Singly Labeled Probes for mRNA Detection

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Solution Overview

Problem

Current methods for detecting mRNA numbers in individual cells are unreliable due to issues with probe synthesis, purification, and specificity, leading to inaccurate counts and difficulty in determining legitimate binding versus non-specific binding events.

Innovation Solution

The use of a plurality of singly fluorescently labeled nucleic acid hybridization probes, with at least 30 probes hybridizing simultaneously to a target mRNA sequence, creating a detectable fluorescent spot through combined fluorescence, while ensuring non-overlapping probe binding regions and using distinct fluorophores for multiplexed probing to enable accurate gene expression profiling.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If multiple fluorophores are coupled to the same oligonucleotide probe, then sensitivity is improved, but synthesis difficulty and purification complexity increase significantly

Engineering Contradiction:

Improvedetection sensitivityVSAvoidprobe synthesis and purification

Core Design Contradiction:

Measurement precisionVSEase of manufacture

Solution Approach 1:

The invention divides the detection system into multiple separate singly-labeled probes instead of using fewer heavily-labeled probes. Each probe carries only one fluorophore, simplifying synthesis and purification, while the collective signal from multiple probes achieves the required detection sensitivity.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The invention combines the signals from multiple singly-labeled probes to achieve the detection sensitivity previously requiring heavily-labeled probes. The fluorescent signals from individual probes accumulate at the target location, producing a detectable signal without the complexity of multi-fluorophore coupling.

Inventive Principle:

Principle #5Merging (Combining)

2Illumination intensity

If fewer probes are used with multiple fluorophores per probe, then signal intensity per probe increases, but the distribution of bound probes widens and specificity decreases

Engineering Contradiction:

Improvefluorescence signal intensityVSAvoidbinding specificity and count accuracy

Core Design Contradiction:

Illumination intensityVSReliability

Solution Approach 1:

The detection system is segmented into many individual probe-target binding events rather than fewer binding events. This segmentation narrows the distribution of bound probes per target and reduces the impact of non-specific binding, improving reliability while maintaining sufficient total signal intensity.

Inventive Principle:

Principle #1Segmentation

3Measurement precision

If heavily labeled probes are used, then single molecule detection sensitivity is achieved, but non-specific binding events increase background noise

Engineering Contradiction:

Improvesingle molecule detection sensitivityVSAvoidbackground noise from non-specific binding

Core Design Contradiction:

Measurement precisionVSObject-generated harmful factors

Solution Approach 1:

The probe system is segmented into multiple singly-labeled probes rather than fewer heavily-labeled probes. This reduces the fluorescent signal carried by each individual probe, thereby reducing the background noise generated by non-specific binding events while maintaining sufficient total signal from specific binding events.

Inventive Principle:

Principle #1Segmentation

4Quantity of substance

If multiple amino groups are introduced into oligonucleotides for fluorophore coupling, then labeling capacity increases, but side reactions such as transamidation occur and coupling efficiency decreases

Engineering Contradiction:

Improvenumber of fluorophore moieties per probeVSAvoidcoupling reaction efficiency

Core Design Contradiction:

Quantity of substanceVSEase of manufacture

Solution Approach 1:

The invention extracts or removes the multiple amino group coupling step from the probe preparation process. Instead of introducing multiple amino groups and performing sequential coupling reactions, the method uses singly-labeled probes that require only a single coupling reaction, eliminating side reactions and simplifying manufacturing.

Inventive Principle:

Principle #2Taking out (Extraction)

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 accurate and reliable detection of individual mRNA molecules, providing precise gene expression analysis and enabling the identification of mRNA species within cells, including in cancer and pathogenic states, with improved sensitivity and specificity compared to existing methods.

Implementation Method 1

nucleic acid hybridization probes, with at least 30 probes hybridizing simultaneously to a target mRNA sequence

Methodology Applied

Scientific EffectNucleic acid hybridization:

Implementation Method 2

singly fluorescently labeled nucleic acid hybridization probes, creating a detectable fluorescent spot through combined fluorescence

Methodology Applied

Scientific EffectFluorescence: Fluorescence

Data Source

PatentEP3133170B1Imaging individual mRNA molecules using multiple singly labeled probes

Publication Date: 2020.03.18 RUTGERS THE STATE UNIV

Figure 1a~1c
Figure 2
Figure 3a~3b

AI summary

The present invention provides a probe set for in situ hybridization that enables detection of individual mRNA molecules. The present invention also provides a kit or a hybridization solution comprising a probe of the invention and a method of detecting a first target sequence of individual mRNA molecules in a cell.