Systems and Methods for Producing RNA Constructs with Increased Translation and Stability
AssigneeThe Board of Trustees of the Leland Stanford Junior University
InventorsMaria Barna, Kathrin Leppek, Gun Woo Byeon
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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.
Claims34
A method to determine RNA translatability, 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;
- transfecting a cell or cell lysate with the pool of RNA molecules;
- performing polysome profiling on the pool of RNA molecules to segregate RNA molecules based on the number of ribosomes bound to the RNA molecule;
- and
- isolating a first 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.
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A method to determine 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 experimental condition;
- and
- isolating the pool of RNA molecules at a specified timepoint to generate a fraction of RNA molecules showing stability under the experimental condition for the specified timepoint.
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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.
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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 selected from the group consisting of:
- a deletion, a random sequence, an ambiguous sequence, and a truncation;
- assessing translatability 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.
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Dependencies are inferred from the claim text.