mRNA quantification methods
Provided herein are methods for determining a concentration of RNA (e.g. mRNA) in a solution. Certain aspects of the disclosure relate to digesting the target RNA polymer by adding a base to a solution of RNA, optionally comprising at least one chemically modified nucleotide, and incubating the mixture to digest the RNA into a plurality of nucleotides. Certain other aspects of the disclosure relate to measuring the absorbance of the digested RNA mixture at 260 nm; while other aspects relate to using the absorbance measurement with a value of a relative percent of each nucleotide in the digestion mixture, and a mass-corrected coefficient of each nucleotide in the mixture to determine a concentration of the RNA. Other aspects of the disclosure relate to measuring the concentration of RNA in a solution using computer-implemented techniques.
1 . A method for determining a concentration of mRNA in a mRNA solution, the method comprising:
measuring an absorbance of a nucleotide solution comprising a plurality of nucleotides; and
determining the concentration of the mRNA in the mRNA solution based on (i) the measured absorbance of the nucleotide solution and (ii) a sequence corrected molar extinction coefficient (SCC) associated with nucleotide monophosphates present in a digested mRNA mixture.
2 . The method of claim 1 , wherein the plurality of nucleotides and/or the nucleotide solution comprise intact mRNA, and wherein the concentration of the mRNA is determined based on a calibration curve generated from the digested mRNA mixture.
3 . The method of claim 1 , wherein the nucleotide solution is the mRNA solution.
4 . The method of claim 1 , wherein the nucleotide solution is not the digested mRNA mixture.
5 . The method of claim 1 , wherein the plurality of nucleotides comprises a plurality of nucleotide monophosphates.
6 . The method of claim 1 , wherein the nucleotide solution is the digested mRNA mixture.
7 . The method of claim 1 , further comprising digesting the mRNA in the mRNA solution to form the digested mRNA mixture.
8 . A method for determining a concentration of mRNA in a solution, comprising:
digesting a mRNA mixture by adding a base to a solution of mRNA and incubating the mixture to digest the mRNA into nucleotide monophosphates;
obtaining a plurality of mass-corrected coefficient values for each type of nucleotide in the digestion mixture;
obtaining a plurality of sequence corrected molar extinction coefficient (SCC) values associated with nucleotide monophosphates present in the digested mRNA mixture based on (i) the plurality of mass-corrected coefficient values for each type of nucleotide in the nucleotide solution and (ii) relative percentage values for each type of nucleotide in the nucleotide solution;
measuring the absorbance of the digested mixture; and,
using the absorbance measurement and SCC to determine a concentration of the mRNA.
9 . A method for preparing a set of sequence corrected molar extinction coefficient (SCC) values for an mRNA comprising:
digesting a mRNA mixture by adding a base to a solution of mRNA and incubating the mixture to digest the mRNA into nucleotide monophosphates;
obtaining a plurality of mass-corrected coefficient values for each type of nucleotide in the digestion mixture and using the plurality of mass-corrected coefficient values; and
obtaining a plurality of sequence corrected molar extinction coefficient (SCC) values associated with nucleotide monophosphates present in the digested mRNA mixture based on (i) the plurality of mass-corrected coefficient values for each type of nucleotide in the nucleotide solution and (ii) relative percentage values for each type of nucleotide in the nucleotide solution.
10 . The method of claim 8 , wherein the determined concentration of the mRNA in the mRNA solution is further based on a pathlength and a dilution factor.
11 . The method of claim 8 , wherein the base comprises sodium hydroxide at a concentration of at least 0.8 N.
12 . The method of claim 8 , wherein the mixture is incubated at a temperature of about 60-70° C. and the absorbance is measured at 260 nm.
13 . The method of claim 12 , further comprising quenching the digestion by adding an acid solution to the mRNA mixture, wherein the acid solution comprises hydrochloric acid.
14 . The method of claim 13 , further comprising adding a buffer solution to the digested mixture, wherein the buffer solution comprises sodium citrate at a concentration of between 2 mM to 250 mM.
15 . The method of claim 8 , wherein the mRNA comprises at least one nucleotide selected from the group consisting of N1-methyl-pseudouridine, 5-methoxy-uridine, and N1-ethyl-pseudouridine.
16 . The method of claim 1 , wherein the mRNA comprises at least one chemically modified nucleotide.
17 . A computer-implemented method for outputting a concentration of mRNA in a mRNA solution, the method comprising:
receiving or inputting an absorbance measurement of a nucleotide solution comprising a plurality of nucleotides;
receiving or inputting one or more of (i) a sequence corrected molar extinction coefficient (SCC) associated with nucleotide monophosphates present in a digested mRNA mixture, (ii) a plurality of mass-corrected coefficient values, wherein the plurality comprises a mass-corrected coefficient value for each type of the nucleotide monophosphates in the digestion mixture, and (iii) relative percent values for each type of the nucleotide monophosphates in the digestion mixture;
determining the concentration of the mRNA based on (i) the absorbance measurement of the nucleotide solution and (ii) the one or more of the SCC, the plurality of mass-corrected coefficient values, and the relative percent values; and
outputting the concentration of the mRNA in the solution.
18 . The method of claim 17 , wherein the plurality of nucleotides and/or nucleotide solution comprises intact mRNA, and wherein concentration of the mRNA is determined based on a calibration curve generated from the digested mRNA mixture.
19 . The method claim 17 , wherein the nucleotide solution is the mRNA solution.
20 . The method of claim 17 , wherein the nucleotide solution is not the digested mRNA mixture.
21 . The method of claim 17 , wherein the plurality of nucleotides comprises a plurality of nucleotide monophosphates.
22 . The method of claim 17 , wherein the nucleotide solution is the digested mRNA mixture.
23 . The method of claim 17 , further comprising digesting the mRNA in the mRNA solution to form the digested mRNA mixture.
24 . The method of claim 17 , wherein the plurality of nucleotides comprises at least one chemically modified nucleotide.
25 . A method for determining a concentration of mRNA in a solution, the method comprising:
at least one computer hardware processor to perform:
obtaining an absorbance measurement of a nucleotide solution comprising a plurality of nucleotides;
obtaining one or more of (i) a sequence corrected molar extinction coefficient (SCC) associated with nucleotide monophosphates present in a digested mRNA mixture, (ii) a plurality of mass-corrected coefficient values, wherein the plurality comprises a mass-corrected coefficient value for each type of the nucleotide monophosphates in the digestion mixture, and (iii) relative percent values for each type of the nucleotide monophosphates in the digestion mixture;
determining the concentration of the mRNA based on (i) the absorbance measurement of the nucleotide solution and (ii) the one or more of the SCC, the plurality of mass-corrected coefficient values, and the relative percent values; and
outputting the concentration of the mRNA in the solution.
26 . The method of claim 25 , further comprising adjusting the volume of the solution to achieve a therapeutic dose of the mRNA in the solution based on the calculated concentration of the mRNA in the solution.
27 . The method of claim 25 , wherein determining of the mRNA concentration using plurality of mass-corrected coefficient values accounts for secondary structure and intramolecular interactions within the mRNA.
28 . The method of claim 25 , wherein the base comprises sodium hydroxide and the absorbance is measured at 260 nm using an ultraviolet visible spectrophotometer.
29 . The method of claim 25 , wherein the spectrophotometer is selected from the group consisting of the Nanodrop 2000 spectrophotometer, the BioTek Synergy spectrophotometer, and the FLUOstar OMEGA.
30 . The method of claim 25 , wherein the plurality of nucleotides comprises at least one chemically modified nucleotide.