# US 9,040,774 B2 · Recombinant DNA constructs encoding ribonuclease cleavage blockers and methods for modulating expression of a target gene

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- Number: US 9,040,774 B2
- Document ID: US9040774B2
- Status: Granted
- Kind: B2
- Application number: 12/999,777
- Filed: 2009-07-01
- Published: 2011-12-01
- Granted: 2015-05-26
- Earliest claimed: 2008-07-01
- Assignees: Monsanto Technology LLC
- Applicants: Sergey I. Ivashuta; Barbara E. Wiggins; Yuanji Zhang
- Inventors: Sergey I. Ivashuta; Barbara E. Wiggins; Yuanji Zhang
- Web page: https://patentgrep.com/patent/US9040774B2

## CPC

- C12N 15/8218: {Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]}
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12N 15/8216: {Methods for controlling, regulating or enhancing expression of transgenes in plant cells}
- C12N 15/8261: {with agronomic (input) traits, e.g. crop yield}
- C12N 2310/141: MicroRNAs, miRNAs
- Y02A 40/146: Genetically Modified [GMO] plants, e.g. transgenic plants
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

This invention provides recombinant DNA constructs and methods for manipulating expression of a target gene that is regulated by a small RNA, by interfering with the binding of the small RNA to its target gene. More specifically, this invention discloses recombinant DNA constructs encoding cleavage blockers, 5-modified cleavage blockers, and translational inhibitors useful for modulating expression of a target gene and methods for their use. Further disclosed are miRNA targets useful for designing recombinant DNA constructs including miRNA-unresponsive transgenes, miRNA decoys, cleavage blockers, 5-modified cleavage blockers, and translational inhibitors, as well as methods for their use, and transgenic eukaryotic cells and organisms containing such constructs.

## Claims

### Claim 1

A recombinant DNA construct comprising a promoter operable in a plant cell, operably linked to DNA encoding a single-stranded cleavage blocker RNA that binds in vivo to a target RNA transcript in said plant cell, at an miRNA recognition site for an endogenous mature miRNA, and forms, through complementary base-pairing, a hybridized segment of between 19 to 24 nucleotides in length at said miRNA recognition site in said target RNA transcript, wherein said hybridized segment comprises:
at least one nucleotide in said single-stranded cleavage blocker RNA that does not match through complementary base-pairing with said miRNA recognition site at positions corresponding to positions 10 or 11 of said endogenous mature miRNA;
and wherein the single-stranded cleavage blocker RNA interferes with the binding of said endogenous mature miRNA to said target RNA transcript at said miRNA recognition site.

### Claim 2 (depends on 1)

The recombinant DNA construct of claim 1, wherein formation of said hybridized segment inhibits cleavage of said target RNA transcript mediated by said endogenous mature miRNA.

### Claim 3 (depends on 1)

A method of modulating expression of a target gene in a plant cell, comprising expressing in said plant cell the recombinant DNA construct of claim 1, wherein said target gene encodes said, target RNA transcript.

### Claim 4 (depends on 3)

The method of claim 3, wherein formation of said hybridized segment inhibits suppression of said at least one target gene by said endogenous mature miRNA.

### Claim 5 (depends on 1)

A non-natural plant chromosome or plastid comprising the recombinant DNA construct of claim 1.

### Claim 6 (depends on 1)

A non-natural transgenic plant cell having in its genome the recombinant DNA construct of claim 1, or a non-natural transgenic plant or a non-natural transgenic plant seed or a non-natural transgenic pollen grain each comprising said non-natural transgenic plant cell.

### Claim 7 (depends on 6)

A non-natural partially transgenic plant, wherein:
a. said non-natural partially transgenic plant comprises the non-natural transgenic plant cell of claim 6 and further comprises non-transgenic tissue; or
b. said non-natural partially transgenic plant comprises a transgenic rootstock comprising the non-natural transgenic plant cell of claim 6 and further comprises a non-transgenic scion.