Target RNA-Primed Rolling Circle Amplification for In Situ Detection
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing RCA-based detection methods for in situ analysis of nucleic acids suffer from low sensitivity, specificity, and detection efficiency due to heterogenous micro-environments and asynchronous amplification processes.
Innovation Solution
The method involves using a guide nucleic acid complexed with an RNA-cutting enzyme to specifically cut target RNA, which is then used to prime rolling circle amplification (RCA) of a circular probe, eliminating the need for separate nucleic acid primers and promoting a more homogeneous amplification system.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If separate nucleic acid primers are used in RCA-based detection methods, then the amplification process can be initiated, but the system becomes heterogeneous and detection efficiency decreases
Solution Approach 1:
The patent extracts and eliminates the separate primer component from the RCA system. Instead of using external primers, the target RNA molecule itself is utilized as the primer through its 3' end, thereby simplifying the amplification system and eliminating the heterogeneity caused by separate primer-target annealing steps.
Solution Approach 2:
The target RNA serves a dual function: it is both the template to be amplified and the primer that initiates amplification. The 3' end of the target RNA automatically primes the RCA reaction without requiring separate primer molecules, making the system self-sufficient and more efficient.
2Measurement precision
If RCA is performed in heterogeneous micro-environments, then amplification can occur, but RCP homogeneity and detection precision deteriorate
Solution Approach 1:
The patent creates a homogeneous amplification environment by eliminating separate primers and using the target RNA's 3' end as the primer. This ensures that all RCA reactions start from identical conditions, producing uniform RCPs with consistent size and intensity, thereby improving detection precision.
3Productivity
If asynchronous RCA amplification occurs, then the detection process can be completed, but sensitivity and detection efficiency are reduced
Solution Approach 1:
The target RNA's 3' end is pre-positioned and ready to serve as the primer before the RCA reaction begins. This preliminary arrangement eliminates the need for separate primer annealing steps and ensures that all amplification reactions start synchronously, improving detection efficiency and reducing time variability.
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 achieves better RCP homogeneity, improved sensitivity and specificity, elevated median intensity, better signal-to-noise ratios, and enhanced localization of detected RCPs.
Implementation Method 1
a guide nucleic acid is used to provide a DNA-RNA or RNA duplex for cutting (e.g., cleavage) by an RNA-cutting enzyme of a target RNA in the duplex
Implementation Method 2
cutting (e.g., cleavage) by an RNA-cutting enzyme of a target RNA in the duplex
Implementation Method 3
The cut target RNA itself can then be used to prime RCA of a circular probe or a circularized probe
Data Source
AI summary
The present disclosure relates in some aspects to methods, systems, and kits for analyzing a biological sample comprising generating a rolling circle amplification product (RCP) using a target ribonucleic acid (RNA) as a primer. In some aspects, an RNA-cutting enzyme and a guide nucleic acid are used to generate a free 3′ end of the target RNA to prime RCA.