Kits and methods for generating 5' capped RNA

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...

AI Technical Summary

Benefits of technology

[0052] During development of embodiments of the present invention, it was determined that vaccinia virus capping enzyme or a poxvirus capping enzyme that has similar enzymatic activities can utilize modified cap nucleotides as substrates to cap uncapped RNA comprising primary RNA transcripts or RNA having a 5′-diphosphate. Thus, in some embodiments, the present invention provides a method for catalyzing formation of a modified-nucleotide-capped RNA comprising the step of contacting an RNA transcript with a capping enzyme system, such as poxvirus capping enzyme, and modified cap nucleotide under conditions permissive to the formation of the modified-nucleotide-capped RNA. In some embodiments of the method, the uncapped RNA comprising primary RNA transcripts or RNA having a 5′-diphosphate is in a biological sample. In some embodiments, the uncapped RNA is mRNA in the biological sample. In some embodiments, the uncapped RNA in the biological sample comprises small primary RNA transcripts that are not mRNA. In some embodiments, the uncapped RNA in the biological sample comprises small nuclear RNA (snRNA), micro RNA (miRNA), or another primary RNA transcript. In some embodiments, the uncapped RNA comprising primary RNA transcripts or RNA having a 5′-diphosphate is synthesized by an RNA polymerase in an in vitro transcription reaction or RNA amplification reaction, or by a replicase in an in vitro replication reaction. In some cases, up to essentially 100% of the uncapped RNA comprising primary RNA transcripts or RNA having a 5′-diphosphate in a population of RNA molecules is capped (e.g., see Examples). Thus, the present invention provides compositions, kits and methods for significantly improving synthesis of capped RNA transcripts (e.g., mRNA). The present invention provides a method, and kits for performing the method, which use a capping enzyme, such as vaccinia capping enzyme or poxvirus capping enzyme, and a modified cap nucleotide to synthesize modified-nucleotide-capped RNA with higher efficiency (e.g., with a capping efficiency approaching 100%) and in higher yields than is obtained by co-transcriptional capping using a dinucleotide cap analog. In some embodiments, the capping efficiency is greater than 80%. In some embodiments, the capping efficiency is greater than 85%. In some embodiments, capping efficiency is greater than 90%. In some embodiments, capping efficiency is greater than 97%. In some embodiments, capping efficiency is greater than of 99%. Thus, the present invention provides compositions, kits and methods for significantly improving synthesis of capped RNA transcripts (e.g., mRNA).

[0061] In other embodiments of the kits and method for synthesizing a modified-nucleotide-capped RNA for in vitro or in vivo translation of proteins or polypeptides, the modified cap nucleotide is 3′-O-methylguanosine-5′-triphosphate or 2′-O-methylguanosine-5′-triphosphate, in which embodiments, the modified-nucleotide-capped RNA synthesized using the kit and method should be the same as the capped RNA obtained in an in vitro transcription reaction using the respective m27,3′-OGpppG or m27,2′-OGpppG ARCA. The present invention is beneficial because such modified-nucleotide-capped RNA molecules having a modified cap nucleoside with a 3′- or 2′-O-methyl group can be synthesized with higher efficiency and in higher yields using a method or kit of the invention than the molecules synthesized by in vitro transcription reaction using the respective ARCA that have been reported to be translated in vitro and in vivo, with higher efficiency than unmodified m7G-capped RNA. In other embodiments in which the modified cap nucleotide is 3′-deoxyguanosine-5′-triphosphate, the modified-nucleotide-capped RNA obtained using the kit or method of the invention is identical to the capped RNA obtained by in vitro transcription in the presence of the m7(3′)dGpppG ARCA, which has also been reported to be translated in vitro with higher efficiency. Thus, the kits and methods of the invention are more desirable in terms of yield, purity, cost and / or time compared to existing techniques for obtaining such capped RNA molecules using a dinucleotide ARCA in an in vitro transcription reaction.

[0083] In some embodiments, the present invention provides methods that comprise the step of translating the modified-nucleotide-capped RNA having either a cap 0 or a cap I structure into protein. Although an understanding of the mechanism is not necessary to practice the present invention and the present invention is not limited to any particular mechanism of action, in some embodiments, compositions and methods of the present invention produce modified-nucleotide-capped RNA (e.g., mRNA) that is translated more efficiently in vitro or in vivo due to the efficiency at which the capping enzyme system is able to cap uncapped RNA comprising primary RNA transcripts or RNA having a 5′-diphosphate, and / or because of the characteristics of the modified-nucleotide-capped RNA to serve as a substrate for translation.

[0085] In some embodiments, the kits and methods use a modified cap nucleotide to synthesize a modified-nucleotide-capped RNA with improved in vitro or in vivo translation properties compared to capped RNA comprising the same sequence but which is uncapped or which comprises the N7-methyl-G cap nucleotide. For example, but without limitation, in some embodiments, the method or kit uses O6-Me-GTP to synthesize modified-nucleotide-capped RNA that results in improved translation, especially in vivo. In other embodiments, a method or kit of the invention uses another modified cap nucleotide that results in levels of in vitro or in vivo translation which are either similar to or higher than what is obtained with capped RNA comprising the standard N7-methyl-G cap nucleotide, in some cases, depending on the cell and other factors. In some embodiments, the modified cap nucleotide provides other benefits in addition to being translated efficiently, such as a method to capture or label the modified-nucleotide-capped RNA (e.g., using a modified cap nucleotide that comprises an amino, azido, or thiol group).

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 the cap analog can never be incorporated to 100% limits the purity of the capped RNA product, necessitating more work to purify the product and increasing the risk that the capped RNA product will still be contaminated with impurities, including unincorporated cap analog.

This is particularly detrimental if the capped RNA is to be used for medical applications, such as for therapeutics, or for clinical research, since the contaminants may produce undesired effects.

Thus, 2′-dGTP appeared to be incorporated into capped RNA, but inefficiently.

However, a capped RNA with a cap I structure could not be synthesized using an m7Gpppm2′-OG cap analog (Pasquinelli, RNA 1: 957, 1995).