Phased Small RNA Constructs for Plant Gene Suppression
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Solution Overview
Problem
Current gene suppression methods in plants, such as RNA interference, often rely on siRNAs, miRNAs, or trans-acting siRNAs, which have limitations in specificity and efficiency, particularly in regulating gene expression without the use of hairpin structures or RNA-dependent RNA polymerases.
Innovation Solution
A novel recombinant DNA construct that encodes a transcript forming a hybridized RNA structure cleaved in phase into phased small RNAs, independent of RNA-dependent RNA polymerase, using DCL4 or its orthologs to produce multiple small double-stranded RNAs for targeted gene suppression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional RNA interference methods using siRNAs or miRNAs are used for gene suppression, then gene expression can be regulated, but the methods require complex structures such as hairpin structures or RNA-dependent RNA polymerases which increase device complexity
Solution Approach 1:
The patent extracts and eliminates the requirement for RNA-dependent RNA polymerases and complex hairpin structures from the gene suppression pathway. By using a simplified linear RNA transcript that directly forms hybridized RNA with target mRNA, the invention removes unnecessary cellular machinery requirements while maintaining effective gene suppression.
Solution Approach 2:
Instead of using the conventional approach where RNA polymerases transcribe DNA into RNA that then forms complex structures, this invention inverts the approach by designing a DNA construct that directly transcribes into a linear RNA transcript capable of forming hybridized RNA without requiring additional processing enzymes or complex folding structures.
2Device complexity
If phased small RNAs are produced independently of RNA-dependent RNA polymerase, then the pathway is simplified, but specificity and efficiency of gene suppression must be maintained without conventional miRNA or siRNA mechanisms
Solution Approach 1:
The patent introduces hybridized RNA as an intermediary structure that mediates between the simple linear RNA transcript and the target mRNA. This hybridized RNA forms through direct base-pairing with the target sequence, providing the necessary specificity without requiring complex processing pathways or additional enzymatic machinery.
Solution Approach 2:
The invention changes the fundamental parameter of RNA structure from complex folded structures (hairpins, duplexes requiring Dicer processing) to simple linear transcripts that directly hybridize with target mRNA. This parameter change simplifies the pathway while maintaining specificity through direct sequence complementarity.
3Productivity
If multiple small double-stranded RNAs are produced from a single transcript, then productivity of gene suppression increases, but the cleavage process must be precisely phased to ensure correct small RNA generation
Solution Approach 1:
The patent segments the linear RNA transcript into multiple adjacent regions that can be independently processed into separate small double-stranded RNAs. Each segment contains a target sequence that can be cleaved independently by Dicer, allowing multiple small RNAs to be produced from a single transcript without requiring complex coordination or precise phasing mechanisms.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach allows for specific and efficient gene suppression in plants, providing protection against pests and pathogens without the need for miRNAs or hairpin structures, and can be used in transgenic plants and seeds to regulate gene expression effectively.
Implementation Method 1
cleaved in phase in vivo by DCL4 or a DCL4-like ribonuclease
Implementation Method 2
encodes a transcript that folds into hybridized RNA
Data Source
AI summary
This invention discloses recombinant DNA constructs encoding phased small RNAs useful in regulating expression of one or more genes of interest. Also disclosed by this invention are transgenic plant cells, plants, and seeds containing a recombinant DNA construct of this invention.