Multiplex Method for Detecting Spatially Overlapping Transcripts
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Solution Overview
Problem
Current multiplex methods for detecting analytes in biological samples are inflexible, expensive, time-consuming, and often provide inaccurate results, with low encoding capacities and inability to differentiate spatially overlapping transcripts due to the diffraction limit of optical microscopes.
Innovation Solution
A novel high-resolution multiplex method involving simultaneous hybridization of target probes for two analytic sets with unique tails and decoder sets, allowing independent dataset generation and combination, enabling detection of spatially overlapping transcripts beyond the diffraction limit through sequential signal-encoding.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If conventional multiplex methods are used to detect multiple analytes, then detection capability is provided, but the methods are inflexible, expensive, time-consuming and provide inaccurate results with low encoding capacities
Solution Approach 1:
The method divides the detection process into multiple sequential rounds, where in each round only a subset of analytes is detected. This segmentation allows complex multiplexing to be achieved through simpler, repeated operations. The probe sets are also segmented into different groups that can be selectively hybridized in different rounds, reducing the complexity of any single detection step while maintaining high overall multiplexing capability.
Solution Approach 2:
The patent employs preliminary hybridization of probe sets to analytes before the actual detection rounds. This preliminary action establishes the foundation for subsequent sequential detection, allowing the system to prepare multiple probe-analyte complexes simultaneously that can then be read out in different rounds. This preliminary positioning enables the method to handle complex multiplexing scenarios without increasing the complexity of individual detection steps.
2Measurement precision
If optical microscopes are used for detection, then detection is possible, but targets with distance below the diffraction limit cannot be differentiated
Solution Approach 1:
The patent introduces decoder oligonucleotides as intermediary molecules that bridge the gap between the probe sets hybridized to analytes and the detectable signals. These decoders contain unique identifier sequences that allow computational differentiation of signals even when analytes are spatially overlapping beyond the diffraction limit. The intermediary decoders enable the system to recover spatial information that would otherwise be lost due to optical diffraction constraints.
Solution Approach 2:
The method transitions from relying solely on spatial separation in three-dimensional space to using an additional dimension of identification through unique nucleotide sequences. By encoding analyte identity in the sequence domain rather than relying purely on spatial position, the system can differentiate targets that are closer than the diffraction limit, effectively adding a fourth dimension (sequence space) to the detection capability.
3Productivity
If directly labeled probe sets are used for detection, then detection is achieved, but several differently tagged probe sets are needed per transcript and denaturation is required after every detection round
Solution Approach 1:
The patent uses unlabeled probe sets that are hybridized to analytes, and then uses separate decoder oligonucleotides that contain the detectable labels. This copying approach separates the binding function (performed by the unlabeled probes) from the detection function (performed by the labeled decoders). The unlabeled probes can remain hybridized to analytes across multiple rounds without needing denaturation, while only the decoder labels need to be removed and replaced between rounds, significantly reducing the time loss compared to denaturing and re-hybridizing labeled probes each time.
Solution Approach 2:
The method extracts the detectable label from the probe set itself and places it on separate decoder oligonucleotides. This extraction allows the probe sets to remain permanently bound to analytes without requiring denaturation, while the labels are taken out and replaced only when needed for different detection rounds. This separation eliminates the time-consuming denaturation step that would otherwise be required after each detection round.
4Loss of information
If high multiplexing is attempted in a single round, then encoding capacity increases, but accuracy decreases due to signal overlap and diffraction limit
Solution Approach 1:
The patent segments the detection of multiple analytes into multiple sequential rounds, with each round detecting a manageable subset of analytes using dedicated probe sets and decoders. This segmentation maintains high encoding capacity across all analytes while ensuring that each individual round operates at optimal accuracy levels with minimal signal overlap. The unique identifier sequences in the decoders allow computational reconstruction of the complete multiplexed data set with high precision.
Solution Approach 2:
The decoder oligonucleotides serve as intermediaries that enable high multiplexing accuracy by providing unique sequence identifiers for each analyte. These decoders allow the system to computationally distinguish between signals from different analytes even when they are spatially overlapping, maintaining high detection accuracy while achieving high encoding capacity through the sequential multi-round approach.
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 multiplexing capability, allows detection of previously invisible transcripts, reduces complexity, and improves signal-to-noise ratio, enabling accurate identification of multiple analytes, including those with high expression levels, in fewer rounds with higher confidence.
Implementation Method 1
contacting the sample with at least a first set of analyte-specific probes for encoding of at least different analytes, each set of analyte-specific probes interacting with a different analyte
Implementation Method 2
contacting the sample with at least a set of decoding oligonucleotides, wherein in each set of decoding oligonucleotides for an individual analyte each decoding oligonucleotide comprises an identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T)
Implementation Method 3
contacting the sample with at least a set of signal oligonucleotides, each signal oligonucleotide comprising a translator connector element (C) comprising a nucleotide sequence which is essentially complementary to at least a section of the nucleotide sequence of a translator element (c)
Data Source
AI summary
The technology provided herein relates to high resolution multiplex methods and kits for detecting different analytes in a sample in parallel by sequential signal-encoding of said analytes, wherein the method allows a differentiation of targets which distance is below the diffraction limit of optical microscopes, that is, targets with spatial optical overlap. The disclosed methods also include in vitro methods for screening, identifying and/or testing a substance and/or drug and in vitro methods for diagnosis of a disease, and an optical multiplexing system.