RNA Stem Loop Linking via PreQ1-TGT for Selective RNA Modification
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Solution Overview
Problem
The transient nature of RNA molecules and challenges in selectively modifying a single RNA of interest among others in cellular conditions have hindered our understanding and manipulation of RNA functions, particularly in covalent RNA-modifying approaches.
Innovation Solution
A method involving the use of a PreQ1 linking compound and a tRNA-guanine transglycosylase (TGT) enzyme to link RNA stem loop oligonucleotides, enabling the covalent attachment of PreQ1 derivatives to specific guanosine bases in RNA molecules, facilitating dimerization and cyclization of sgRNA molecules, and modulating gene expression through CRISPR-associated endonuclease delivery.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If covalent RNA-modifying approaches are used to selectively modify a single RNA of interest, then RNA function manipulation capability is improved, but selectivity and specificity among other RNAs in cellular conditions deteriorates
Solution Approach 1:
The patent introduces an intermediary system consisting of a modified nucleotide (preQ1), a specific enzyme (tRNA-guanine transglycosylase or TGT), and a guide RNA that together enable selective modification. The preQ1 nucleotide serves as an intermediary substrate that the TGT enzyme recognizes and incorporates only at specific target sites guided by the guide RNA, thus achieving high selectivity while enabling covalent modification of the target RNA among many other RNAs in the cell
2Stability of the object's composition
If the transient nature of RNA molecules is addressed by covalent modification, then RNA stability and studyability are improved, but the complexity of selectively modifying specific RNA increases
Solution Approach 1:
The system employs the cell's own enzymatic machinery (TGT enzyme) and natural nucleotide metabolism pathways to achieve covalent RNA modification. The preQ1 nucleotide is a naturally occurring modified guanine that the cellular enzyme TGT recognizes and incorporates into RNA at specific sites. This self-service approach leverages existing cellular components rather than requiring complex external modification systems, thereby achieving stable covalent modification while keeping the system relatively simple
Solution Approach 2:
The guide RNA serves as an intermediary that directs the TGT enzyme to specific target RNAs. This intermediary component enables the system to distinguish between different RNA molecules and modify only the intended target, reducing the complexity of achieving selective modification while providing stable covalent attachment of the preQ1 nucleotide to the target RNA
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 precise modification and dimerization of RNA molecules, enhancing gene editing efficiency and enabling single-cell resolution and multiplexed photo-activation of CRISPR-Cas9 gene editing.
Implementation Method 1
contacting the first RNA stem loop oligonucleotide and the second RNA stem loop oligonucleotide with a PreQ1 linking compound and a tRNA-guanine transglycosylase (TGT) enzyme
Data Source
AI summary
Provided herein are, inter alia, methods and compositions for linking RNA stem loops. The methods include linking a first RNA stem loop and a second RNA stem loop by way of a preQ1 linking compound.