Sequential Hybridization Barcoding for Low-Noise Nucleic Acid Profiling
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Solution Overview
Problem
Existing methods for profiling transcripts or DNA loci in cells, particularly single cells, are costly, labor-intensive, prone to artifacts, and introduce noise and bias due to low efficiency in mRNA to DNA conversion and require distinct fluorophores for scale-up, leading to high data complexity.
Innovation Solution
A sequential barcoding scheme involving multiple rounds of contacting cells with detectably labeled oligonucleotides, each round using a different detectable moiety, followed by imaging and optional removal, to multiplex target detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If single cell RNA-seq or qPCR are used for transcript profiling, then gene expression can be measured, but the process requires cell isolation and multi-well format which increases cost and labor intensity
Solution Approach 1:
The patent segments the transcript profiling process into multiple sequential hybridization rounds, each targeting specific transcripts with unique barcode combinations. This allows multiplexed detection of many transcripts simultaneously in a single cell without requiring physical cell isolation or multi-well formats, thereby reducing operational complexity while maintaining measurement precision
Solution Approach 2:
The patent introduces a temporal dimension by performing sequential hybridization rounds at different time points, where each round adds another layer of barcode information. This transforms a spatial problem (multiplexing many targets in one step) into a temporal solution (multiple steps building information layer by layer), enabling high-dimensional transcript profiling without increasing operational complexity
2Productivity
If enzymatic reactions are used to convert mRNA to DNA template, then sequencing can be performed, but the conversion efficiency is low (1% for RT and 10% for PLA) introducing noise and bias
Solution Approach 1:
The patent extracts and eliminates the problematic enzymatic conversion step (reverse transcription and ligation) from the workflow. Instead of converting mRNA to DNA through inefficient enzymatic reactions, the method uses direct hybridization of DNA barcodes to mRNA targets followed by detection, thereby achieving near 100% conversion efficiency without introducing enzymatic noise or bias
Solution Approach 2:
The patent introduces a DNA barcode oligonucleotide as an intermediary that directly hybridizes to the mRNA target. This intermediary approach replaces the inefficient enzymatic conversion pathway with a simple hybridization-based capture mechanism, maintaining measurement accuracy while dramatically improving productivity
3Adaptability or versatility
If spectral mRNA barcoding with smFISH is used, then multiple targets can be detected, but distinct fluorophores are required for each target which limits the number of barcodes and increases data complexity
Solution Approach 1:
The patent makes the DNA barcode oligonucleotides universal by using the same set of barcodes across multiple hybridization rounds and different targets. Each barcode combination encodes information about which targets were present, allowing the system to detect many more targets than the number of physical fluorophores used. This universality reduces device complexity while increasing adaptability
Solution Approach 2:
The patent adds a combinatorial dimension to the detection system by using combinations of barcodes in different hybridization rounds to encode target identity. Instead of requiring one fluorophore per target, the system uses temporal and combinatorial coding, where the sequence and combination of barcode appearances across rounds encodes which targets were detected, dramatically reducing data complexity
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 allows for efficient, low-noise profiling of multiple targets in cells, reducing costs and labor while enhancing data analysis simplicity.
Implementation Method 1
performing a first contacting step that involves contacting a cell comprising a plurality of nucleic acids with a first plurality of detectably labeled oligonucleotides, each of which targets a nucleic acid
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. In some embodiments, through sequential barcoding, the present invention provides methods for high-throughput profiling of a large number of targets, such as transcripts and/or DNA loci.