Detectably Labeled Magnetic Particles for In Situ Nucleic Acid Analysis
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Solution Overview
Problem
Current methods for analyzing biological samples using single molecule fluorescent in situ hybridization face challenges such as dim and diffuse signals, and the laborious process of removing initial probes to apply a second probe, which limits signal intensity and efficiency.
Innovation Solution
The use of detectably labeled magnetic particles comprising a magnetic core, a detectable label, and oligonucleotides with a reporter hybridization sequence that hybridize to target nucleic acid molecules, allowing for enhanced signal detection and simultaneous removal of probes through magnetic fields and photo-crosslinking.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If single molecule fluorescent in situ hybridization is used to detect analytes, then the method can determine expression levels of RNA, but the signals become dim and diffuse
Solution Approach 1:
The patent combines multiple intermediate probes bound to a target nucleic acid molecule with a single detectably labeled magnetic particle. The magnetic particle comprises a magnetic core, a detectable label (such as a fluorophore), and oligonucleotides with reporter hybridization sequences. This merging concentrates multiple probe-binding events into a single bright magnetic particle signal, resolving the contradiction between detection capability and signal brightness.
Solution Approach 2:
The patent uses detectable labels such as fluorophores attached to magnetic particles that emit bright optical signals when excited. This allows the system to transition from dim single-molecule fluorescent signals to bright magnetic particle-associated fluorescent signals, improving signal brightness while maintaining measurement precision.
2Adaptability or versatility
If chemical or enzymatic means are used to remove initial probes for applying a second probe, then probe replacement can be achieved, but the process becomes laborious and time-consuming
Solution Approach 1:
The patent replaces chemical or enzymatic probe removal methods with magnetic field-based removal. The magnetic particle can be removed from the sample by applying a magnetic field, which quickly and easily removes both the magnetic particle and any bound intermediate probes without requiring laborious chemical or enzymatic treatment. This resolves the contradiction between probe replacement capability and time consumption.
3Illumination intensity
If numerous molecular signals are colocalized on a target to enhance signal brightness, then signal intensity increases, but signal diffusion occurs
Solution Approach 1:
The magnetic particle acts as an intermediary that binds to multiple intermediate probes through oligonucleotide hybridization. This intermediary structure concentrates the binding events at a single location (the magnetic particle) rather than allowing signals to diffuse across the target molecule. The magnetic particle serves as a focal point that maintains signal localization while providing enhanced brightness through the attached detectable label.
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 improves signal intensity and compactness by leveraging proximity-enforced hybridization, enabling brighter and more localized signals, and facilitates rapid probe removal, thus overcoming the limitations of existing methods.
Implementation Method 1
the reporter hybridization sequence is complementary to a reporter sequence present in a plurality of intermediate probes bound directly or indirectly to a target nucleic acid molecule in the biological sample, whereby the magnetic particle hybridizes to the reporter sequence via one or more of the oligonucleotide molecules and is thereby associated with the target nucleic acid molecule
Implementation Method 2
detecting the detectably labeled magnetic particle at a location in the biological sample
Implementation Method 3
In some embodiments, CNVK oligos can be used to photo-crosslink the detectably labeled magnetic particle to intermediate probes for simultaneous removal of the magnetic particle and intermediate probes within a time frame on the order of seconds
Data Source
AI summary
The present disclosure relates in some aspects to methods and compositions for analyzing a biological sample. In some aspects, provided herein are detectably labeled magnetic particles, methods, and kits for analyzing a biological sample (e.g., for detecting a target nucleic acid at a location in the biological sample).