Caged Antisense Oligonucleotide for RNA Capture in Fixed Tissue
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for isolating and analyzing mRNA from single cells, especially in live tissue, are limited by the inability to effectively detect or compare nucleic acid molecules longitudinally or among different cells or subcellular compartments, and fixed tissue samples are compromised due to RNA crosslinking, lacking comprehensive RNA fingerprint analysis.
Innovation Solution
A caged molecule comprising an antisense oligonucleotide linked to a ruthenium-based photolinker is used, which, upon irradiation, exposes the oligonucleotide to hybridize with nucleic acids, allowing for the capture and isolation of various RNA species, including microRNAs and mRNA, from both live and fixed tissue samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If fixed tissue samples are used for molecular analysis, then tissue preservation is improved, but RNA analysis capability deteriorates due to crosslinking
Solution Approach 1:
The patent introduces a caged molecule that is pre-administered to the tissue sample before fixation. This molecule remains inactive during the fixation process and only becomes active after fixation is complete, allowing RNA capture to occur on fixed tissue without being compromised by the fixation-induced crosslinking.
Solution Approach 2:
The caged molecule acts as an intermediary that bridges the gap between fixed tissue and RNA analysis. The photolinker serves as a mediator that can be activated by light to release the active capturing moiety, enabling RNA capture on fixed tissue without direct interference from the fixation process.
2Measurement precision
If conventional mRNA isolation tools are used, then mRNA detection is improved, but the ability to detect all RNA species and perform multiplexed analysis deteriorates
Solution Approach 1:
The caged molecule is designed with a universal capturing mechanism that can bind to all RNA species through the exposed moiety, not just polyA+ mRNA. The system can be configured with different capturing moieties to detect various RNA types (mRNA, microRNA, lincRNA, etc.) and can perform multiplexed analysis by detecting multiple targets simultaneously within the same cell.
Solution Approach 2:
The patent changes the state of the capturing molecule from caged (inactive) to uncaged (active) through photolinker activation. This parameter change enables the molecule to transition from a delivery-optimized state to a detection-optimized state, allowing it to capture and detect diverse RNA species with high versatility.
3Measurement precision
If single cells are isolated for analysis, then molecular fingerprinting is improved, but the systemic regulation context is lost
Solution Approach 1:
The caged molecule is administered to the tissue sample before isolation or sectioning, allowing it to distribute throughout the tissue and reach target cells in their native context. This preliminary administration ensures that when cells are later isolated or sectioned, the capturing moieties are already in position to capture RNA molecules, preserving the systemic context information.
4Productivity
If polyA+ mRNA enrichment is used, then mRNA isolation efficiency is improved, but the ability to capture all RNA species deteriorates
Solution Approach 1:
The caged molecule employs a universal capturing mechanism that does not rely on polyA tail enrichment. Instead, it uses a photolinker-caged moiety that can be designed to capture various RNA species through different mechanisms (hybridization, binding to specific sequences or structures), enabling total RNA capture while maintaining high efficiency.
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 comprehensive detection and analysis of nucleic acid fingerprints from single cells and subcellular compartments, overcoming the limitations of existing methods by capturing a wide range of RNA species and maintaining RNA integrity in fixed samples.
Implementation Method 1
irradiation of ruthenium-based photolinker exposes the antisense oligonucleotide thereby allowing the antisense oligonucleotide to hybridize with a nucleic acid
Data Source
AI summary
Compositions and methods of capturing one or more nucleic acid molecules of a cell or subcellular compartment are described. In certain aspects, the compositions comprise a caged molecule comprising one or more photolinkers and an antisense oligonucleotide, which when uncaged hybridizes to a target nucleic acid molecule.