Riboswitch RNA Scaffolds for Cellular Aptamer Stability
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for developing small molecule binding RNA aptamers face challenges in transitioning from in vitro selection to functional aptamers that can reliably operate within cellular environments, as many RNA-based aptamers struggle to integrate and function effectively in cells.
Innovation Solution
The use of scaffolds derived from riboswitches and small ribozymes, such as the Bacillus subtilis xpt-pbuX guanine riboswitch, Vibrio cholerae cyclic di-GMP riboswitch, and Schistosoma mansoni hammerhead ribozyme, to create a library of oligonucleotides with destabilizing nucleotides and specific structural motifs, facilitating the selection of aptamers that can bind ligands like 5-hydroxytryptophan and couple with readout modules for functional RNA devices.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If traditional in vitro selection methods are used to generate RNA aptamers, then a diverse library of aptamers can be obtained, but many of these aptamers cannot be easily integrated into devices or do not reliably function in a cellular context
Solution Approach 1:
The patent applies local quality by incorporating destabilizing nucleotides (mismatches, bulges, G·U wobble base pairs) at specific local positions within the RNA aptamer sequences. These localized structural modifications enable the aptamers to adapt to cellular environments while maintaining ligand binding capability, resolving the contradiction between versatility and reliability in cellular contexts.
Solution Approach 2:
The patent segments the RNA aptamer into distinct functional domains including a ligand-binding domain and a readout domain. This segmentation allows independent optimization of each domain - the ligand-binding domain maintains high affinity through natural selection, while the readout domain is engineered for cellular compatibility and device integration, thereby improving both adaptability and reliability.
2Strength
If RNA aptamers are selected for high ligand binding affinity, then robust small molecule binding is achieved, but integration into functional RNA devices becomes difficult
Solution Approach 1:
The patent divides the RNA aptamer into segmented functional domains: a ligand-binding domain responsible for high affinity binding and a separate readout domain for device functionality. This segmentation reduces integration complexity by allowing modular assembly while preserving strong ligand binding capabilities.
Solution Approach 2:
The patent designs RNA aptamers with universal structural features (helix domains, hairpin domains, junctions) that can be integrated into various RNA device architectures. The standardized domain structure enables these high-affinity aptamers to function across multiple device types, reducing integration complexity while maintaining binding strength.
3Stability of the object's composition
If destabilizing nucleotides are introduced to improve cellular compatibility, then aptamer stability in cells increases, but selection and characterization becomes more challenging
Solution Approach 1:
The patent introduces destabilizing nucleotides at specific local positions rather than throughout the entire sequence. This localized approach improves cellular stability while minimizing interference with overall aptamer structure and function, making characterization more tractable compared to global destabilization strategies.
Solution Approach 2:
The patent uses scaffold RNA structures as intermediaries during selection and characterization. These scaffolds provide a stable framework that mediates between the destabilizing nucleotides and the detection system, enabling easier characterization of aptamers with modified stability properties while maintaining their cellular compatibility.
Data Source
AI summary
Provided herein are libraries of scaffolds derived from riboswitches and small ribozymes and their methods of use. The scaffolds of the invention yield aptamers that are easily identified and characterized by virtue of the structural scaffold. The nature of the scaffold predisposes these RNAs for coupling to readout domains to engineer biosensors that function in vitro and in vivo. Biosensors, synthetic RNA agents and synthetic DNA agents, and their methods of use, are also provided.