Multi-Input miRNA Sensing Circuits for Cell-Type Classification
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Solution Overview
Problem
Existing technologies lack efficient methods to classify individual cell types in complex biological samples based on unique microRNA (miRNA) profiles for cell phenotype and developmental stage detection.
Innovation Solution
The development of sequestrons that utilize sensor circuits regulated by miRNAs to express output molecules under specific conditions, incorporating constitutive promoters and endoribonucleases or ribozymes to translate output molecules only in the absence of inhibitory miRNAs, allowing rapid response to miRNA profile changes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional RNA sensing methods are used, then detection capability is achieved, but cell health is compromised due to toxic effects
Solution Approach 1:
The patent introduces constitutive ERNs as intermediary molecules that indirectly sense RNA through protein binding rather than direct RNA-ERN hybridization. This mediator approach (proteins binding to both target RNA and constitutive ERN) reduces toxic effects on cells while maintaining detection capability, resolving the contradiction between measurement precision and object-affected harmful factors
Solution Approach 2:
The patent replaces the conventional mechanical/chemical hybridization-based sensing mechanism with a protein-binding-based sensing mechanism. Instead of relying on RNA-ERN hybridization which causes cellular stress, the system uses proteins to bind target RNA and subsequently interact with constitutive ERNs, substituting the harmful mechanical hybridization process with a more biocompatible protein-mediated process
2Measurement precision
If multiple individual RNA targets are sensed separately, then detection accuracy is maintained, but system complexity increases
Solution Approach 1:
The patent creates a universal constitutive ERN system that can sense multiple different RNA targets through a common mechanism. Different proteins specific to each target RNA bind to their respective targets and simultaneously interact with the same constitutive ERN pool, enabling multi-target detection with a single universal sensing molecule type, thus reducing system complexity while maintaining detection accuracy
Solution Approach 2:
The patent segments the sensing function into two independent components: target-specific protein recognition modules and a universal constitutive ERN reporting system. This segmentation allows each protein to be optimized for its specific target while sharing the common ERN sensing machinery, reducing overall system complexity compared to having separate sensing systems for each target
3Adaptability or versatility
If endogenous RNA levels are monitored, then physiological relevance is achieved, but signal reliability is reduced due to cellular stress responses
Solution Approach 1:
The patent utilizes the cell's own constitutive ERNs (endogenous RNAs that are always present) as the sensing reporter molecules. These self-service ERNs are naturally abundant and stable, providing reliable signals that reflect true physiological RNA levels without being confounded by cellular stress responses, thus simultaneously achieving physiological relevance and signal reliability
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 rapid and accurate classification of cell types by expressing output molecules in response to specific miRNA profiles, facilitating disease diagnosis and treatment through detectable or therapeutic molecules.
Implementation Method 1
constitutive ERNs that can base pair with one or more different target RNAs
Implementation Method 2
a protein that binds to both the target RNA and the constitutive ERN
Data Source
Figure 1A~1C
Figure 2A~2C
Figure 3A~3E
AI summary
Provided herein are sequestrons for detecting an miRNA profile indicative of a cell state and expressing an output molecule in cells having such an miRNA profile. Also provided are methods of using sequestrons provided herein.