Method for screening regulatory element for increasing mrna translation, novel regulatory element resulting from method, and use thereof
AssigneesSeoul National University R&Db Foundation, Institute for Basic Science
InventorsVic Narry KIM, Jenny SEO, Soo-Jin JUNG
Life of this patent
Estimating the term…
Abstract
The present disclosure relates to a method of screening a regulatory element for enhancing mRNA translation, a novel regulatory element resulting from the method, and uses thereof. Through the screening method of the present disclosure, a novel regulatory element capable of enhancing mRNA translation may be obtained. Furthermore, the novel regulatory element may increase the expression of a target protein and as such, may be applied to various fields, depending on the intended use of the target protein.
Claims16
A method for screening a regulatory element for enhancing mRNA translation, the method comprising:
- preparing a plurality of oligonucleotides by tiling a viral genome;
- preparing a pool of vectors, each including one of the oligonucleotides, wherein each vector includes a reporter gene and includes one of the oligonucleotide in a 3′ UTR thereof;
- introducing each vector into a cell;
- fractionating the polysomes of the cell into free mRNA, monosome, light polysome (LP), medium polysome (MP), and heavy polysome (HP), performing sequencing, and calculating, for each oligonucleotide, a value of Equation (1) and a mean ribosome load (MRL):
- =
- Log
-
- 2
-
- (
- HP
- /
- free
-
- mRNA
- )
- -
- Mean
-
- ribsome
-
- load
-
- (
- MRL
- )
- =
- 1
- ×
- p
-
- (
- Monosome
- )
- +
- 2.5
- ×
- p
-
- (
- LP
- )
- +
- 6
- ×
- p
-
- (
- MP
- )
- +
- 11
- ×
- p
-
- (
- HP
- )
- Equation
-
- (
- 1
- )
- where p(X) is a proportion of sequencing reads for each fraction X, and
- selecting, as a regulatory element for enhancing mRNA translation, an oligonucleotide for which the value of Equation (1) exceeds 0.2 and the MRL exceeds 4.5.
Dependencies are inferred from the claim text.