Encoding Probe Architecture for Nucleic Acid Detection
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Solution Overview
Problem
Existing in situ analyte detection methods face challenges with signals of small size or weak intensity not reaching detection thresholds and signals of large size or high intensity causing optical overcrowding, leading to compromised sensitivity in genomic, transcriptomic, and proteomic profiling.
Innovation Solution
A method involving encoding probes that hybridize to target sequences, undergo rolling circle amplification (RCA), and generate detectable signals, allowing for sensitive detection and amplification of target nucleic acids with single-nucleotide resolution, including SNPs and splice variants, while tuning signal intensity based on autofluorescence levels.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If signal intensity is increased to improve detection sensitivity, then weak signals can be detected, but optical overcrowding occurs causing signal distortion
Solution Approach 1:
The probe is divided into multiple distinct functional regions: a binding region that specifically binds to the target analyte, a spacer region that provides physical separation, and a reporter region that generates the detectable signal. This segmentation allows the signal to be generated at a distance from the binding site, reducing optical overcrowding while maintaining detection sensitivity.
Solution Approach 2:
A spacer region acts as an intermediary element between the target-binding region and the signal-generating reporter region. This spacer provides physical separation that prevents the reporter from interfering with the binding interaction, thereby reducing optical overcrowding effects while still allowing signal transmission.
2Measurement precision
If probe size is increased to enhance signal intensity, then detection sensitivity improves, but spatial resolution and image quality deteriorate
Solution Approach 1:
The probe architecture segments the signal generation function from the binding function, placing the reporter region at a distance from the target binding site via a spacer. This allows the probe to maintain a compact footprint for high spatial resolution while the reporter provides sufficient signal intensity for sensitive detection.
Solution Approach 2:
The probe design extends the signal generation into a different spatial dimension by using the spacer to position the reporter in three-dimensional space away from the binding interface. This dimensional separation allows the probe to occupy minimal space on the target while generating adequate signal intensity.
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
Enhances the sensitivity and quality of image analysis by enabling the detection of target nucleic acids and variants with high specificity and intensity control, overcoming the limitations of existing methods.
Implementation Method 1
the first encoding probe and each second encoding probe are capable of hybridizing to a first target sequence and a second target sequence, respectively
Implementation Method 2
generating a rolling circle amplification (RCA) product of the circularized first encoding probe
Data Source
AI summary
In some aspects disclosed herein are methods and compositions for detecting an analyte such as a target nucleic acid in a biological sample, said method comprising generating and analyzing a detectable signal associated with the target nucleic acid and a separate signal associated with a region of interest in the target nucleic acid.