Systems and Methods for Producing RNA Constructs with Increased Translation and Stability

Abstract

Systems and methods for enhancing RNA translatability and stability are disclosed. Some embodiments describe RNA molecules exhibiting increased translatability and/or stability. Additional embodiments describe methods for screening RNA molecules for increased translatability and/or stability. Various embodiments utilize screening methods, including degenerative sequences to identify sequences or regions that increase the translatability and/or stability of RNA molecules.

1 - 33 . (canceled) 
     
     
         34 . A method for identifying RNA molecules possessing increased translatability and 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;   assessing translatability of the pool of RNA molecules by:
 transfecting a cell or cell lysate with a first subset of the pool of RNA molecules; 
 performing polysome profiling on the first subset of the pool of RNA molecules to segregate RNA molecules based on the number of ribosomes bound to the RNA molecule; and 
 isolating a fraction from the polysome profile to generate a first set of RNA molecules showing a first level of ribosomes bound to the RNA molecules in the set of RNA molecules; and 
   assessing stability of the pool of RNA molecules by:
 treating a second subset of the pool of RNA molecules under an experimental condition; and 
 isolating a fraction from the second subset the pool of RNA molecules at a specified timepoint to generate a second set of RNA molecules showing stability under the experimental condition for the specified timepoint. 
   
     
     
         35 . The method of  claim 34 , further comprising sequencing the barcode sequence of the first set of RNA molecules and the second set of RNA molecules to identify the presence of each RNA molecule in each fraction of RNA molecules. 
     
     
         36 . The method of  claim 35 , further comprising determining translatability and stability of the RNA molecules associated with each barcode sequence in the first set of RNA molecules and the second set of RNA molecules by identifying the prevalence of each barcode in each fraction of RNA molecules. 
     
     
         37 . The method of  claim 34 , wherein the barcoding sequence is selected from SEQ ID NOs: 115-1380. 
     
     
         38 . The method of  claim 34 , wherein the profiling sequence is selected from SEQ ID NOs: 1381-1382. 
     
     
         39 . A method to select for RNA elements, comprising:
 obtaining a library of RNA molecules, wherein each RNA molecule comprises a coding sequence, a 5′ untranslated region (5′UTR), and a 3′ untranslated region (3′UTR), wherein one of the coding sequence, the 5′UTR, or the 3′UTR comprises a degenerate region;   assessing a property of the library of RNA molecules, wherein the property is selected from the group consisting of translatability, in vivo stability, and in vitro stability; and   selecting an RNA molecule from the library of RNA molecules showing increase in the property over other RNA molecules in the library of RNA molecules.   
     
     
         40 . The method of  claim 39 , further comprising sequencing the selected RNA molecule. 
     
     
         41 . The method of  claim 39 , wherein the selected RNA molecule is a pool of RNA molecules. 
     
     
         42 . The method of  claim 41 , further comprising:
 reassessing the property of the pool of RNA molecules; and   selecting an RNA molecule from the pool of RNA molecules showing increase in the property over other RNA molecules in the pool of RNA molecules.   
     
     
         43 . The method of  claim 42 , further comprising sequencing the selected RNA molecule from the pool of RNA molecules. 
     
     
         44 . The method of  claim 39 , wherein the property is translatability. 
     
     
         45 . The method of  claim 39 , wherein the degenerate region is selected from the group consisting of: a deletion, a random sequence, an ambiguous sequence, and a truncation. 
     
     
         46 . The method of  claim 34 , wherein the RNA molecules are transfected into a collection of cells. 
     
     
         47 . The method of  claim 46 , wherein the collection of cells is selected from mammalian cells, yeast cells, bacteria cells, and plant cells. 
     
     
         48 . The method of  claim 34 , wherein the RNA molecules are added to a cell lysate. 
     
     
         49 . The method of  claim 34 , wherein polysome profiling comprises adding a cell lysate to a sucrose gradient and centrifuging the sucrose gradient to segregate the RNA molecules. 
     
     
         50 . The method of  claim 34 , further comprising isolating a second fraction from the polysome profile to generate a second set of RNA molecules showing a second level of ribosomes bound to the RNA molecules in the set of RNA molecules, wherein the first level and second level represent different amounts of bound ribosomes. 
     
     
         51 . The method of  claim 50 , further comprising sequencing the barcode sequence of each RNA molecule in the first set of RNA molecules and the second set of RNA molecules to identify the presence of each RNA molecule in the first set of RNA molecules and the second set of RNA molecules. 
     
     
         52 . The method of  claim 34 , 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. 
     
     
         53 . The method of  claim 34 , further comprising size selecting for full-length RNA molecules.