Blocking Strand Nucleic Acid Probe Specificity
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Solution Overview
Problem
Current methods for analyzing short nucleic acid sequences, such as single nucleotide polymorphisms (SNPs), suffer from low assay specificity and high rates of false positive results due to poor ligase discrimination and template-independent ligation, especially when targeting RNA molecules.
Innovation Solution
The use of a probe and a blocking strand that are designed to hybridize specifically to a target nucleic acid, where the blocking strand is displaced only when the probe's interrogatory region is complementary to the target sequence, allowing for precise ligation and amplification of the region of interest.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional ligation-based methods are used to detect short nucleic acid sequences, then the detection process can be performed, but the assay specificity is low and false positive rates are high due to poor ligase discrimination and template-independent ligation
Solution Approach 1:
A blocking strand is introduced as an intermediary element that competes with the target nucleic acid for binding to the probe. The blocking strand contains a blocking sequence that hybridizes to the interrogatory region, preventing premature ligation. Only when the target nucleic acid displaces the blocking strand through specific complementary binding does ligation proceed, thereby eliminating template-independent ligation and reducing false positives.
Solution Approach 2:
The blocking strand performs a preliminary anti-action by pre-hybridizing to the probe's interrogatory region, creating a blocked state that prevents unwanted ligation events. This preliminary blocking action counteracts the tendency of ligases to perform template-independent ligation, ensuring that ligation only occurs when the specific target sequence is present to displace the blocking strand.
2Reliability
If the probe hybridization region is blocked by a blocking strand, then specificity is improved, but the hybridization region is initially unavailable for binding to the target nucleic acid
Solution Approach 1:
The system transitions from a static blocked state to a dynamic equilibrium where the blocking strand and target nucleic acid compete for binding to the probe. The blocking strand initially occupies the hybridization region, but the system dynamically responds to the presence of the target sequence, allowing the blocking strand to be displaced when specific binding conditions are met, thus making the hybridization region available only when needed.
Solution Approach 2:
The blocking strand is pre-bound to the probe before target addition, creating a preliminary blocked configuration. This preliminary action ensures that the probe is protected from non-specific binding and ligation events. When the target nucleic acid is introduced, it triggers the displacement of the blocking strand, converting the preliminary blocked state into an active detection state only under specific conditions.
3Measurement precision
If blocking strands are used to prevent template-independent ligation, then ligation specificity is improved, but the assay complexity increases due to additional components and steps
Solution Approach 1:
The blocking strand and probe are designed to work as an integrated system where the blocking sequence is part of the overall probe structure. The blocking strand combines multiple functions: it blocks the hybridization region, provides a competing binding element, and enables specific displacement by the target. This merging of functions into a single oligonucleotide component reduces the need for separate blocking agents and simplifies the overall assay protocol.
Solution Approach 2:
The blocking strand serves multiple functions simultaneously: it acts as a blocking agent to prevent non-specific ligation, a competing binder to ensure specific target recognition, and a displacement element that enables signal generation. This multi-functionality eliminates the need for separate components for each function, reducing assay complexity while maintaining high ligation specificity.
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 increases specificity and reduces background noise, enabling accurate detection of short sequences like SNPs with improved sensitivity and reliability, even when targeting RNA without the need for cDNA synthesis.
Implementation Method 1
the blocking strand is hybridized to a hybridization region in the probe or the target nucleic acid, thereby blocking the hybridization region from hybridizing to a complementary hybridization region
Implementation Method 2
allowing hybridization between the probe and the target nucleic acid, wherein if the interrogatory region is complementary to the region of interest, the blocking strand is displaced and the hybridization region is available for hybridizing
Implementation Method 3
ligating the probe hybridized to the target nucleic acid to itself or to another probe hybridized to the target nucleic acid
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 detecting a region of interest in a target nucleic acid, wherein hybridization between an interrogatory region of a probe and a region of interest of the target nucleic acid is blocked by a blocking strand unless the interrogatory region is complementary to the region of interest. In some aspects, the methods provided herein increase specificity of detecting a region of interest in a target nucleic acid (e.g., a SNP in an RNA molecule). In some aspects, the presence, amount, and/or identity of a region of interest in a target nucleic acid is analyzed in situ. Also provided are polynucleotides, sets of polynucleotides, compositions, and kits for use in accordance with the methods, for example for RNA-targeting padlock probe-mediated SNP detection.