RNA Templated Ligation Probes for Specificity
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Solution Overview
Problem
Current methods for analyzing nucleic acids, particularly RNA, suffer from reduced specificity and sensitivity, leading to inaccurate detection and sequencing due to poor ligation efficiencies and fidelity, especially in RNA-templated ligation reactions.
Innovation Solution
The use of modified probes or probe sets with a duplex region positioned between hybridization regions, which are cleaved by nucleases to generate ligatable ends, ensuring only hybridized and processed probes are ligated, thereby improving ligation specificity and accuracy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional probes are used for nucleic acid detection, then the detection process is simple, but ligation specificity and sensitivity are reduced leading to false positives
Solution Approach 1:
The probe is divided into distinct functional regions: a hybridization region that binds to the target nucleic acid, a stem-loop structure that blocks the ligatable end, and a ligatable end that is released only after specific cleavage. This segmentation ensures that ligation occurs only when the probe is correctly hybridized to the target, improving specificity while maintaining manageable structural complexity.
Solution Approach 2:
The probe is pre-designed with a stem-loop structure that blocks the ligatable end before hybridization. This preliminary blocking mechanism ensures that ligation can only occur after the probe has successfully hybridized to the target and the stem-loop is cleaved, preventing false positives from non-specific binding.
2Manufacturing precision
If RNA-templated ligation is used for RNA detection, then direct RNA analysis is enabled, but ligation fidelity is poor leading to sequencing errors
Solution Approach 1:
The stem-loop structure acts as an intermediary element that mediates between the hybridization event and ligation. It must be cleaved by a nuclease only after successful hybridization, creating a controlled intermediate state that ensures high fidelity ligation while maintaining efficient detection through the released ligatable ends.
3Reliability
If probes without blocking structures are used, then ligation is faster and simpler, but non-specific ligation occurs reducing detection accuracy
Solution Approach 1:
The stem-loop structure performs preliminary anti-action by blocking the ligatable end before hybridization. This prevents non-specific ligation events, and the block is removed only after successful target binding, ensuring high detection accuracy while the rapid cleavage step minimizes time loss.
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 the specificity and sensitivity of nucleic acid detection and sequencing by ensuring that only correctly hybridized probes are ligated, reducing false positives and improving the accuracy of RNA sequencing.
Implementation Method 1
cleaving the probe or probe set, e.g., with a nuclease, to generate a first ligatable end and release the duplex region or a portion thereof
Implementation Method 2
a first hybridization region capable of hybridizing to a first target sequence in the target nucleic acid
Implementation Method 3
ligating the first ligatable end to a second ligatable end in the probe or probe set to generate a ligated probe
Data Source
AI summary
The present disclosure relates in some aspects to methods for analyzing a target nucleic acid in a biological sample. In some aspects, provided herein are methods and compositions for improving the specificity of ligation in situ in biological samples, as well as in single cell analysis and spatial applications of RNA templated ligation reactions.