Combinatorial Nucleic Acid Probes for Error-Corrected mRNA Imaging
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Solution Overview
Problem
Single-molecule fluorescent in situ hybridization (smFISH) methods suffer from low throughput due to a lack of distinguishable probes and high costs associated with producing large amounts of labeled probes, limiting the ability to efficiently detect and analyze mRNA molecules within cells.
Innovation Solution
The use of a composition comprising nucleic acid probes with a target sequence and multiple read sequences distributed to form an error-correcting code, along with methods involving primary and secondary nucleic acid probes to determine binding patterns and apply error correction, enabling high-resolution imaging of nucleic acids within cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If single-molecule fluorescent in situ hybridization (smFISH) is used to detect individual mRNA molecules, then detection precision and spatial distribution detail are improved, but throughput remains low due to limited distinguishable probes
Solution Approach 1:
The probe is segmented into two functional portions: a target sequence portion for specific binding to mRNA and a read sequence portion for combinatorial identification. This segmentation allows multiple probes to be distinguished through their read sequences, enabling high-throughput detection while maintaining single-molecule precision.
Solution Approach 2:
The read sequences serve multiple functions: they act as barcodes for probe identification, enable combinatorial coding to distinguish numerous probes with limited labels, and participate in error-correcting code formation. This multi-functionality resolves the contradiction by allowing a small set of distinguishable probes to detect many different mRNA targets simultaneously.
2Measurement precision
If large amounts of labeled probes are produced to achieve high efficiency staining, then detection sensitivity is improved, but cost increases significantly
Solution Approach 1:
Instead of producing large amounts of differently labeled probes, the invention uses a small set of probe designs that are computationally 'copied' into combinatorial combinations through read sequence assignment. Error-correcting codes provide redundancy that allows reliable detection without requiring excessive probe quantities, reducing cost while maintaining sensitivity.
Solution Approach 2:
The invention changes the identification parameter from fluorescent label intensity to combinatorial read sequence patterns. This parameter change allows numerical distinction of probes through code words rather than requiring proportional increases in labeled probe amounts, thereby reducing material costs while maintaining detection sensitivity.
3Productivity
If multiple nucleic acid probes are used to detect different mRNA molecules, then throughput is improved, but misidentification errors increase without error correction
Solution Approach 1:
Error-correcting codes are built into the read sequence design beforehand to cushion against potential misidentification errors. The redundant structure of error-correcting codes allows the system to tolerate and correct errors that may occur during detection, maintaining reliability while enabling high-throughput multi-probe detection.
Solution Approach 2:
The error-correcting code structure provides feedback mechanisms through its redundant design. When detection data is decoded, the error-correcting properties provide feedback that validates or corrects probe identification, ensuring accurate mapping of detected signals to specific mRNA targets even in high-throughput scenarios.
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 the simultaneous detection of a large number of nucleic acids using a small number of labels, improving throughput and reducing misidentification errors through error-correcting codes, thereby enhancing the analysis of nucleic acid distribution and expression within cells.
Implementation Method 1
determining binding of the nucleic acid probes within the sample
Data Source
AI summary
The present invention generally relates to systems and methods for imaging or determining nucleic acids, for instance, within cells. In some embodiments, the transcriptome of a cell may be determined. Certain embodiments are directed to determining nucleic acids, such as mRNA, within cells at relatively high resolutions. In some embodiments, a plurality of nucleic acid probes may be applied to a sample, and their binding within the sample determined, e.g., using fluorescence, to determine locations of the nucleic acid probes within the sample. In some embodiments, codewords may be based on the binding of the plurality of nucleic acid probes, and in some cases, the codewords may define an error-correcting code to reduce or prevent misidentification of the nucleic acids. In certain cases, a relatively large number of different targets may be identified using a relatively small number of labels, e.g., by using various combinatorial approaches.