Combinatorial Capture Probe Pairs for Specific Nucleic Acid Immobilization
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Solution Overview
Problem
Existing methods for capturing target nucleic acids on solid supports lack specificity and efficiency, often capturing closely or distantly related sequences, which compromises the purity and efficiency of the capture process.
Innovation Solution
The use of combinatorial capture probe pairs, each comprising a target binding region and a tag region with a stem region, allows for the hybridization of capture oligonucleotides to a target nucleic acid, forming a combined capture sequence that stably binds to an oligonucleotide on a solid support, enhancing specificity and efficiency of immobilization.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If single natural deoxyoligonucleotides are used to capture target sequences, then the capture process is simple, but the specificity and purity of target capture are compromised due to capturing of closely or distantly related sequences
Solution Approach 1:
The capture system is divided into multiple components: a first capture oligonucleotide with a first capture sequence, a second capture oligonucleotide with a second capture sequence, and a bridging oligonucleotide that connects them. Each component has a specific function, and their combination provides high specificity through multiple binding sites while maintaining manageable individual structures.
Solution Approach 2:
Multiple capture sequences are combined in a single capture complex. The first and second capture oligonucleotides are brought into proximity by the bridging oligonucleotide, creating a multi-valent capture system that simultaneously engages multiple target sequences, thereby enhancing specificity and reducing off-target binding.
2Productivity
If single capture oligonucleotides are used, then the assay procedure is straightforward, but the efficiency and speed of target immobilization are insufficient
Solution Approach 1:
The first and second capture oligonucleotides are pre-formed with their respective target binding regions and capture sequences. The bridging oligonucleotide is designed in advance to complement both capture sequences. This preliminary preparation allows for rapid assembly of the complete capture complex during the assay, improving efficiency without sacrificing the multi-component design.
Solution Approach 2:
The bridging oligonucleotide serves as an intermediary that connects the first and second capture oligonucleotides. It contains sequences complementary to both capture sequences, facilitating their association and enabling the formation of a stable multi-component capture complex that efficiently immobilizes target nucleic acids.
3Reliability
If capture regions have low individual affinity, then the probe design is flexible, but stable binding to the solid support oligonucleotide cannot be achieved under assay conditions
Solution Approach 1:
Multiple capture sequences (first and second capture sequences) are combined in proximity through the bridging oligonucleotide. Individually, these capture sequences have low affinity, but when brought together, they collectively provide sufficient binding strength to stably associate with the solid support oligonucleotide, achieving both flexibility and stability.
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 significantly enhances the specificity and efficiency of target nucleic acid capture, allowing for the immobilization of specific sequences with high precision and speed, even in complex samples, while minimizing non-specific interactions.
Implementation Method 1
The target binding regions comprise nucleic acid sequences that allow them to hybridize to adjacent regions on the target nucleic acid
Implementation Method 2
The stem regions have nucleic acid sequences that are complementary to each other and the capture regions each comprise a sequence that when positioned adjacent to one another produce a combined nucleic acid sequence
Implementation Method 3
This sequence is then hybridized to the oligonucleotide bound to the solid support (or in solution, which is subsequently bound to a solid support) thereby immobilizing the target nucleic acid on the solid support
Data Source
AI summary
The present invention provides methods for immobilizing target nucleic acids on a solid support utilizing combinatorial capture probe pairs. These pairs contain first and second capture oligonucleotides that each comprise a target binding region, a capture region and a stem region positioned between the target binding and capture regions. The target binding regions comprise nucleic acid sequences that allow them to hybridize to adjacent regions on the target nucleic acid. The stem regions have nucleic acid sequences that are complementary to each other and the capture regions each comprise a sequence that when positioned adjacent to one another produce a combined nucleic acid sequence that is complementary to a portion of an oligonucleotide bound to a solid support. When the first and second capture oligonucleotides are annealed to the target nucleic acid, the stem regions are brought together allowing them to hybridize, which in turn brings the capture regions together to produce a combined nucleic acid sequence. This combined nucleic acid sequence is then able to hybridize to the oligonucleotide bound to the solid support thereby immobilizing the target nucleic acid.