Sequential Pseudo-Color Probing for Single-Molecule Transcript Detection
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Solution Overview
Problem
Existing methods for transcriptomic profiling, such as single cell RNA-seq and qPCR, are labor-intensive, costly, and prone to artifacts, while in situ sequencing and smFISH face inefficiencies and noise due to low conversion rates and high data complexity.
Innovation Solution
A method involving sequential barcoding with n rounds of hybridization, using probes with unique barcodes and detectable visual signals, followed by error correction, to multiplex molecular targets and generate unique codes for each target.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If in situ sequencing uses enzymatic reactions to convert mRNA into DNA template, then transcriptomic profiling can be performed, but conversion efficiency is low (1-10%) introducing significant noise and bias
Solution Approach 1:
The patent replaces enzymatic conversion (chemical/biological system) with direct hybridization of DNA probes to mRNA (physical binding system). This substitution eliminates the inefficient enzymatic steps while maintaining specific detection capability through complementary base pairing between probes and target transcripts.
Solution Approach 2:
The patent uses multiple DNA probes that hybridize to the mRNA template to create detectable signal copies. Each probe binds to a specific sequence on the mRNA, and the collective binding events generate sufficient signal for detection without requiring enzymatic amplification or conversion.
2Adaptability or versatility
If smFISH uses multiple fluorophores for barcoding, then more genes can be profiled, but data analysis complexity increases significantly
Solution Approach 1:
The patent segments the barcoding process into multiple sequential hybridization rounds, where each round uses a single fluorophore to write one bit of the barcode. This temporal segmentation replaces spatial multiplexing with sequential operations, reducing the complexity of simultaneously processing multiple fluorophores while maintaining high multiplexing capacity.
Solution Approach 2:
The patent employs periodic hybridization cycles where probes are sequentially introduced, imaged, and removed. Each cycle contributes one element to the final barcode, creating a time-based multiplexing scheme that simplifies data analysis compared to simultaneous multi-color imaging.
3Adaptability or versatility
If sequential hybridization barcoding is performed with multiple rounds, then more molecular targets can be identified, but the process becomes more complex and time-consuming
Solution Approach 1:
The patent performs preliminary hybridization of DNA probes to mRNA targets before imaging. The probes remain bound during the imaging process, allowing rapid acquisition of barcode information without requiring time-consuming enzymatic reactions or signal amplification steps during the actual measurement phase.
Solution Approach 2:
The hybridized probes serve as both the detection element and the barcode writer simultaneously. The probes' intrinsic fluorescence properties provide the signal, eliminating the need for separate labeling or amplification steps, thereby reducing overall process time while maintaining versatility.
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
Enables accurate, high-throughput transcriptomic profiling with single molecule sensitivity, reducing noise and bias, and simplifying data analysis.
Implementation Method 1
n sequential barcoding rounds (where n≥2), wherein each barcoding round comprises m serial hybridizations of probes collectively bound to the N molecular targets
Implementation Method 2
each probe is capable of generating at least one detectable visual signal representing binding of the probe to a molecular target
Data Source
AI summary
The present invention, among other things, provides technologies for detecting and/or quantifying nucleic acids in cells, tissues, organs, or organisms. Pre-designed barcodes are associated with specific molecular targets through sequential hybridization experiments. A pseudo-color based barcoding scheme is described that overcomes the limitations in the previous generation of the technology. The current method can be applied to both in vitro and in situ analysis.