In Situ ssDNA Probe Ligation for Single-Cell RNA Profiling
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Solution Overview
Problem
Current single-cell RNA sequencing (scRNAseq) methods face inefficiencies in detecting RNA transcripts, particularly for low copy number RNA, due to template switching issues during reverse transcription, leading to dropout or noisy measurements.
Innovation Solution
The method involves in situ hybridization of single-stranded DNA probe pairs to adjacent sequences in target RNA within fixed and permeabilized cells, followed by ligation and split-pool barcoding to generate cell-specific barcoded long probe polynucleotides, which are then sequenced.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If reverse transcription with template switching is used to convert RNA to DNA, then polyadenylated RNA can be analyzed in an unbiased way, but detection efficiency drops to 5-45% due to inefficiency of the template switching reaction
Solution Approach 1:
The invention extracts and eliminates the problematic template switching step from the reverse transcription process. Instead of relying on template switching to convert RNA to DNA, the method uses direct in situ hybridization with DNA probes that bind to RNA targets, followed by probe ligation and sequencing. This removes the source of inefficiency while maintaining the ability to analyze polyadenylated RNA.
Solution Approach 2:
The invention introduces DNA probes as intermediary molecules that mediate the detection of RNA targets. These probes hybridize to the RNA in situ, and their presence is detected through ligation and sequencing. This intermediary approach bypasses the inefficient template switching reaction while enabling reliable detection of low copy number RNA transcripts.
2Reliability
If probe-based methods are used to increase detection efficiency, then detection efficiency improves, but complex oligo hybridization and ligation steps are required leading to low probe detection efficiency and high background
Solution Approach 1:
The invention segments the probe into two separate oligonucleotides that hybridize to adjacent sequences on the target RNA. This segmentation allows each probe component to be simpler and more specific, reducing non-specific binding and background signal while maintaining high detection efficiency. The segmented probes can be individually optimized without the complexity of designing and handling a single complex probe.
3Measurement precision
If microfluidic partitioning is used to profile single cells, then single-cell resolution is achieved, but the number of cells that can be profiled is limited and costly instrumentation is required
Solution Approach 1:
The invention uses combinatorial indexing to create digital copies of cell barcodes that can be sequenced in bulk. Instead of physically partitioning and analyzing each cell individually through microfluidics, the method attaches unique molecular identifiers to RNA molecules from multiple cells simultaneously, then uses high-throughput sequencing to read and distinguish the barcodes. This copying approach enables profiling of millions of cells with single-cell resolution while eliminating the need for costly microfluidic instrumentation.
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 enhances detection efficiency and measurement confidence, allowing for the accurate profiling of RNA in cells without the need for reverse transcription or microfluidic partitioning, thereby overcoming limitations of existing scRNAseq methods.
Implementation Method 1
in the bulk solution, diffusing single-stranded (ss) DNA probe pairs into the cells and annealing the ssDNA probe pairs to RNA in the cells
Implementation Method 2
ligating in the cells annealed probe pairs such that adjacent annealed 5′ binding probes and annealed 3′ binding probes are ligated to form one long probe
Implementation Method 3
hybridizing a double-stranded (ds) barcoding oligonucleotide comprising (i) a first overhang sequence and (ii) a central double-stranded sequence having a barcode sequence
Implementation Method 4
ligating a strand of the ds barcoding oligonucleotide to the 3′ or 5′ end of the long probe to form barcoded ligated products
Data Source
AI summary
Provided are methods and compositions for in situ detection of RNA in cells that does not require either the use of reverse transcriptase nor use of droplets. The methods can comprise annealing, in fixed and permeabilized cells, a pair of polynucleotide probes to adjacent sequences in a target RNA, which are subsequently ligated. A cell-specific barcode sequence can be subsequently synthesized in the cell using split-pool rounds to add barcode sequences to the ligated probe pair sequences in the cells, wherein an effect of multiple rounds of the split pooling is that ligated probe pair sequences in different cells have unique barcodes that are cell-specific.