3D DNA Nanostructures for Single Molecule Quantification
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Solution Overview
Problem
Current gene expression profiling (GEP) methods face challenges in achieving simultaneous, high-specificity, and high-sensitivity analysis of multiple genes while being cost-effective, fast, and easy to use, due to issues such as enzymatic errors, long processing times, and complex, expensive equipment.
Innovation Solution
A method utilizing 3D DNA nanostructures and micro-well arrays for the direct detection and quantification of target mRNA molecules, which involves introducing host bodies with target structures into micro-wells, binding 3D DNA nanostructures specifically to the target structures, and measuring fluorescence signals to distinguish bound from unbound nanostructures.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Difficulty of detecting and measuring
If enzymatic reactions (reverse transcription and PCR amplification) are used for GEP analysis, then the detection capability is improved, but systematic errors occur and quantification becomes inaccurate
Solution Approach 1:
The patent replaces enzymatic reactions (chemical/biological processes) with direct hybridization and fluorescence detection (physical/optical processes). This substitution eliminates systematic errors from enzymatic amplification while maintaining detection capability through direct labeling of target molecules with fluorescent reporters.
Solution Approach 2:
The patent uses fluorescent DNA reporters as copies that hybridize to target mRNA molecules. These reporter molecules carry fluorescent labels that enable direct detection and quantification without requiring enzymatic amplification of the original target, thus avoiding amplification errors.
2Productivity
If microarray-based methods are used for parallel detection, then the number of detectable sequences increases, but the process time becomes too long for clinical applications
Solution Approach 1:
The patent extracts the fluorescent reporter molecules from the complex microarray surface hybridization process. By using free-floating fluorescent reporters that hybridize to targets in solution, the method eliminates the time-consuming surface binding and washing steps inherent to microarray methods, dramatically reducing process time while maintaining parallel detection capability.
3Measurement precision
If dPCR is used for accurate quantification, then the measurement precision improves, but the method is limited to typically two targets
Solution Approach 1:
The patent creates a universal detection system where multiple fluorescent reporters with different spectral properties can simultaneously detect multiple different target mRNA sequences. Each reporter-target combination provides specific quantification, enabling accurate measurement of many genes in parallel within a single reaction mixture, thus achieving both precision and versatility.
4Measurement precision
If nCounter system is used for enzyme-free detection, then the quantification accuracy improves, but complex purification steps and expensive equipment are required
Solution Approach 1:
The patent uses simple, inexpensive fluorescently labeled DNA reporters that can be synthesized commercially at low cost. These disposable-like reporters are used in straightforward hybridization reactions that require only basic laboratory equipment, eliminating the need for expensive specialized instruments while maintaining accurate quantification.
5Difficulty of detecting and measuring
If fluorescent reporters are stretched after surface immobilization for barcode reading, then the detection capability is improved, but about 80% of target molecules are excluded by unidentifiable barcodes
Solution Approach 1:
The patent inverts the conventional approach by reading the fluorescent barcode signals while the reporters are still in solution or loosely associated with the target, before any stretching or surface immobilization is required. This reversal ensures that all hybridized reporters remain identifiable, eliminating the 80% loss of unidentifiable barcodes while maintaining detection capability.
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 precise, rapid, and cost-effective quantification of multiple target molecules with reduced false positives and negatives, allowing for efficient molecular and phenotypic analysis of individual cells or structures.
Implementation Method 1
The 3D DNA nanostructures and the parameters of the fluorescence measurement are selected such that the at least one measured fluorescence signal of the identification structure formed in a) differs from the fluorescence signal of each of the at least two isolated 3D DNA nanostructures
Implementation Method 2
each of the 3D DNA nanostructures having one or more internal fluorescent dye molecules; Detection of the target structure by measuring at least one fluorescence signal
Data Source
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AI summary
The present invention relates to a method and a DNA nanostructure for the detection of a target structure. In particular, the present invention relates to a DNA nanostructure which, through a suitable choice of its shape and the placement of marker molecules attached to it, ensures a preferably linear dependence of the number of marker molecules and the measurement signal, independent of the spatial arrangement of several such DNA nanostructures. Furthermore, the invention relates to the use of these DNA nanostructures and other nanoreporters, preferably in combination with adapters, which bind specifically to target molecules, in a method for the preferably simultaneous quantification of a plurality of target molecules by a multiplex technique. The method particularly relates to single-cell analysis.