Reporter Oligonucleotide Protection in Immune Profiling
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Solution Overview
Problem
Current methods for detecting and characterizing antigen-B cell interactions are hindered by DNase II-mediated degradation of reporter oligonucleotides within B cells, reducing the sensitivity of immune profiling workflows for antibody discovery.
Innovation Solution
Inhibition of DNase II-mediated degradation using pH-increasing reagents, DNase II inhibitory peptides, or phosphorothioate linkages in reporter oligonucleotides, which are incorporated into the B cell system to protect the nucleic acids from lysosomal degradation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If reporter oligonucleotide is used to detect antigen-B cell interactions, then detection capability is improved, but DNase II-mediated degradation reduces sensitivity
Solution Approach 1:
The patent introduces an inhibitory peptide as an intermediary substance that binds to DNase II and blocks its degradative activity. This peptide mediator protects the reporter oligonucleotide from degradation without interfering with the antigen-B cell interaction detection, thereby resolving the contradiction between detection sensitivity and signal stability
Solution Approach 2:
The patent converts the harmful effect of DNase II degradation into a beneficial protective mechanism by using the inhibitory peptide to specifically block DNase II activity. The peptide transforms the degradation threat into a protected state, allowing the reporter oligonucleotide to remain stable while maintaining detection capability
2Productivity
If B cell internalization is enhanced for better antigen processing, then antigen presentation is improved, but reporter oligonucleotide degradation increases
Solution Approach 1:
The patent applies preliminary anti-action by pre-treating the system with inhibitory peptide that blocks DNase II activity before the B cell internalizes the antigen-reporter complex. This preventive measure ensures that when internalization occurs and lysosomal degradation would normally follow, the reporter oligonucleotide is already protected, thus maintaining integrity while allowing efficient antigen processing
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 enhances the sensitivity of immune profiling by reducing DNase II-mediated degradation, allowing for more accurate detection and characterization of antigen-B cell interactions and improved antibody discovery processes.
Implementation Method 1
a reagent that increases the pH in the partition. The DNase II mediated-degradation of the reporter oligonucleotide in the partition is inhibited by the pH-increasing reagent
Implementation Method 2
a DNase II inhibitory peptide coupled to the target antigen. the DNase II mediated-degradation of the reporter oligonucleotide in the partition is inhibited by DNase II inhibitory peptide
Implementation Method 3
one or more phosphorothioate linkages is incorporated in the sequence of the reporter oligonucleotide. the DNase II mediated-degradation of nucleic acids in the partition is inhibited by the one or more phosphorothioate linkages
Data Source
AI summary
Provided herein, inter alia, are methods and compositions for inhibiting DNase II-mediated degradation of reporter nucleic acid molecules coupled to antigens of interest, and thus improving sensitivity of single-cell immune profiling workflows,