ExFISH Oligonucleotide Linker for RNA Retention in Expansion Microscopy
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Solution Overview
Problem
Current methods for nanoscale-resolution imaging of RNA in intact tissues face challenges in achieving the necessary precision to pinpoint associations with cellular compartments or proteins, as existing techniques like Expansion Microscopy (ExM) struggle to retain native proteins and require custom reagents, limiting their widespread adoption and ability to image RNA effectively.
Innovation Solution
The development of ExFISH, which involves synthesizing a small molecule linker to covalently attach RNA to the ExM gel, enabling RNA fluorescent in situ hybridization with single molecule precision, allowing for the identification of transcripts in situ and revealing nanoscale structures of long non-coding RNAs and localizing neural mRNAs to individual dendritic spines.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If Expansion Microscopy (ExM) is used to achieve nanoscale-resolution imaging, then imaging resolution is improved, but the ability to retain native proteins and nucleic acids deteriorates
Solution Approach 1:
The patent introduces an oligonucleotide linker as an intermediary molecule that bridges the target protein/nucleic acid and the ExM gel matrix. This linker contains a functional group that covalently attaches to the target and another group that incorporates into the gel, thereby mediating the retention of native biomolecules during expansion while maintaining nanoscale resolution imaging capability
Solution Approach 2:
The patent creates a composite structure by integrating oligonucleotide linkers with the ExM gel matrix, forming a hybrid material system. This composite approach allows the gel to retain native biomolecules through the oligonucleotide anchoring mechanism while preserving the optical properties necessary for super-resolution imaging
2Reliability
If custom reagents are used to enable RNA anchoring to ExM gel, then RNA retention is improved, but ease of manufacture and widespread adoption deteriorates
Solution Approach 1:
The patent designs a universal oligonucleotide linker platform that can anchor various types of RNA molecules to the ExM gel matrix through a standardized mechanism. This universal approach replaces the need for custom-synthesized RNA-specific reagents with a broadly applicable oligonucleotide-based system that can be readily purchased and used for different RNA targets
Solution Approach 2:
The patent uses oligonucleotide linkers that can be synthesized through standard oligonucleotide synthesis methods, creating reusable molecular templates that can be easily replicated and purchased from commercial suppliers, thereby replacing the need for custom protein or RNA reagent synthesis
3Productivity
If genetically encoded fluorophores are imaged without antibody labeling, then imaging speed is improved, but the original ExM protocol requires antibody labeling which limits direct imaging capability
Solution Approach 1:
The patent introduces oligonucleotide linkers as intermediaries that directly attach to genetically encoded fluorophores or RNA molecules of interest, enabling their incorporation into the ExM gel matrix without requiring additional antibody labeling steps. This intermediary approach allows direct imaging of genetically encoded markers while maintaining nanoscale resolution
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
ExFISH enables robust RNA anchoring and imaging with nanoscale precision, supporting single molecule RNA readout and multiplexed RNA imaging in intact tissues, overcoming previous limitations of ExM by allowing for the retention of nucleic acids and proteins within the gel, and facilitating sequential hybridization and multiplexing strategies.
Implementation Method 1
The sample is contacted with a label which comprises an oligonucleotide that can hybridise with a complementary sequence (a target nucleic acid) on an affinity tag
Implementation Method 2
a swellable polyelectrolyte gel was synthesized in the sample, so that it incorporated the labels. Finally, the sample was treated with a nonspecific protease to homogenize its mechanical properties, followed by dialysis in water to mediate uniform physical expansion of the polymer-specimen composite
Data Source
Figure 1A
Figure 1B~1C
Figure 1D
AI summary
The invention enables in situ genomic and transcriptomic assessment of nucleic acids to be conducted in biological specimens that have been physically expanded. The invention leverages the techniques for expansion microscopy (ExM) to provide new methods for in situ genomic and transcriptomic assessment of nucleic in a new process referred to herein as "expansion fluorescent in situ hybridization" (ExFISH).