Acrydite-Anchored RNA Probes for RNase-Resistant Imaging
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Solution Overview
Problem
Existing RNA imaging technologies, such as MERFISH, face challenges due to the vulnerability of RNA species to chemical insults and the ubiquitous presence of ribonuclease (RNase), which leads to RNA degradation during prolonged imaging processes. Current RNase inhibitors are costly and require frequent replenishment.
Innovation Solution
The method involves anchoring a primary nucleic acid probe with an acrydite moiety within a polymer matrix, where the probe hybridizes with the target nucleic acid and forms a covalent bond with the matrix. This approach immobilizes the nucleic acid probe, making it resistant to RNase degradation, and allows for subsequent imaging of the target nucleic acids.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If RNA imaging is performed using conventional methods (e.g., MERFISH), then spatial transcriptomic profiling can be achieved, but RNA degradation occurs due to RNase activity during prolonged imaging
Solution Approach 1:
The patent introduces an anchoring agent as an intermediary substance that forms a covalent bond with the RNA probe and anchors it to the polymer matrix. This intermediary protects the RNA from RNase degradation while allowing the imaging function to proceed, thus resolving the contradiction between maintaining RNA signal stability and achieving accurate spatial transcriptomic profiling
Solution Approach 2:
The patent applies preliminary action by embedding the RNA probe in a polymer matrix and forming covalent bonds with anchoring agents before the imaging process begins. This pre-protection mechanism ensures RNA stability throughout the prolonged imaging period, preventing degradation before it can occur
2Reliability
If chemical RNase inhibitors are used to prevent RNA degradation, then RNA stability is improved, but cost increases and frequent replenishment is required
Solution Approach 1:
The patent implements self-service by designing a system where the RNA probe itself becomes anchored to the matrix through covalent bonding with anchoring agents. The anchored probe continuously protects itself from RNase degradation without requiring external RNase inhibitors or frequent replenishment, thereby eliminating the complexity associated with chemical inhibitor management
Solution Approach 2:
The patent segments the protection mechanism from the RNA probe by introducing separate anchoring agents that covalently bind to the probe and anchor it to the matrix. This segmentation allows the RNA to focus on its imaging function while the anchoring system handles protection, eliminating the need for continuous chemical inhibitor addition
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 method provides an RNase-insensitive approach for imaging nucleic acids, enhancing the stability and durability of RNA signals during imaging processes, thereby improving the accuracy and reliability of RNA profiling in biological samples.
Implementation Method 1
the primary probe comprises an acrydite moiety and hybridizes with a target nucleic acid
Implementation Method 2
the anchoring agent forms a covalent bond with the primary nucleic acid probe; embedding the biological sample in a polymer matrix wherein the primary nucleic acid probe and the anchoring agent each form a covalent bond with the polymer matrix
Data Source
AI summary
The present disclosure is generally directed to anchoring primary nucleic acid probes in a polymer matrix, which are configured to hybridize to cellular nucleic acid, embedding in a polymer matrix, clearing cellular components including the cellular nucleic acid and imaging. The disclosure describes methods for imaging cellular RNA in an RNase insensitive method.