RNA Immobilization via 5' Phosphate Attachment
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Solution Overview
Problem
Existing methods for analyzing biological samples often result in the loss of nucleic acid analytes, particularly fragmented RNA, during sample preparation, leading to incomplete characterization of disease states.
Innovation Solution
The method involves anchoring or immobilizing ribonucleic acids, including fragmented ones, to exogenous or endogenous molecules in a biological sample using an attachment agent. This attachment agent contains reactive moieties that bind to the RNA's 5′-phosphate group and attachment moieties that attach covalently or noncovalently to a matrix-forming agent, forming a three-dimensional polymerized matrix that embeds the sample and immobilizes the RNA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If standard sample preparation protocols (permeabilization and de-crosslinking) are used to enable analysis and imaging, then RNA accessibility is improved, but nucleic acid analytes are lost
Solution Approach 1:
The patent applies preliminary action by modifying the 5' ends of RNA molecules with capture probes before sample preparation procedures are performed. This pre-modification ensures that RNA molecules are anchored to solid supports in advance, preventing their loss during subsequent permeabilization and de-crosslinking steps while maintaining their accessibility for analysis
2Stability of the object's composition
If limited sample treatment is performed to preserve RNA, then RNA integrity is maintained, but target analytes are blocked by proteins and ribosomes
Solution Approach 1:
The patent uses capture probes as intermediary molecules that hybridize to target RNA sequences. These probes serve as mediators between the RNA and the detection system, allowing RNA to remain intact and protected while the probes provide accessible binding sites for downstream detection methods, effectively bypassing the blocking effect of proteins and ribosomes
3Quantity of substance
If hybridization with complementary nucleic acid probes and cross-linking is used to capture analytes, then target capture is improved, but RNA analytes are still lost during sample preparation
Solution Approach 1:
The patent merges the target capture function with the protection function by using capture probes that are covalently attached to solid supports. This combination ensures that when RNA hybridizes to the probes, it is simultaneously captured and protected from loss during sample preparation, eliminating the need for separate capture and protection steps
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 downstream analysis of ribonucleic acid analytes by preventing their removal or destruction during sample preparation, improving the capture of longer RNA sequences, and enhancing the sensitivity of detection methods such as probe hybridization and sequencing.
Implementation Method 1
forming a covalent bond between the reactive moiety of the attachment agent and the RNA
Implementation Method 2
forming a three-dimensional polymerized matrix from the matrix-forming agent, thereby embedding the biological sample and immobilizing the RNA
Data Source
AI summary
The present disclosure relates in some aspects to methods and compositions for repairing and/or immobilizing ribonucleic acid analytes in biological samples, and more specifically fragmented ribonucleic acids. A workflow is provided for polishing and repairing fragmented ribonucleic acids in a biological sample, for improving nucleic acid quality for downstream applications, such as in situ, spatial array, or single-cell based analysis. Ribonucleic acid analytes may be tethered covalently or non-covalently to a matrix-forming agent, for example, matrix-forming agent that can form a three-dimensional polymerized matrix.