TNA-Based Probe for miRNA Detection in Living Cells
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Solution Overview
Problem
Current methods for detecting and imaging microRNAs (miRNAs) in living cells are limited by complex RNA extraction processes, high experimental costs, low sensitivity, and poor reproducibility, and existing probes face challenges such as enzymatic degradation, nuclear sequestration, and cytotoxicity, restricting their applicability in clinical and biotechnological settings.
Innovation Solution
Development of threose nucleic acid (TNA)-based probes with fluorophore-labeled sense strands and quencher-labeled recognition strands that hybridize with target miRNAs, enabling rapid, selective, and sensitive fluorescence detection and imaging, capable of distinguishing one to two base mismatches, with enhanced nuclease and thermal stability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional methods (qRT-PCR, northern blotting, microarrays, NGS) are used for miRNA detection, then sensitivity and accuracy can be achieved, but experimental complexity, cost, and time requirements increase significantly
Solution Approach 1:
The invention extracts and utilizes only the essential hybridization function of nucleic acids by employing TNA molecules that can specifically bind to miRNA targets through base pairing, eliminating the need for complex RNA extraction, reverse transcription, and amplification steps required by conventional methods while maintaining detection sensitivity
Solution Approach 2:
The invention changes the chemical parameter of the probe material from natural nucleic acids (DNA/RNA) to synthetic threose nucleic acid (TNA), which provides enhanced stability and binding affinity while simplifying the detection protocol and reducing experimental complexity
2Difficulty of detecting and measuring
If DNA-based probes are used for miRNA detection in living cells, then detection capability is achieved, but enzymatic degradation, nuclear sequestration, and cytotoxicity occur
Solution Approach 1:
The invention uses composite material TNA, which combines the structural features of nucleic acids with the stability of synthetic polymers, providing resistance to enzymatic degradation and improved cellular compatibility while maintaining target binding capability
Solution Approach 2:
The TNA probes are designed as stable, non-degradable molecules that can function repeatedly in cellular environments without being degraded by nucleases, eliminating the need for continuous replenishment and ensuring reliable long-term detection
3Stability of the object's composition
If LNA-based probes are used for miRNA detection, then thermal stability and binding affinity are improved, but sequence limitations and synthesis constraints reduce versatility
Solution Approach 1:
The TNA probe system provides universal applicability to all miRNA sequences without the synthesis constraints that limit LNA probes, allowing design and synthesis of probes for any target sequence while maintaining high thermal stability and binding affinity
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 TNA-based probes provide rapid, cost-effective, and sensitive miRNA detection and imaging with high specificity and selectivity, enabling real-time monitoring of miRNA expression in living cells without cytotoxicity, overcoming limitations of existing probes and methods.
Implementation Method 1
hybridization-based probes that are designed to leverage Watson-Crick base pairing for detecting complementary nucleic acid sequences
Implementation Method 2
The recognition nucleic acid strands are usually functionalized with fluorescence resonance energy transfer pairs, fluorescent dye quencher pairs
Implementation Method 3
enhanced nuclease and thermal stability
Implementation Method 4
exhibit high thermal stability and strong binding affinity and specificity toward target RNAs
Data Source
AI summary
The present invention provides a TNA-based probe for detecting and imaging a target miRNA in living cells. TNA-based probe is composed of a fluorophore-labeled TNA reporter strand partially hybridizing to a quencher-labeled TNA recognition strand which is designed to be antisense to the target RNA transcript via pair pairing. Upon cellular entry without the need of harmful transfection treatment, the quencher-labeled TNA recognition strand binds to targeted transcript, and these target binding events displace the reporter strand from the quencher, resulting in a discrete “turning-on” of the fluorescence. The extent of fluorescence enhancement is quantifiably related to the target RNA expression level. Additionally, the TNA-based probe shows rapid detection response, excellent selectivity and specificity toward target miRNAs and is able to distinguish the target molecules with 1-2 base mismatches.