Systems and Methods to Assess RNA Stability

Abstract

Systems and methods for assessing mRNA in vivo and/or in vitro stability are disclosed. Some embodiments methods obtain RNA indexed or barcoded RNA molecules which are then tested against various conditions including stability inside of cells, stability in cell lysate, and stability in solution (e.g., for storage and/or transportation). Additional embodiments describe methods to determine degradation points with single base resolution.

1 . A method to determine in vitro RNA stability, comprising:
 obtaining a pool of RNA molecules, wherein each RNA molecule is uniquely encoded with a barcoding sequence and each barcoding sequence is flanked by at least one profiling sequence;   treating the pool of RNA molecules under an in vitro experimental condition;   isolating the pool of RNA molecules at a specified timepoint to generate a fraction of RNA molecules showing stability under the in vitro experimental condition for the specified timepoint; and   selecting for stable RNA molecules in the fraction of RNA molecules by performing a nuclease digestion to digest degraded and damaged RNA molecules in the fraction of RNA molecules.   
     
     
         2 . The method of  claim 1 , further comprising sequencing the barcode sequence of each RNA molecule in the fraction to identify the presence of each RNA molecule in the fraction of RNA molecules. 
     
     
         3 . The method of  claim 2 , further comprising determining stability of the RNA molecules associated with each barcode sequence in the fraction by identifying the prevalence of each barcode in the fraction. 
     
     
         4 . The method of  claim 1 , wherein the in vitro treatment condition is selected from temperature, pH, presence of certain molecules, presence of certain ions, concentration of certain molecules, concentration of certain ions, irradiation, buffer type, and buffer concentration. 
     
     
         5 . The method of  claim 1 , further comprising size selecting for full-length RNA molecules. 
     
     
         6 . The method of  claim 5 , wherein size selecting comprises one or more of agarose gel electrophoresis, polyacrylamide gel electrophoresis, and capillary electrophoresis. 
     
     
         7 . The method of  claim 5 , wherein size selecting comprises treating the RNA molecules with a 5′-3′ nuclease that is inhibited by the presence of a 5′ cap moiety. 
     
     
         8 . The method of  claim 7 , wherein the nuclease is XRN1. 
     
     
         9 . The method of  claim 1 , wherein
 the isolating step further comprises isolating the pool of RNA molecules at a second specified timepoint to generate a second fraction of RNA molecules showing stability under the in vitro experimental condition for the specified timepoint; and   the selecting for stable RNA molecules step further comprises selecting for stable RNA molecules in the second fraction of RNA molecules by performing a nuclease digestion to digest degraded and damaged RNA molecules in the fraction of RNA molecules.   
     
     
         10 . The method of  claim 9 , further comprising integrating a computational filter to remove sequences showing anomalous stability. 
     
     
         11 . The method of  claim 10 , wherein the computational filter constructs a single-exponential curve and calculates a difference between experimental fraction intact at a time point and the expected intact fraction for each RNA molecule in the pool of RNA molecules. 
     
     
         12 . The method of  claim 11 , wherein an RNA molecule is ignored from stability, if the residual fraction is greater than a particular threshold. 
     
     
         13 . A method to identify a degradation site within an RNA molecule, comprising:
 obtaining a pool of RNA molecules, wherein the RNA molecules encode for a sequence of interest;   treating the pool of RNA molecules under an experimental condition to degrade the RNA molecules in the pool of RNA molecules;   isolating the pool of RNA molecules at a specified timepoint;   ligating an adapter to one end of the degraded RNA molecules in the pool of RNA molecules; and   sequencing the ligated and degraded RNA molecules in the pool of RNA molecules to identify the degradation locations in the pool of RNA molecules.   
     
     
         14 . The method of  claim 13 , wherein the adapter is ligated to the 5′ end of the degraded RNA molecules. 
     
     
         15 . The method of  claim 13 , wherein the treatment condition is selected from temperature, pH, presence of certain molecules, presence of certain ions, concentration of certain molecules, concentration of certain ions, irradiation, buffer type, and buffer concentration. 
     
     
         16 . The method of  claim 13 , wherein the pool of RNA molecules comprises a plurality of sequences of interest, wherein each sequence of interest is uniquely encoded with a barcoding sequence and each barcoding sequence is flanked by at least one profiling sequence.