Single-Cell Nucleic Acid Detection via Oligo Hybridization
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Solution Overview
Problem
Current methods for single-cell RNA sequencing face challenges such as limited RNA availability, RNA degradation, and biases introduced by reverse transcription and amplification steps, which limit the accuracy and sensitivity of transcriptome analysis.
Innovation Solution
The method involves hybridizing single-stranded DNA oligonucleotide probes to nucleic acids within a compartment, removing non-specific probes, and identifying specifically hybridized probes by sequencing or amplification, allowing for direct detection and quantification of nucleic acids without sequential probe hybridization or RNA isolation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If reverse transcription and PCR amplification steps are used to detect RNA, then sensitivity is improved, but biases are introduced and accuracy deteriorates
Solution Approach 1:
The patent extracts and eliminates the reverse transcription step from the workflow, directly detecting RNA molecules through hybridization of DNA probes to RNA targets. This removes the source of reverse transcription biases while maintaining detection sensitivity through direct probe binding and sequencing of the probe-RNA hybrids.
Solution Approach 2:
Instead of converting RNA to cDNA through reverse transcription and then amplifying, the patent inverts the approach by directly hybridizing DNA probes to RNA targets and sequencing the probes themselves. This inversion eliminates the need for reverse transcription and subsequent amplification biases, while still enabling sensitive detection through the probe sequencing step.
2Measurement precision
If sequential probe hybridization is used to increase specificity, then detection specificity is improved, but procedure complexity increases
Solution Approach 1:
The patent segments the detection process into two distinct stages: (1) hybridization of multiple DNA probes to RNA targets in parallel, and (2) sequencing or amplification of the hybridized probes for identification. This segmentation allows simultaneous hybridization of many probes without sequential complexity, while maintaining high specificity through probe sequence identification in the second stage.
3Measurement precision
If RNA isolation steps are performed, then nucleic acid detection is enabled, but RNA degradation occurs and loss of information increases
Solution Approach 1:
The patent performs preliminary hybridization of DNA probes to RNA targets within the intact cell or compartment before any RNA isolation or degradation can occur. By capturing the RNA-probe hybrids in their native cellular environment, the method preserves RNA integrity and prevents degradation that would occur during traditional isolation procedures.
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 enables sensitive detection of low-abundance nucleic acids and their spatial mapping, overcoming limitations of existing methods by providing a high-throughput, unbiased analysis of RNA species and their abundance in single cells.
Implementation Method 1
hybridizing a plurality of single-stranded (ss)DNA oligonucleotide probes to complementary nucleic acid molecules
Data Source
AI summary
The present invention relates to the field of nucleic acid sequencing at the single cell level, e.g., single-cell RNA sequencing (scRNA-seq). In particular, the invention provides a method of detecting nucleic acid in a fixated or non-fixated nucleic acid-containing compartment such as a eukaryotic cell or nucleus thereof, by hybridizing a plurality of single-stranded (ss)DNA oligonucleotide probes to complementary nucleic acid molecules within said compartment; removing ssDNA oligonucleotide probes from the compartment that have not specifically hybridized to nucleic acid; and identifying the ssDNA oligonucleotide probes specifically hybridized to nucleic acid molecules within said compartment by sequencing or amplification, thereby determining the corresponding nucleic acids present in said compartment. The method does not require a step of sequential ssDNA probe hybridization to the same target nucleic acid as a means for increased specificity or sensitivity, and preferably further does not require steps of RNA isolation and cDNA generation. The method of the invention has the potential to detect substantially every known and/or unknown nucleic acid species, in particular RNA, e.g., protein-encoding mRNAs as well as non-coding RNAs. The method further enables spatial mapping of detected nucleic acids, wherein the compartment is sectioned or dissociated into a single cell suspension prior to probe hybridization to obtain a collection of fractions and thus nucleic acid molecules are separated from each other depending on their localization or which cell type they belonged to. Spatial mapping of detected nucleic acids may be combined with the detection of at least one DNA locus, at least one protein, or with the analysis of chromatin condensation. The method of the invention is designated oligo-seq.