Sequential Hybridization Barcoding for Multiplex RNA Detection
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Solution Overview
Problem
Existing methods for profiling transcripts in cells, such as single cell RNA-seq and qPCR, are labor-intensive, costly, and prone to artifacts, while in situ sequencing technologies are inefficient, and spectral mRNA barcoding technologies are limited in barcode generation and 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 in situ sequencing technologies use enzymatic reactions to convert mRNA into DNA template, then transcript profiling can be performed in cells, but the conversion efficiency is highly inefficient (only 1% for RT and 10% for PLA)
Solution Approach 1:
The patent extracts and eliminates the inefficient enzymatic conversion step (reverse transcription and ligation) from the sequencing workflow. Instead of converting mRNA to DNA template through low-efficiency enzymes, the invention directly sequences RNA molecules using template switching oligonucleotides that enable PCR amplification of RNA without requiring high-efficiency enzymatic conversion, thereby resolving the contradiction between detection accuracy and conversion efficiency
Solution Approach 2:
The patent introduces template switching oligonucleotides as an intermediary mechanism. These oligonucleotides facilitate the transition from RNA to amplifiable DNA templates through a template switching reaction during reverse transcription, improving the overall conversion efficiency by providing an alternative pathway that doesn't rely on the inefficient PLA step, thus addressing the productivity bottleneck while maintaining measurement precision
2Adaptability or versatility
If spectral mRNA barcoding technologies utilize single molecule fluorescence in situ hybridization, then multiple targets can be detected, but distinct fluorophores are required for scale up and the number of barcodes is limited
Solution Approach 1:
The patent applies universality by using a single fluorophore (or limited set of fluorophores) for multiple barcoding cycles instead of requiring distinct fluorophores for each target. The template switching approach enables the same fluorescent label to be used across multiple sequencing rounds, with barcode information encoded in the sequence data rather than in multiple fluorophore channels, thereby reducing device complexity while maintaining or enhancing multiplexing capability
Solution Approach 2:
The patent transitions from encoding information in the fluorophore dimension (spectral barcoding) to encoding information in the sequence dimension (digital barcoding). By using template switching to capture full-length transcripts with unique molecular identifiers, the system moves from a limited spectral palette to potentially unlimited sequence-based barcodes, effectively adding a new dimension for multiplexing that isn't constrained by the number of available fluorophores
3Measurement precision
If smFISH uses two or more colors for a target, then transcript detection is enhanced, but high density of objects in the image increases the complexity of data analysis
Solution Approach 1:
The patent uses PCR amplification to generate multiple copies of the transcript-DNA complex after template switching. Instead of relying on multiple fluorophores to enhance signal, the method amplifies the genetic material itself, creating sufficient signal for detection with a single fluorophore. This copying approach enhances measurement precision through signal amplification while avoiding the data analysis complexity associated with multi-color imaging and spectral unmixing
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 and accurate profiling of multiple targets in cells with reduced noise and bias, enabling detection of transcripts and DNA loci with improved resolution and data analysis.
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.