Method for quantitative monitoring mRNA capping efficiency
A method for quantifying mRNA capping efficiency includes combining a sample, an enzyme mixture, and an isotopic standard solution in a buffer solution to create an incubation mixture, the enzyme mixture including a non-specific, single-stranded nuclease and an acid phosphatase and the isotopic standard including isotopically labeled m7G and isotopically labeled 2′-O-methylated nucleoside; incubating the mixture; and analyzing the mixture using liquid chromatography-mass spectrometry to determine at least one of a capping efficiency and a 2-O-methyltransferase efficiency.
1 . A method for quantifying mRNA capping efficiency comprising:
combining an mRNA sample, an enzyme mixture, and an isotopic standard solution in a buffer solution to create an incubation mixture, the enzyme mixture including a non-specific, single-stranded nuclease and an acid phosphatase and the isotopic standard including isotopically labeled m7G and isotopically labeled 2′-0-methylated nucleoside (Am, Gm, Cm, or Um);
incubating the mixture; and
analyzing the mixture using liquid chromatography-mass spectrometry to determine at least one of a capping efficiency and a 2′-0-methyltransferase efficiency.
2 . The method of claim 1 incubating the mixture is performed at a temperature of between about 30° C. and 70° C.
3 . The method of claim 2 incubating the mixture is performed at a temperature of between about 35° C. and 45° C.
4 . The method of claim 1 wherein the non-specific, single-stranded nuclease includes Nuclease P1.
5 . The method of claim 1 wherein the acid phosphatase is not inhibited by adenosine monophosphate.
6 . The method of claim 1 wherein the acid phosphatase includes Sweet Potato Acid Phosphatase.
7 . The method of claim 1 wherein the enzyme mixture further includes a site-specific, single-stranded RNA endonuclease.
8 . The method of claim 7 wherein the site-specific, single-stranded RNA endonuclease cleaves RNA 3′ of a guanosine.
9 . The method of claim 7 wherein the site-specific, single-stranded RNA endonuclease includes Ribonuclease T1.
10 . The method of claim 9 incubating the mixture is performed at a temperature of between about 30° C. and 50° C.
11 . The method of claim 1 further comprising solubilizing a lyophilized enzyme mixture with the sample buffer to obtain the enzyme mixture.
12 . The method of claim 1 further comprising solubilizing a lyophilized standards mixture with the sample buffer to obtain the standards mixture.
13 . The method of claim 1 , wherein the isotopically labeled m7G and the isotopically labeled 2′-O-methylated nucleoside includes deuterium labeled m7G(D) and deuterium labeled 2′-O-methylated nucleoside (Am(D), Gm(D), Cm(D), or Um(D)).
14 . The method of claim 1 , wherein the isotopically labeled m7G and the isotopically labeled 2′-O-methylated nucleoside are N 15 labeled or C 13 labeled.
15 . The method of claim 1 wherein the isotopically labeled m7G and the isotopically labeled 2′-O-methylated nucleoside in the standards mixture are in a molar ratio of between about 2:1 to about 1:2.
16 . The method of claim 15 wherein the standards mixture includes equimolar amounts of isotopically labeled m7G and isotopically labeled 2′-O-methylated nucleoside.
17 . The method of claim 1 wherein incubation mixture includes an amount of isotopically labeled m7G of between about 1.0 nmol and 20 nmol.
18 . The method of claim 17 wherein incubation mixture includes an amount of isotopically labeled 2′-O-methylated nucleoside of between about 1.0 nmol and about 20 nmol.
19 . The method of claim 1 wherein the buffer includes ZnCl 2 in a concentration range of about 0.001 mM and about 90 mM.
20 . The method of claim 19 wherein the buffer includes ZnCl2 in a concentration range of about 0.05 mM and about 0.2 mM.
21 . The method of claim 1 wherein the incubation mixture includes less than 100 mM of divalent cations.
22 . The method of claim 1 wherein the buffer has a pH of between about 2 to about 7.
23 . The method of claim 22 wherein the buffer has a pH of between about 4 to about 6.
24 . The method of claim 1 wherein analyzing the mixture using liquid chromatography-mass spectrometry further includes separating the mixture using a chromatography column, obtaining m/z and intensity data using the mass spectrometer, identifying and integrating the peaks for m7G and isotopically labeled m7G, peaks for 2′-O-methylated nucleoside and isotopically labeled 2′-O-methylated nucleoside, or both; calculating a m7G: isotopically labeled m7G peak ratio, a 2′-0-methylated nucleoside: isotopically labeled 2′-O-methylated nucleoside peak ratio, or both; and determining the capping efficiency based on the m7G: isotopically labeled m7G peak ratio, the 2-0-methyltransferase efficiency based on the 2′-O-methylated nucleoside: isotopically labeled 2′-O-methylated nucleoside peak ratio or both.