Circular Nucleic Acid Probes for Rolling Circle Replication
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Solution Overview
Problem
Current rolling circle nucleic acid detection techniques are inadequate for efficiently detecting single-stranded nucleic acid sequences, including RNA, due to limitations in site-specific cleavage and localization, particularly for RNA targets where existing methods result in low yield and dislocation of probes from their hybridization site.
Innovation Solution
The introduction of novel circular nucleic acid probes, such as the turtle probe and slicer probe, which incorporate endonuclease activity and self-templated ligation capabilities, enabling site-specific cleavage and localization of target nucleic acids, allowing for improved detection of RNA and DNA sequences through rolling circle replication.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional rolling circle detection techniques are used on RNA targets, then detection can be performed, but the probe dislocates from the hybridization site resulting in low yield and poor localization
Solution Approach 1:
The probe incorporates endonuclease activity directly into its structure, enabling it to autonomously cleave the target RNA at the hybridization site without requiring external enzymes. This self-service capability ensures the probe remains localized and functional, resolving the dislocation problem while maintaining high detection yield
Solution Approach 2:
The invention merges the probe function with endonuclease catalytic activity into a single integrated molecule. By combining hybridization capability and cleavage function in one probe structure, the system achieves both reliable localization and high productivity simultaneously
2Measurement precision
If endonuclease activity is incorporated into circular probes, then site-specific cleavage and localization are achieved, but probe design and manufacturing complexity increases
Solution Approach 1:
The probe is designed as a modular circular structure with distinct functional segments: a target-complementary sequence for hybridization, an endonuclease catalytic core for cleavage, and optional identifier elements. This segmentation allows precise control over cleavage specificity while simplifying the design process through standardized modular components
Solution Approach 2:
The endonuclease activity is localized specifically to the region where the probe hybridizes to the target, ensuring cleavage occurs only at the intended site. This local concentration of catalytic activity provides high measurement precision while the modular design keeps overall complexity manageable
3Measurement precision
If intramolecular structures are added to improve hybridization/ligation performance, then detection sensitivity increases, but probe structure becomes more complex
Solution Approach 1:
The probe utilizes a circular topology that brings the 5' and 3' ends into proximity, enabling efficient ligation and enhancing hybridization performance. The circular structure inherently provides the necessary intramolecular geometry without requiring additional complex elements, achieving high detection sensitivity with minimal structural complexity
Solution Approach 2:
The probe design incorporates identifier elements and functional regions nested within the circular structure, allowing multiple functions to be integrated in a compact arrangement. This nesting approach increases detection sensitivity through enhanced hybridization while keeping the overall probe structure efficient and manageable
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
These probes enhance the detection sensitivity and specificity of nucleic acid sequences by ensuring the probe remains localized at the target site, enabling efficient rolling circle replication and amplification, even on RNA targets where traditional methods fail.
Implementation Method 1
one or more elements internally in the target complementary sequence, dividing it into two or more parts, having endonuclease activity
Implementation Method 2
a target-complementary sequence capable of hybridising to a target nucleic acid
Implementation Method 3
performing rolling circle replication from the 3'-end with the circular probe as template
Data Source
Figure 1A~1H
Figure 2A~2F
Figure 3A~3B
AI summary
The present invention provides oligonucleotides and methods for efficient detection of target nucleic acids using rolling circle replication. In one aspect, the oligonucleotides of the invention are characteristic in that they can be circularised without an external ligation template. In another aspect, the oligonucleotides of the invention are characteristic in that they can generate a free 3'end of the target nucleic acid necessary for rolling circle replication. The oligonucleotides and detection methods of the invention are useful e.g. as research tool and for diagnosis.