Recombinant DNA Cleavage Blockers for RNase III Resistance
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Solution Overview
Problem
Current technologies face challenges in modulating gene expression effectively through RNA-mediated pathways, particularly in resisting RNase III cleavage and translating target genes, due to limitations in designing recombinant DNA constructs that interact with miRNAs and other small RNAs.
Innovation Solution
Development of recombinant DNA constructs that process to single-stranded RNAs which bind to target gene transcripts, forming hybridized segments that confer resistance to RNase III cleavage or inhibit translation, thereby modulating gene expression by using miRNA recognition sites and specific nucleotide sequences to prevent cleavage or translation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If recombinant DNA constructs are designed to interact with miRNAs and small RNAs, then gene expression modulation is improved, but the complexity of designing and constructing these DNA molecules increases
Solution Approach 1:
The recombinant DNA construct is divided into distinct functional segments: a promoter region, a coding region for the cleavage blocker or translational inhibitor, and a terminator. This segmentation allows independent optimization of each component's function while simplifying the overall design process.
Solution Approach 2:
The patent introduces intermediary RNA molecules (cleavage blockers and translational inhibitors) that mediate between the recombinant DNA construct and the target gene transcript. These intermediaries bind to miRNA recognition sites and prevent RNase III cleavage or inhibit translation, thereby achieving gene expression modulation without directly modifying the target gene.
2Reliability
If single-stranded RNAs bind to target gene transcripts to form hybridized segments, then resistance to RNase III cleavage is improved, but the precision of binding specificity must be maintained
Solution Approach 1:
The recombinant DNA construct incorporates specific nucleotide sequences in the coding region that encode for local structural features in the resulting RNA. These local features create specific binding interfaces that confer RNase III resistance while maintaining overall binding specificity to the target transcript.
Solution Approach 2:
The patent utilizes parameter changes in the nucleotide sequence composition and secondary structure of the RNA to optimize binding specificity. By adjusting parameters such as GC content, stem-loop structures, and hairpin configurations, the RNA achieves precise binding to the target gene transcript while resisting RNase III cleavage.
3Reliability
If miRNA recognition sites are used to prevent cleavage or translation, then gene expression control is improved, but the complexity of selecting and designing appropriate recognition sites increases
Solution Approach 1:
The recombinant DNA construct is designed to be universally applicable across different plant species and target genes by utilizing conserved miRNA recognition sites. The same construct framework can be adapted to target different genes by simply changing the coding region sequence while maintaining the promoter and terminator elements.
Solution Approach 2:
The patent performs preliminary identification and selection of appropriate miRNA recognition sites before constructing the recombinant DNA molecule. By pre-selecting recognition sites with known binding characteristics and resistance properties, the design process is simplified and the reliability of gene expression control is improved.
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
The recombinant DNA constructs effectively increase or decrease target gene expression by resisting RNase III cleavage or inhibiting translation, providing a method to regulate gene expression specifically and improve crop yields by expressing cleavage blockers or translational inhibitors in plants.
Implementation Method 1
single-stranded RNA that binds to the transcript of at least one target gene to form a hybridized segment of at least partially double-stranded RNA
Implementation Method 2
imparts to the transcript resistance to cleavage by an RNase III ribonuclease within or in the vicinity of the hybridized segment
Implementation Method 3
the binding of the single-stranded RNA to the transcript (and the formation of the hybridized segment) inhibits translation of the transcript
Data Source
AI summary
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.