Small RNA Circularization for Sensitive Detection
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Solution Overview
Problem
Current methods for detecting and quantifying small RNAs, such as miRNAs, face challenges due to their small size, lack of modification sites, and instability, leading to poor sensitivity and throughput in existing detection techniques.
Innovation Solution
The method involves circularizing target RNAs by ligation of their 5′- and 3′-ends, followed by rolling circle amplification to produce multimer nucleic acids that can be labeled or subjected to PCR, allowing for efficient and sequence-specific amplification and detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional detection methods are used for small RNAs, then the process is simple, but sensitivity and throughput are poor
Solution Approach 1:
The patent applies preliminary action by circularizing the small RNA molecules before detection. This pre-processing step transforms the small RNAs into a configuration that enables subsequent amplification, thereby enhancing detection sensitivity without requiring complex detection procedures. The circularization step prepares the molecules for efficient rolling circle amplification in later stages.
Solution Approach 2:
The patent employs copying through rolling circle amplification, where the circularized small RNA serves as a template to generate multiple copies of itself. This amplification process dramatically increases the amount of target molecule available for detection, improving sensitivity while maintaining a relatively simple detection workflow. The copied molecules can then be detected using standard methodologies.
2Productivity
If conventional methods are used, then the number of steps is reduced, but simultaneous detection of multiple targets is limited
Solution Approach 1:
The patent implements universality by designing a circularization approach that works for multiple types of small RNAs simultaneously. The same circularization and rolling circle amplification process can detect different small RNA sequences in parallel, enabling multiplexing capability. This universal method allows simultaneous detection of multiple targets without requiring separate procedures for each RNA type.
Solution Approach 2:
The patent merges multiple detection functions into a single integrated workflow. By combining circularization, rolling circle amplification, and detection steps into one streamlined process, the patent achieves high throughput while reducing the number of separate steps and reagents required. Multiple targets can be detected in the same reaction mixture.
3Measurement precision
If standard detection protocols are used, then reagent requirements are minimized, but detection accuracy for modified RNAs is poor
Solution Approach 1:
The patent applies parameter changes by modifying the chemical structure of the small RNA through circularization. This structural transformation creates a new molecular configuration that is more suitable for detection, improving accuracy for modified RNAs such as those with 2′OH and 2′-OMe groups. The circularized form presents different physical and chemical properties that enhance detectability without requiring additional reagents.
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 enhances sensitivity and accuracy of small RNA detection, reduces the number of steps and reagents required, and enables simultaneous detection of multiple target RNAs, even those with 2′OH and 2′-OMe modifications at their 3′ ends.
Implementation Method 1
circularization of target RNA by ligation of its 5′- and 3′-ends
Implementation Method 2
synthesis of multimer nucleic acid (MNA) comprising multiple repeats of sequences that are complementary to the target RNA by rolling circle amplification (RCA)
Data Source
AI summary
Currently, the circularization of small RNAs is broadly regarded as an obstacle in ligation-related assays and explicitly avoided while short lengths of linear RNA targets is broadly recognized as a factor limiting use of conventional primers in PCR-related assays. In contrast, the disclosed invention capitalizes on circularization of small RNA targets or their conjugates with oligonucleotide adapters. The circular RNA templates provide amplification of the target sequences via synthesis of multimer nucleic acids that can be either labeled for direct detection or subjected to PCR amplification and detection. Structure of small circular RNAs and corresponding multimeric nucleic acids provide certain advantages over current methods including flexibility in design of conventional RT and PCR primers as well as use of 5′-overlapping dimer-primers for efficient and sequence-specific amplification of short target sequences. Our invention also reduces number of steps and reagents while increasing sensitivity and accuracy of detection of small RNAs with both 2′OH and 2′-OMe at their 3′ ends. Our invention increase sensitivity and specificity of detection of microRNAs and other small RNAs with both 2′OH and 2′-OMe at their 3′ ends while allowing us to distinguish these two forms from each other.