Aptamer-Modulated RNase P Cleavage for Ligand-Controlled Gene Expression
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Solution Overview
Problem
Existing methods for regulating gene expression, such as those using external guide sequences (EGS) for RNase P cleavage, require exogenous sequence expression and lack efficient control over target gene expression in response to specific ligands.
Innovation Solution
A polynucleotide cassette comprising an RNase P substrate sequence linked to a riboswitch with an aptamer, where the aptamer forms a stem upon ligand binding to prevent RNase P cleavage, allowing controlled gene expression through aptamer-mediated ribonuclease cleavage.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If RNAi-based approaches are used to achieve gene silencing, then gene expression can be downregulated, but off-target effects and immunogenicity occur
Solution Approach 1:
The patent uses aptamers as intermediary molecules that specifically bind to target mRNAs (such as c-MET mRNA) to induce cleavage by RNase P. This aptamer-mediated approach serves as an alternative intermediary between the silencing agent and target RNA, avoiding the off-target binding and immunogenicity problems associated with RNAi while maintaining specific gene silencing efficacy through the high specificity of aptamer-RNA interactions
2Ease of operation
If small molecules are used for gene regulation, then they can penetrate cell membranes easily, but they lack sequence specificity
Solution Approach 1:
The patent employs peptide nucleic acids (PNAs) as composite molecules that combine the membrane-penetrating properties of small molecules with the sequence-specific binding capability of nucleic acids. PNAs have peptide-like backbones that facilitate cell membrane penetration while containing nucleic acid-like binding regions that provide high sequence specificity for target mRNA recognition and cleavage site targeting
3Measurement precision
If large molecules are used for gene regulation, then they can achieve high specificity, but they have difficulty penetrating cell membranes
Solution Approach 1:
The patent modifies the molecular parameters of regulatory agents by using peptide nucleic acids with altered backbone chemistry (peptide-like instead of sugar-phosphate), which changes the molecular size and charge characteristics to enable cell membrane penetration while preserving the sequence-specific binding properties of nucleic acid-based molecules
4Ease of manufacture
If traditional methods are used to achieve gene silencing, then established protocols are available, but delivery systems are complex and require multiple components
Solution Approach 1:
The patent extracts and utilizes the catalytic activity of endogenous RNase P enzyme as the active silencing component, eliminating the need for complex delivery systems that must protect and deliver synthetic catalytic molecules. By harnessing the cell's own RNase P enzyme and directing it to specific targets through aptamer-PNA conjugates, the system simplifies the delivery requirement to primarily nuclear localization signals while maintaining effective gene silencing
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
Enables precise modulation of gene expression by allowing increased expression upon ligand binding and decreased expression when ligand is absent, enhancing safety and control in therapeutic applications.
Implementation Method 1
An aptamer of Formula (I) or a conjugate of a compound of Formula (I) with a compound of Formula (II) or a conjugate of a compound of Formula (II) with a compound of Formula (III), wherein the aptamer, compound of Formula (II), or compound of Formula (III) directs RNase P to a target sequence
Implementation Method 2
directs RNase P to a target sequence to cleave an mRNA
Data Source
Figure 1a~1b
Figure 2a~2b
Figure 2c
AI summary
The present disclosure provides polynucleotide constructs for the modulation of target gene expression by aptamer-mediated ribonuclease cleavage of the target gene RNA and methods of using the constructs to modulate gene expression in response to the presence or absence of a ligand that binds the aptamer. The polynucleotide constructs contains a ribonuclease substrate sequence (e.g., an RNase P substrate) and a riboswitch comprising an effector region and an aptamer such that when the aptamer binds a ligand, target gene expression occurs.