Crosslinked Rolling Circle Amplification for Uniform Signal Mapping
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Solution Overview
Problem
Existing rolling circle amplification (RCA) methods lack uniformity in the size and intensity of signals due to heterogeneous analyte abundance and variations in reaction locations, leading to overlapping and masking of smaller signal spots, which complicates spatial mapping of target nucleic acids.
Innovation Solution
The method involves crosslinking a circular nucleic acid template to a second nucleic acid strand, followed by de-crosslinking, to synchronize polymerase activity and ensure uniform amplification, using crosslinkable moieties like vinylcarbazone-based moieties and UV irradiation for crosslinking and de-crosslinking.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If rolling circle amplification is performed without crosslinking, then the amplification process is simple and fast, but the uniformity of signal size and intensity is poor
Solution Approach 1:
The patent applies preliminary action by crosslinking the primer to the circular template before initiating rolling circle amplification. This pre-crosslinking step ensures that the primer remains stably bound to the template throughout the amplification process, preventing primer dissociation and ensuring uniform signal generation across all amplification sites.
Solution Approach 2:
The patent uses UV crosslinking as an intermediary mechanism to create covalent bonds between the primer and template. This intermediary chemical bonding process acts as a mediator that stabilizes the primer-template interaction, ensuring consistent amplification efficiency and signal uniformity without requiring complex additional reagents or equipment.
2Manufacturing precision
If crosslinking is performed to synchronize polymerase activity, then uniformity of amplification products is improved, but additional steps and time are required
Solution Approach 1:
The patent applies periodic action through the de-crosslinking step, where UV crosslinks are temporarily formed to synchronize polymerase activity during amplification, then subsequently removed to allow product release. This periodic bonding and unbonding mechanism enables uniform amplification while ultimately releasing the products for detection, with the entire process optimized to minimize time loss.
3Productivity
If de-crosslinking is performed to continue amplification, then complete rolling circle amplification is achieved, but the process complexity increases
Solution Approach 1:
The patent replaces mechanical or chemical complex de-crosslinking methods with simple UV irradiation. The same UV light source used for crosslinking can be applied again to de-crosslink the primer-template complex after amplification, allowing complete rolling circle amplification to proceed while minimizing additional process complexity. This substitution leverages the reversibility of photochemical crosslinking.
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 signal uniformity by synchronizing amplification, reducing differences in product sizes and intensities, thereby improving the spatial mapping and detection of target nucleic acids in situ.
Implementation Method 1
the circular nucleic acid template is crosslinked to a second nucleic acid strand... using crosslinkable moieties like vinylcarbazone-based moieties and UV irradiation for crosslinking
Implementation Method 2
the method further comprises de-crosslinking the circular nucleic acid template from the second nucleic acid strand
Data Source
AI summary
The present disclosure in some aspects relates to methods, compositions, and kits for rolling circle amplification (RCA) comprising extending a nucleic acid priming sequence hybridized to a circular nucleic acid template using a polymerase to generate an extended priming sequence, wherein the circular nucleic acid template is crosslinked to a second nucleic acid strand; de-crosslinking the circular nucleic acid template from the second nucleic acid strand; and extending the extended priming sequence to generate a rolling circle amplification product (RCP).