Methods and Compositions Relating to Gene Silencing

Inactive Publication Date: 2008-08-28
NEW ENGLAND BIOLABS
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0043]FIG. 6A shows that a GFP hsiRNA mixture effectively silences green fluorescent protein (GFP) expression in HEK-293 cells using fluorescence microscopy. (i) control in which cells have been transfected with a plasmid containing GFP cDNA; and (ii) cells transfected with a plasmid containing GFP cDNA and hsiRNA corresponding to GFP (Example III).
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Problems solved by technology

A standard method for generating siRNA relies on an inherently expensive chemical synthesis of a pre-determined short sequence.
Problems associated with using crude cell extracts containing a putative cleavage enzyme are for example, that it is unclear what proteins in the mixture of proteins are necessary and sufficient to generate the observed effect.
In addition, the extract is relatively inefficient at cleaving large double-stranded RNA with only a relatively small amount of the starting material being cleaved to the desired size in vitro even under extended incubation times. (Paddison et al., Proc. Natl. Acad. Sci.
Limitations of this approach include the cost of baculovirus expression systems, the i

Method used

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  • Methods and Compositions Relating to Gene Silencing
  • Methods and Compositions Relating to Gene Silencing
  • Methods and Compositions Relating to Gene Silencing

Examples

Experimental program
Comparison scheme
Effect test

example i

Preparation of an hsiRNA Mixture

[0131]Determining the Effect of Manganese Ions on Cleavage of dsRNA by RNaseIII.

[0132]Full length double-stranded RNA (dsRNA) corresponding to the gene of interest, in this Example hu PKR, was generated using the HiScribe M RNAi Transcription Kit (New England Biolabs, Inc., Beverly, Mass.). Methods for creating double-stranded RNA are described in detail in Example VII.

[0133]A 0.4 kb double-stranded RNA molecule (0.25 μg) was digested with 30 units E. coli RNaseIII (0.9 μg) (New England Biolabs, Inc., Beverly, Mass.) in 20 μl of buffer consisting of 100 mM NaCl, 50 mM Tris-HCl, 5, 10, 20 or 50 mM MnCl2 or 10 mM MgCl2 (control), 1 mM dithiothreitol (pH 7.5@25° C.) at 37° C. Samples containing 50-100 mM MnCl2 are also tested to provide complete digestion of the long double-stranded RNA.

[0134]Digestion products of RNaseIII in the presence of various concentrations of manganese ion were enriched in the size range of 18-25 bp. The mixture of fragments obta...

example ii

Preparation of hsiRNA Using Various Divalent Metal Ions

[0144]To determine the effect of various divalent cations on the cleavage products of RNaseIII, the following experiment was undertaken: 1 μg of a large double-stranded RNA molecule (800 bp) was digested with each of two concentrations of E. coli RNaseIII (0.04 μg / μl or 0.02 μg / μl) at pH 7.5 (25° C.) in 50 μL of buffer containing 100 mM NaCl, 50 mM Tris-HCl, 1 mM dithiothreitol and either 10 mM MgCl2 at 37° C. (lanes 1 and 2), 10 mM MnCl2 (lanes 3 and 4) 10 mM CoCl2 (lanes 5 and 6) or 10 mM NiSO4 (lanes 7 and 8) for 30 minutes. The results are shown in FIG. 2. A double-stranded RNA product having an approximate size of 22 bp (within a range of 20 bp-40 bp) was produced by complete digestion of the large double-stranded RNA in the presence of 0.04 μg / μl RNaseIII and 10 mM manganese ions. Digestion with 0.04 μg / μl RNaseIII in the presence of 10 mM Mg2+ buffer absent manganese ions produced fragments which were smaller than the des...

example iii

Short Double-Stranded RNA Cleavage Products of RNaseIII Digestion Contain Sequences Representing the Entire Parent Sequence

[0145]The DNA template for transcription of p53 (1.1 kb fragment encoding amino acids 100-393) was digested with the restriction enzyme AciI and the resulting fragments separated on an agarose gel. The gel was ethidium-stained, photographed and subsequently transferred to a nylon membrane (Hybond® N+, Amersham, Piscataway N.J.).

[0146]Double-stranded RNA synthesized by in vitro transcription of the 1.1 kb fragment was digested with RNaseIII at a final concentration of 0.04 μg / μl in the presence of 10 mM Mn++ at pH 7.5 and 25° C. for 30 minutes as described in Example II and the products were separated on a 20% native polyacrylamide gel.

[0147]The products of the digestion (the hsiRNA mixture) were visualized by ethidium bromide staining and the fraction corresponding to about 21 bp was excised in a small gel slice and purified by electro-elution for 20 min in a sm...

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Abstract

A method for obtaining a mixture of heterogenous short double-stranded RNA molecules suitable for use in gene silencing (hsiRNA) by subjecting large double-stranded RNA to enzymatic cleavage under specified conditions. The resulting mixture consistently includes enhanced representation of fragments having a size of 21-22 nucleotides absent any fractionation step. The fragments contain sequences that collectively span the entire length of the large double-stranded RNA from which they are derived. Double-stranded RNA with sequences that individually represent segments of a target mRNA may be analyzed using the methods described herein to identify the most active subset of hsiRNA fragments or individual siRNA fragments for achieving gene silencing for any gene or transcribed sequences. A method is additionally provided for preparing and cloning DNA encoding selected siRNA, hsiRNA mixtures or hairpin sequences to provide a continuous supply of a gene silencing reagent derived from any long double-stranded RNA.

Description

CROSS REFERENCE[0001]This application is a Continuation of U.S. application Ser. No. 10 / 622,240 filed Jul. 18, 2003, which claims priority from U.S. Provisional Application Ser. No. 60 / 402,769 filed Aug. 12, 2002, U.S. Provisional Application Ser. No. 60 / 407,543 filed Aug. 30, 2002 and U.S. Provisional Application Ser. No. 60 / 467,541 filed May 2, 2003. These Applications are herein incorporated by reference.BACKGROUND OF THE INVENTION[0002]RNA interference (RNAi) employing short double-stranded RNA (siRNA) is a powerful tool for silencing gene expression in mammalian cells (see for example, U.S. Pat. No. 6,506,559, International Publication No. WO 01 / 29058, International Publication No. WO 01 / 68836, International Publication No. WO 01 / 75164, U.S. Publication No. 20020114784, U.S. Publication No. 20030125281, U.S. Publication No. 2002162126, U.S. Publication No. 20030108923, U.S. Publication No. 20020173478, Fire, et al. Nature 391:806-811 (1998); Yang, et al., Mol. Cell. Biol. 21:78...

Claims

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Application Information

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IPC IPC(8): C12N9/18C12N15/09A61K31/713A61K35/76A61K48/00A61P31/18A61P35/00A61P35/02A61P37/04C12N15/11C12Q1/02C12Q1/42C12Q1/68
CPCC12N15/111C12N2310/14C12Y301/26003C12N2330/31C12N2330/30A61P31/18A61P35/00A61P35/02A61P37/04
Inventor TZERTZINIS, GEORGEFEEHERY, GEORGETUCKEY, CORINNANOREN, CHRISTOPHERMCREYNOLDS, LARRYZHANG, YINHUA
Owner NEW ENGLAND BIOLABS
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