kinase R (PKR). The enhanced potency of the longer duplexes is attributed to the fact that they are substrates of the Dicer endonuclease, directly linking the production of siRNAs to incorporation in the RNA-induced silencing complex. These results provide an alternative strategy for eliciting RNAi-mediated target cleavage using low concentrations of synthetic RNA as substrates for cellular Dicer-mediated cleavage.

https://www.idtdna.com/Scitools/Documents/Kim2005NBTv23p222.pdf

Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy.

Structural requirements of double-stranded RNA for the activation of 2',5'-oligo(A) polymerase and protein kinase of interferon-treated HeLa cells.

EXPOSURE of cells to the antiviral agent interferon inhibits virus replication1. In interferon-treated cells the adsorption, penetration and uncoating of infecting viruses is not affected, but the synthesis of virus-specific mRNA and viral proteins is inhibited2,3. We have studied a mechanism for degradation of mRNA which may be activated in interferon-treated cells by the presence of double-stranded RNA (dsRNA) of viral origin. Lengyel et al. have reported that an endonuclease present in extracts of cells treated with interferon is activated by dsRNA and ATP4–6. The endonuclease degrades mRNA by a process which can be divided into two phases: an activation phase requiring the presence of both ATP and dsRNA but not of mRNA, and an endonucleolytic phase, which does not require either ATP or dsRNA6. Kerr et al. have at the same time reported that protein synthesis is inhibited in extracts of cells treated with interferon after incubation with dsRNA and ATP7. In these incubation conditions a low molecular weight inhibitor is formed from ATP8,9. The mechanism of action of this inhibitor, the structure of which has recently been determined as pppA2′p5′A2′p5′A (pppApApA)10, is not known. Our results indicate that this trinucleotide mediates the activation of a nuclease which degrades mRNA and may therefore inhibit protein synthesis in this way.

Interferon action may be mediated by activation of a nuclease by pppA2'p5'A2'p5'A.

Synthesis of 2'5'-oligo(A) in extracts of interferon-treated HeLa cells.

Extracts of interferon-treated HeLa cells adsorbed to poly(I) . poly(C)-agarose have been used to synthesize 2'5'oligo(A). This oligonucleotide has been characterized by enzymatic digestion with alkaline phosphatase, snake venom phosphodiesterase, T2 ribonuclease and chromatography on DEAE, and PEI-cellulose. The oligonucleotide inhibits protein synthesis in vitro and activates an endonuclease present in extracts of control and interferon-treated cells. The metabolic stability of 2'5'oligo(A) has been investigated in these cell extracts. The oligonucleotide undergoes rapid degradation, particularly in the absence of ATP and of an energy regenerating system. Furthermore, the 2'5'oligo(A)-activated endonuclease reverts to an inactive state under these conditions, but can be reactivated upon further addition of 2'5'oligo(A). A possible role for the degradation of 2'5'oligo(A) in the mechanism of interferon action is discussed.

Metabolic stability of 2' 5'oligo (A) and activity of 2' 5'oligo (A)-dependent endonuclease in extracts of interferon-treated and control HeLa cells.

Activation of 2',5'-oligo(A) polymerase and protein kinase of interferon-treated HeLa cells by 2'-O-methylated poly (inosinic acid) . poly(cytidylic acid), Correlations with interferon-inducing activity.

Michael A. Minks

Papers

Structural requirements of double-stranded RNA for the activation of 2',5'-oligo(A) polymerase and protein kinase of interferon-treated HeLa cells.

Interferon action may be mediated by activation of a nuclease by pppA2'p5'A2'p5'A.

Synthesis of 2'5'-oligo(A) in extracts of interferon-treated HeLa cells.

Metabolic stability of 2' 5'oligo (A) and activity of 2' 5'oligo (A)-dependent endonuclease in extracts of interferon-treated and control HeLa cells.

Activation of 2',5'-oligo(A) polymerase and protein kinase of interferon-treated HeLa cells by 2'-O-methylated poly (inosinic acid) . poly(cytidylic acid), Correlations with interferon-inducing activity.