Nucleic Acid Nanostructure Barcode Probes for Multiplexed Detection
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Solution Overview
Problem
Current molecular barcode strategies face limitations in addressability and readability due to the diffraction limit of visible light, making it difficult to multiplex and detect multiple analytes simultaneously using standard fluorescent microscopy.
Innovation Solution
Nucleic acid nanostructures, such as DNA nanostructures, are used to create barcode probes with fluorescently labeled regions spaced beyond the diffraction limit, allowing for the creation of robust and addressable barcodes that can be read using standard fluorescent microscopes, even under sub-optimal labeling and imaging conditions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If fluorescently labeled regions are spaced within the diffraction limit, then the barcode can be read using standard fluorescent microscopy, but the measurement precision and addressability of individual regions deteriorates
Solution Approach 1:
The patent transitions from two-dimensional planar barcodes to three-dimensional hierarchical structures. Fluorescently labeled regions are arranged in multiple layers at different z-heights, allowing standard microscopy to capture the overall structure while super-resolution techniques can resolve individual regions within each layer. This dimensional expansion enables both readability and precise addressability simultaneously.
Solution Approach 2:
The barcode is divided into multiple hierarchical segments: outer structural elements that define the overall pattern visible to standard microscopy, and inner fluorescently labeled regions that can be individually addressed. This segmentation allows different resolution levels to serve different functions - the outer structure provides robust readability while the inner segments enable precise identification.
2Adaptability or versatility
If multiple fluorophores are used to increase multiplexing capability, then the number of detectable analytes increases, but the spectral overlap and difficulty in distinguishing individual signals worsens
Solution Approach 1:
Instead of relying solely on spectral differentiation in the wavelength dimension, the patent encodes information in the spatial dimension by arranging fluorescently labeled regions in unique geometric patterns and hierarchical positions. This allows multiple analytes to be distinguished by their spatial barcodes rather than requiring spectrally distinct fluorophores for each target.
Solution Approach 2:
The patent employs asymmetric arrangements of fluorescently labeled regions within the hierarchical structure. Each analyte is associated with a unique asymmetric pattern of labeled regions, creating distinct spatial signatures that are easily distinguishable even when using fluorophores with overlapping spectra. The asymmetric positioning provides robust signal differentiation.
3Quantity of substance
If fluorescent labels are placed closer together to increase information density, then more analytes can be encoded in a smaller space, but the diffraction limit prevents accurate detection of individual regions
Solution Approach 1:
The patent resolves the diffraction limit constraint by organizing fluorescently labeled regions in three-dimensional hierarchical layers. Regions that would be indistinguishable in two dimensions are separated in the z-dimension, allowing standard microscopy to capture the layered structure while enabling super-resolution methods to resolve individual regions within each layer, thereby maintaining high information density with accurate detection.
Solution Approach 2:
The barcode structure implements a nested hierarchy where multiple levels of information are embedded within each other. Outer structural elements contain inner fluorescently labeled regions, which in turn may contain further subdivided elements. This nested organization allows high information density to be achieved without compromising the detectability of individual regions at any hierarchical level.
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 the simultaneous detection of multiple analytes with increased multiplexing capability and robustness, as the spatial pattern of fluorescently labeled regions can be accurately detected, even when labeled regions are within the diffraction limit, using techniques like super-resolution microscopy.
Implementation Method 1
fluorescently labeled regions that may be stably or transiently bound (and thus labeled) with fluorophore-bearing oligonucleotides
Implementation Method 2
The barcode probes may further comprise a target binding moiety
Implementation Method 3
The nature of the fluorescent signals (e.g., the wavelength or 'color', intensity, etc.) and the pattern (or orientation, or arrangement or geometry) of such signals on the barcode can also be used to identify particular analytes
Data Source
AI summary
Provided herein are, inter alia, barcode probes comprised of transiently or stably fluorescently labeled nucleic acid nanostructures that are fully addressable and able to be read using standard fluorescent microscope and methods of use thereof including methods of use as detectable labels for probes.