Boronic Acid RNA Tethering for Fragmented Analyte Preservation
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Solution Overview
Problem
Current methods for analyzing RNA in biological samples often result in the loss of nucleic acid analytes during sample preparation, particularly fragmented RNAs, due to issues like protein blocking and the need for large probe quantities, leading to gaps in understanding disease states.
Innovation Solution
The method involves anchoring or immobilizing ribonucleic acids, especially fragmented ones, using a boronic acid moiety that covalently reacts with 2′,3′ vicinal diols, forming a covalent bond with a matrix-forming agent to create a three-dimensional polymerized matrix, allowing for downstream analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If standard sample preparation protocols (permeabilization and de-crosslinking) are used to enable RNA analysis and imaging, then analysis capability is improved, but nucleic acid analytes are lost
Solution Approach 1:
The patent applies preliminary action by performing crosslinking of RNA to proteins or other molecules in the biological sample before sample preparation steps. This pre-crosslinking ensures that RNA analytes are anchored to the sample matrix, preventing their loss during subsequent permeabilization and de-crosslinking steps. The crosslinking is performed in situ, maintaining spatial orientation while securing the RNA for downstream analysis.
2Loss of substance
If limited sample treatment is performed to preserve RNA intact, then RNA preservation is improved, but target analytes are blocked by proteins and ribosomes
Solution Approach 1:
The patent uses crosslinking reagents as intermediaries to bridge RNA analytes and the sample matrix (proteins, cellular structures). These crosslinkers form covalent bonds that attach RNA to surrounding molecules, making blocked analytes accessible for detection. The crosslinking creates a stable complex that allows subsequent probe hybridization and imaging without requiring extensive sample treatment that would compromise RNA integrity.
3Measurement precision
If large quantities of probe materials are used to overcome blocking, then signal detection is improved, but cost and complexity increase
Solution Approach 1:
By performing crosslinking before probe hybridization, the patent ensures that RNA analytes are already secured to the sample matrix in their spatial context. This preliminary anchoring increases the efficiency of probe binding, as probes can hybridize to accessible crosslinked RNA without being lost during washing or processing steps. Consequently, lower probe quantities are required to achieve sufficient signal detection, reducing cost and simplifying the assay.
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 effectively preserves and analyzes fragmented RNAs, reducing losses and enhancing the characterization of disease states by maintaining the spatial orientation of RNA molecules within the sample.
Implementation Method 1
contacting a biological sample comprising a ribonucleic acid (RNA) with an attachment agent comprising a boronic acid moiety capable of covalently reacting with at least one 2′,3′ vicinal diol of 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 in the three-dimensional polymerized matrix
Data Source
AI summary
The present disclosure relates in some aspects to methods and boronic acid compositions for immobilizing RNA analytes in biological samples, and more specifically fragmented RNAs. RNA analytes may be tethered covalently or non-covalently to exogenous or endogenous molecules in a biological sample, for example, cross-linked directly to a polymerized three-dimensional matrix.