RNA polyphosphatase compositions, kits, and uses thereof
The present invention relates to the discovery of RNA 5′ polyphosphatase enzymes not previously described in the art, methods for discovery of said enzymes, compositions of said enzymes, methods for making said enzymes, and various methods and kits for using said enzymes for biomedical research, for human and non-human diagnostics, for production of therapeutic products, and for other applications. In particular, some embodiments provide compositions, kits and methods for employing RNA polyphosphatases for isolation, purification, production, and assay of capped RNA using a biological sample or a sample from an in vitro capping reaction wherein the sample also contains RNA that is not capped. Other embodiments provide compositions, kits and methods wherein RNA polyphosphatases comprise signal-amplifying enzymes for analyte-specific assays.
1. A method for obtaining, isolating, or purifying capped RNA in a sample that contains said capped RNA and at least one uncapped RNA, the method comprising the steps of:
(1) providing: (i) a sample that contains capped RNA and at least one uncapped RNA that has a 5′ triphosphate group or a 5′ diphosphate group, (ii) an RNA polyphosphatase that is active in the presence of EDTA and is inhibited in the presence of 1 mM or greater Mg 2+ , and (iii) a 5′ exoribonuclease;
(2) contacting the sample with the RNA polyphosphatase thereby forming a first reaction mixture such that the RNA that has a 5′ triphosphate group or a 5′ diphosphate group is converted to RNA that has a 5′ monophosphate group in said first reaction mixture; and
(3) contacting said first reaction mixture with said 5′ exoribonuclease under conditions such that said RNA that has a 5′ monophosphate group in said first reaction mixture is digested by the 5′ exoribonuclease, but said capped RNA in said first reaction mixture is not digested by the 5′ exoribonuclease, thereby obtaining, isolating, or purifying said capped RNA.
2. The method of claim 1 , wherein said RNA polyphosphatase is E. coli 5′-RNA Polyphosphatase ( E. coli RPP I) or Shigella 5′-polyphosphatase I (Shigella RPP I), and said contacting the sample with the RNA polyphosphatase is performed in the absence of Mg 2+ .
3. The method of claim 1 , further comprising transforming an eukaryotic cell with the capped RNA that is obtained, isolated, or purified from step (3).
4. The method of claim 1 , wherein said eukaryotic cell is an antigen-presenting cell (APC) selected from the group consisting of: a dendritic cell, a macrophage, an epithelial cell, and an artificial APC.
5. The method of claim 1 , wherein the capped RNA is from a biological sample or is from an in vitro capping reaction selected from the group consisting of: (i) a co-transcriptional capping reaction comprising an RNA polymerase and a dinucleotide cap analog, and (ii) a post-transcriptional capping reaction that employs a capping enzyme system comprising RNA guanyltransferase enzymatic activity.
6. The method of claim 1 , wherein the capped RNA comprises bacterial mRNA that is capped in vitro using a capping enzyme system comprising RNA guanyltransferase enzymatic activity.
7. The method of claim 1 , further comprising: prior to step (2), quantifying the amount of total RNA in the sample; and step (4), after step (3), comprising:
(i) quantifying the amount of RNA that is not digested by the 5′ exoribonuclease in step (3), thereby quantifying the amount of said capped RNA in the sample; and/or
(ii) quantifying the amount of RNA that is digested by the 5′ exoribonuclease in step (3), thereby quantifying the amount of said uncapped RNA in the sample.
8. The method of claim 7 , wherein the sample provided in step (1) additionally comprises RNA that has a 5′ monophosphate group and the method further comprises quantifying the amount of said RNA that has a 5′ monophosphate group in the sample, wherein, prior to step (2), the method additionally comprises the sub-steps of:
contacting the sample provided in step (1) with the 5′ exoribonuclease under conditions such that RNA in the sample that has a 5′ monophosphate group is digested by the 5′ exoribonuclease but said capped RNA and said uncapped RNA that has a 5′ triphosphate group or a 5′ diphosphate group is not digested by the 5′ exoribonuclease, and quantifying the amount of RNA that is digested by the 5′ exoribonuclease or the amount of RNA that is not digested by the 5′ exoribonuclease in the sample, whereby the amount of RNA in the sample that is digested by the 5′ exoribonuclease in the sub-steps indicates the amount of RNA that has a 5′ monophosphate group in the sample.
9. The method of claim 7 , wherein the sample provided in step (1) additionally comprises RNA that has a 5′ hydroxyl group, wherein the method additionally comprises providing a polynucleotide kinase and ATP in step (1), and the method further comprises the steps of:
(5) contacting the sample from step (1) with the polynucleotide kinase and the ATP under conditions for sufficient time and forming a second reaction mixture, wherein said RNA that has a 5′ hydroxyl group is phosphorylated to RNA that has a 5′ monophosphate group in said second reaction mixture;
(6) contacting said second reaction mixture with the 5′ exoribonuclease under conditions such that said RNA that has a 5′ monophosphate group is digested by the 5′ exoribonuclease, but said capped RNA and said uncapped RNA that has a 5′ triphosphate group or a 5′ diphosphate group are not digested by the 5′ exoribonuclease; and
(7) quantifying the amount of RNA that is digested by the 5′ exoribonuclease or the amount of RNA that is not digested by the 5′ exoribonuclease in step (6), whereby the amount of RNA in the sample that is digested by the 5′ exoribonuclease in step (6) indicates the amount of RNA that has a 5′ hydroxyl group in the sample.