Barcoded Binding Moieties for Single-Cell Protein Quantification
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Solution Overview
Problem
Current methods lack the capability to measure cytoplasmic and nuclear protein levels in single cells, which is essential for understanding cellular heterogeneity and gene regulation, despite advancements in genomic assays for RNA transcripts and chromatin accessibility.
Innovation Solution
The development of oligonucleotide-target binding moiety conjugates, where specific binding moieties are conjugated to oligonucleotides with barcode sequences, coated with nucleic acid binding proteins to facilitate entry into cells and reduce non-specific binding, allowing for sensitive and specific quantification of cellular proteins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If oligonucleotides are used for target binding, then specificity is improved, but non-specific binding interactions with cellular molecules increase
Solution Approach 1:
The patent uses a nucleic acid binding protein as an intermediary layer between the oligonucleotide and cellular molecules. This protein coating blocks non-specific binding interactions while preserving the specific target binding capability of the oligonucleotide-conjugated binding moiety, thereby resolving the contradiction between binding specificity and non-specific binding.
2Measurement precision
If binding moiety-oligonucleotide conjugates are used for protein quantification, then sensitivity is improved, but the ability to measure cytoplasmic and nuclear proteins is limited
Solution Approach 1:
The patent creates a universal platform where binding moieties (such as antibodies) are conjugated to oligonucleotides, enabling the same basic conjugate structure to target and quantify diverse protein types including cytoplasmic, nuclear, and membrane proteins. The nucleic acid binding protein coating enhances this universality by facilitating entry into different cellular compartments while maintaining the specific binding capability for various protein targets.
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
Enables highly sensitive and specific quantification of cellular proteins, including cytoplasmic and nuclear proteins, by using barcode sequences for amplification and sequencing, combining with other genomic assays for comprehensive single-cell analysis.
Implementation Method 1
The oligonucleotide components of the conjugates are coated with a nucleic acid binding protein, e.g., a sequence non-specific nucleic acid binding protein, or two or more nucleic acid binding proteins, that block the non-specific binding interactions of the oligonucleotide with cellular molecules
Implementation Method 2
each conjugate comprises a target binding moiety that specifically binds to a cellular protein to be quantified
Data Source
AI summary
The invention provides compositions and methods for quantifying cellular molecules of interest, e.g., intracellular proteins, using oligonucleotide-target binding moiety conjugates.