Single-Cell RNA Translation Profiling with In Situ Ribosome Detection
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Solution Overview
Problem
Transcriptional profiles in single cells do not consistently correlate with proteomic profiles, indicating the need for improved methods to quantify protein production directly and accurately, as mRNA levels are an imperfect proxy for protein production and can be biased in defining cellular states.
Innovation Solution
A method for in situ ribosome profiling using proximity ligation with oligonucleotide probes and ribosome-specific antibodies to amplify and sequence actively translated mRNAs, providing precise spatial information on RNA translation status.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If mRNA levels are used as a proxy to estimate protein production, then the measurement process is simple and high-throughput, but the accuracy and reliability of protein production quantification deteriorates
Solution Approach 1:
The patent introduces ribosomes as an intermediary marker to indirectly quantify protein production. Instead of directly measuring proteins (which is difficult) or using mRNA as a proxy (which is inaccurate), the method uses ribosomes bound to mRNA as a measurable intermediate that correlates with active translation and protein production, thereby resolving the accuracy-throughput contradiction
Solution Approach 2:
The patent replaces complex direct proteomic analysis methods with a nucleic acid-based approach. By using RNA-FISH and digital imaging to detect ribosome-bound mRNA, the method substitutes complex protein quantification with simpler, high-throughput optical detection while maintaining accuracy through ribosome-specific targeting
2Measurement precision
If direct single-cell proteomic methods are developed, then the accuracy of protein production quantification improves, but the device complexity and difficulty of detection increase
Solution Approach 1:
The patent uses ribosomes as a convenient intermediary that is easier to detect than proteins themselves. Ribosomes can be targeted with specific antibodies or probes, providing a measurable signal that reflects protein production status without requiring direct protein detection, thus reducing system complexity while maintaining accuracy
Solution Approach 2:
The patent creates a detectable copy or proxy of the protein production state through ribosome-bound mRNA detection. By detecting the ribosome-mRNA complex rather than the protein itself, the method provides an accessible molecular copy that carries information about protein production without requiring direct protein measurement capabilities
3Adaptability or versatility
If spatial information of RNA translation is obtained through antibody staining, then the functional relevance of cellular data improves, but the measurement precision and signal detection difficulty increase
Solution Approach 1:
The patent merges multiple detection approaches by combining ribosome-specific antibodies with fluorescently conjugated probes in a single assay. This combination allows simultaneous detection of translation status and spatial location, integrating functional information with positional data while using the fluorescent signal to enhance detectability
Solution Approach 2:
The patent employs fluorescent labeling of ribosome-bound mRNA complexes, utilizing color/fluorescence changes as a detectable signal. The fluorescent conjugate on the antibody or probe provides a strong, easily detectable signal that indicates the presence and location of actively translating mRNA, thereby reducing detection difficulty while maintaining spatial precision
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 high-throughput characterization of RNA translation status, bridging the gap between transcriptome and proteome, and facilitating disease diagnosis, treatment, and drug discovery by accurately quantifying protein production in single cells.
Implementation Method 1
a second probe comprises a portion that recognizes the ribosome and an oligonucleotide portion that is complementary to a portion of the first probe
Implementation Method 2
performing rolling circle amplification to amplify the circular oligonucleotide using the second probe as a primer to produce one or more concatenated amplicons
Implementation Method 3
ligating the 5' end and the 3' end of the first probe together to produce a circular oligonucleotide
Data Source
Figure 1
Figure 2A~2B
Figure 2C~2D
AI summary
The present disclosure provides methods and systems for profiling RNAs being translated in a cell. Also provided by the present disclosure are methods for diagnosing a disease or disorder in a subject based on a profile of the RNAs being translated in a cell, including cells within an intact tissue. Methods of screening for or testing a candidate agent capable of modulating translation of one or more RNAs are also provided by the present disclosure. The present disclosure also provides methods for treating a disease or disorder in a subject in need thereof. Pairs of probes and sets of probes comprising oligonucleotide portions, which may be useful for performing the methods described herein, are also described by the present disclosure. Additionally, the present disclosure provides kits comprising any of the probes described herein.