Nucleic Acid Probe Coding for Multiplex In-Cell Detection
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Solution Overview
Problem
Single-molecule fluorescent in situ hybridization (smFISH) has 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 multiple genes simultaneously.
Innovation Solution
A composition and method using a plurality of nucleic acid probes with error-correcting codes, where primary probes bind to target nucleic acids and secondary probes determine binding patterns to form codewords, allowing for error correction and high-resolution imaging of nucleic acids within cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional smFISH method is used to detect multiple genes, then detection accuracy is maintained, but throughput is limited and cost increases
Solution Approach 1:
The probe system is segmented into two functional components: primary probes that bind to target nucleic acids and secondary probes that provide distinguishable labels. This segmentation allows a small set of expensive labeled secondary probes to work with a large set of unlabeled primary probes, dramatically reducing the total cost of probe combinations while maintaining detection capability for many genes simultaneously.
Solution Approach 2:
Unlabeled primary probes serve as intermediaries that bridge the target nucleic acids and the labeled secondary probes. The primary probes bind specifically to targets and provide binding sites for secondary probes, enabling the system to detect multiple targets using a limited set of labeled components, thus improving throughput while controlling costs.
2Productivity
If more distinguishable probes are used to increase throughput, then detection of multiple genes improves, but probe complexity and cost increase
Solution Approach 1:
The probe system divides functionality into two segments: primary probes for target recognition and secondary probes for signal generation. This segmentation allows the system to achieve high throughput through combinatorial pairing of a limited number of each probe type, rather than requiring a large number of fully distinguishable probes, thereby reducing overall system complexity.
Solution Approach 2:
Secondary probes are designed to be universal and can bind to multiple different primary probes through their common binding motif. This multi-functionality allows a small set of labeled secondary probes to work with a large set of primary probes targeting different genes, achieving high throughput without proportionally increasing probe complexity or cost.
3Productivity
If traditional labeling methods are used, then detection accuracy is maintained, but the number of simultaneously detectable genes is limited
Solution Approach 1:
The system uses multiple copies of a small set of labeled secondary probes that can be reused across many different primary probe-target complexes. Instead of requiring unique labeled probes for each gene, the same labeled secondary probes are copied and used repeatedly with different primary probes, enabling detection of many genes with minimal labeled probe material.
Solution Approach 2:
The system changes the parameter of probe labeling from 'one label per target' to 'shared labels across multiple targets'. By allowing secondary probes to be common across multiple primary probe types, the system dramatically reduces the total amount of labeled probe material needed while increasing the number of simultaneously detectable genes.
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 the simultaneous detection of a large number of nucleic acids at high resolution and reduced cost by using a small number of labels, improving throughput and accuracy in determining nucleic acid distributions.
Implementation Method 1
nucleic acid probes, at least some of which comprise a first portion comprising a target sequence
Implementation Method 2
single-molecule fluorescent in situ hybridization (smFISH) is a powerful method for detecting individual mRNA molecules
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.