Kits and methods for generating 5' capped RNA

a technology of rna and rna molecules, applied in the field of efficient generating 5′ capped rna, can solve the problems of reverse-capped rna molecules that are not properly imported into the nucleus, reverse-capped u1 rnas in the cytoplasm are not properly translated, and the translation of in vitro-synthesized mrnas having such reverse caps is impaired

Inactive Publication Date: 2007-12-06
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Such reverse-capped RNA molecules behaved abnormally.
Similarly, cytoplasmic reverse-capped U1 RNAs in the cytoplasm were not properly imported into the nucleus.
Thus, translation of in vitro-synthesized mRNAs having such reverse caps is impaired.
However, although RNA can be capped by in vitro transcription of a DNA template in the presence of an ARCA, this approach has several drawbacks.
First, the chemical syntheses of ARCAs (e.g., see Jemielity, J et al., RNA: 1108, 2003) are difficult (˜6 synthetic steps), time-consuming (˜12 weeks) and expensive.
Also, once the ARCA is obtained, the in vitro transcription reaction is wasteful and inefficient.
Still further, the fact that

Method used

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Examples

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examples

[0176] The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.

A. Methods

Method 1. Reaction Mixture for Evaluation of Nucleoside-5′-Triphosphates as Substrates for Capping Enzyme: Synthesis of Capped RNA Having a Cap 0 Structure, but Lacking an N7-Methyl Group in the Cap Nucleotide

[0177] In order to evaluate the ability of a nucleoside-5′-triphosphate to be used as a modified cap nucleotide substrate by capping enzyme for synthesis of modified-nucleotide-capped RNA having a cap 0 structure lacking a 7-methylguanine in the cap nucleotide, capping enzyme reactions were prepared that contained: 1 μg of a 51-base primary RNA transcript (prepared by in vitro transcription of a T7 promoter-containing double-stranded DNA template using a AmpliScribe™ T7-Flash™ Transcription Kit according to the protocol provided with the kit from EPICE...

experiment 2

oside-5′-Triphosphates with Base Modifications as Substrates for Capping Enzyme, and Uses of Modified-Nucleotide-Capped RNA Obtained therefrom.

[0195] 2A. ATP, N7-methyl-GTP (m7GTP), 2′,3′-ddGTP, 7-deaza-GTP, N1-methyl-GTP, 3′-amino-2′,3′-ddGTP, 3′-azido-2′,3′-ddGTP, and O6-methyl-GTP were each used as the nucleoside-5′-triphosphate in a capping enzyme reaction mixture set up as described in Method 1, and the capping enzyme reaction was carried out as described in Method 3 and analyzed as described in Method 4. ATP, N7-methyl-GTP, 2′,3′-ddGTP, and 7-deaza-GTP were not substrates for the capping enzyme system, since the 51-base primary RNA transcript was not converted by the capping enzyme system into a 52-base capped RNA. However, N1-methyl-GTP, 3′-amino-2′,3′-ddGTP, 3′-azido-2′,3′-ddGTP, and O6-methyl-GTP were substrates for the capping enzyme system. The capping enzyme system quantitatively converted the 51-base primary RNA transcript to a 52-base capped RNA in a 30-minute reaction...

experiment 3

oside-5′-Triphosphates Having 2′ and / or 3′ Modifications of the Sugar Moiety as Substrates for Capping Enzyme, and Uses of Modified-Nucleotide-Capped RNA Obtained therefrom.

[0199] 3A. The following nucleoside-5′-triphosphates having 2′ and / or 3′ modifications of the sugar moiety were each used as the modified cap nucleotide in a capping enzyme reaction mixture set up as described in Method 1, and the capping enzyme reaction was carried out as described in Method 3 and analyzed as described in Method 4: 2′,3′-dideoxy-GTP (i.e., 2′,3′-ddGTP); 2′-dGTP; 2′-OMe-GTP; 3′-OMe-GTP; 2′-F-dGTP; 2′-amino-2′-dGTP (i.e., 2′-amino-dGTP); and 2′-azido-2′-dGTP (i.e., 2′-azido-dGTP). The 2′,3′-ddGTP was not a substrate for the capping enzyme system, since the 51-base primary RNA transcript was not converted by the capping enzyme system into a 52-base capped RNA. However, all of the remaining nucleoside-5′-triphosphates having 2′ and / or 3′ modifications of the sugar moiety were substrates for the capp...

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Abstract

The present invention relates to kits and methods for efficiently generating 5′ capped RNA having a modified cap nucleotide and for use of such modified-nucleotide-capped RNA molecules. The invention is used to obtain novel compositions of such modified-nucleotide-capped RNA molecules. In particular, the present invention provides kits and methods for capping RNA using a modified cap nucleotide and a capping enzyme system, such as poxvirus capping enzyme. The present invention finds use for in vitro production of 5′-capped RNA having a modified cap nucleotide and for in vitro or in vivo production of polypeptides by in vitro or in vivo translation of such modified-nucleotide-capped RNA for a variety of research, therapeutic, and commercial applications. The invention also provides methods and kits for capturing or isolating uncapped RNA comprising primary RNA transcripts or RNA having a 5′-diphosphate, such as RNA synthesized in vitro or obtained from a biological source, including prokaryotic mRNA that is in a mixture with other prokaryotic and/or eukaryotic nucleic acids. The method for capturing modified-nucleotide-capped RNA also provides methods and kits for obtaining only type-specific or condition-specific modified-nucleotide-capped RNA by cap-dependent subtraction of that portion of the captured modified-nucleotide-capped RNA in cells of one type or condition that is the same as RNA in cells of another type or condition. The invention further provides methods and kits for using a capping enzyme system and modified cap nucleotides for labeling uncapped RNA comprising primary RNA transcripts or RNA having a 5′-diphosphate with detectable dye or enzyme moieties.

Description

[0001] The present invention claims priority U.S. Provisional Patent Application Ser. No. 60 / 792,220, filed Apr. 14, 2006, the entire disclosure of which is herein incorporated by reference in its entirety.FIELD OF THE INVENTION [0002] The present invention relates to kits and methods for efficiently generating 5′ capped RNA having a modified cap nucleotide and uses of such modified-nucleotide-capped RNA molecules. The invention can be used to obtain novel compositions of such modified-nucleotide-capped RNA molecules. In particular, the present invention provides kits and methods for capping RNA using a modified cap nucleotide and a capping enzyme system, such as vaccinia virus capping enzyme. The present invention finds use for in vitro production of 5′-capped RNA having a modified cap nucleotide and for in vitro or in vivo production of polypeptides by in vitro or in vivo translation of such modified-nucleotide-capped RNA for a variety of research, therapeutic, and commercial appl...

Claims

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Application Information

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IPC IPC(8): C12P19/34C07H21/00C07H21/02C12N5/04C12P21/00C12N5/10C12N9/00C12N9/10C12N15/11C12N15/113
CPCC12N15/111C12N15/113C12N2310/317C12N15/1096C12P19/34C12N15/1072C12N15/1075C12N2320/51C12N9/1007C12N9/1241C12Y201/01056C12Y201/01057C12Y207/07019C12Y207/0705
Inventor JENDRISAK, JEROMEMEIS, RONALDDAHL, GARY
Owner CELLSCRIPT
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