RCA Identifier Sequencing for Multiplexed In Situ Signal Decoding
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Solution Overview
Problem
Plex-scalability of in situ detection methods is limited by optical crowding, where signals from large or high-intensity features overlap with smaller or weaker signals, compromising signal detection and decoding quality, and existing methods require complex probe pools, increasing cost and time.
Innovation Solution
Base-by-base sequencing methods using sequencing primers that hybridize to identifier sequences, generating signal code sequences through nucleotide incorporation or binding events, allowing for decoding of multiple analytes by minimizing ON and OFF signal detection in sequential cycles.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Illumination intensity
If rolling circle amplification (RCA) products, nucleic acid probes, or nucleic acid complexes are used for in situ detection, then signal intensity increases, but optical crowding occurs where signals overlap with and mask other signals from features in close proximity
Solution Approach 1:
The patent segments the identifier sequence into multiple blocks, where each block is detected in a separate imaging cycle. This temporal segmentation allows signals from different spatial locations to be acquired sequentially rather than simultaneously, preventing optical crowding while maintaining signal intensity. Each block detection generates a portion of the final decoded identifier sequence.
Solution Approach 2:
The patent employs periodic imaging cycles to detect different blocks of identifier sequences at different time points. By periodically cycling through detection of multiple blocks across multiple imaging cycles, the system resolves signals that would otherwise overlap spatially, thereby eliminating optical crowding while preserving signal intensity through repeated measurements.
2Adaptability or versatility
If complex pools of oligonucleotide probes are used for multiplexed detection, then the ability to detect multiple analytes increases, but cost and time for detection and decoding increase
Solution Approach 1:
The patent employs universal sequencing primers that can bind to and sequence any identifier sequence regardless of which analyte it represents. This universal primer approach replaces the need for analyte-specific probes for each target, enabling multiplexed detection of many analytes using a single standardized sequencing workflow, thereby reducing both cost and time.
Solution Approach 2:
The patent uses identifier sequences (barcodes) that are copied onto RCA products or probe complexes. Instead of using complex pools of different probes for each analyte, the system copies simple identifier sequences onto detection complexes, which are then universally sequenced. This copying approach simplifies the probe design and reduces the complexity of probe pools required for multiplexed detection.
3Object-affected harmful factors
If small size features or weak intensity signals are used for detection, then optical crowding is reduced, but signals may not reach detection threshold
Solution Approach 1:
The patent merges multiple copies of the same identifier sequence onto a single RCA product or probe complex. By combining multiple signal-generating copies at each spatial location, the system maintains strong signals that exceed detection thresholds while using the temporal block-segmentation approach to prevent optical crowding between different spatial locations.
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
Enhances the quality of signal detection and decoding in multiplexed assays by reducing optical crowding and simplifying probe use, thereby improving the accuracy and efficiency of in situ analyte analysis.
Implementation Method 1
a sequencing primer hybridizes to a priming site 3′ to an identifier sequence
Implementation Method 2
the sequencing primer can be extended by a polymerase in a base-by-base fashion using a sequence of the identifier sequence as a template
Data Source
AI summary
Methods and compositions for performing a rolling circle amplification (RCA) reaction using circularized template comprising identifier sequences are provided. Sequencing is performed on the RCA product using a polymerase to incorporate a plurality of cognate nucleotides into the sequencing primer or an extension product thereof to generate an extension product.