Barcode-Free Single Vesicle Protein RNA Analysis
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Solution Overview
Problem
Current methods for analyzing the protein and RNA content of single extracellular vesicles face challenges such as limited sensitivity, poor multiplexing capability, and the need for barcoded beads, which result in wasteful and technically challenging sample preparation processes.
Innovation Solution
A method and system that uses unique molecular identifiers (UMIs) on oligonucleotide labels introduced into the solution with single vesicles, eliminating the need for barcodes by immunoprobing, emulsifying, and using PCR reagents to amplify and sequence the DNA tags, allowing for the characterization of protein and nucleic acid content without barcodes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If barcoded beads are used for single vesicle analysis, then multiplexing capability and sensitivity are improved, but sample preparation complexity and cost increase
Solution Approach 1:
The patent extracts and eliminates the barcoded bead component from the system, replacing it with direct DNA tagging of antibodies and aptamers. This removal of the bead carrier simplifies sample preparation while maintaining the ability to perform multiplexed single vesicle analysis through sequence-based identification
Solution Approach 2:
The patent creates universal DNA tagging systems that can be applied to multiple antibodies and aptamers simultaneously. The standardized UMI and antibody/aptamer label structure allows any combination of probes to be used without requiring different bead types, enabling flexible multiplexing without increasing preparation complexity
2Adaptability or versatility
If barcoded beads are used for single vesicle analysis, then multiplexing capability is improved, but the number of reagents and cost increase
Solution Approach 1:
The patent merges the functions of barcodes, antibodies, and capture elements into a single DNA-tagged antibody or aptamer molecule. This consolidation eliminates the need for separate bead carriers and reduces the number of distinct reagent components required while maintaining full multiplexing capability
Solution Approach 2:
The patent uses DNA sequence information as a copyable identifier system. Instead of requiring physical barcode beads for each probe type, the system uses sequencable DNA tags that can be digitally replicated and identified, reducing physical reagent quantity while maintaining multiplexing capacity
3Adaptability or versatility
If barcoded beads are used for single vesicle analysis, then ability to characterize protein and RNA is improved, but workflow complexity increases
Solution Approach 1:
The patent performs preliminary tagging of antibodies and aptamers with DNA sequences and UMIs before the single vesicle analysis experiment. This pre-preparation of universal DNA-tagged probes simplifies the actual workflow by eliminating the need to handle and load barcoded beads during the experiment, while maintaining the ability to simultaneously characterize multiple proteins and RNAs
Solution Approach 2:
The patent introduces DNA tags as an intermediary between the biological probes (antibodies/aptamers) and the sequencing detection system. This DNA intermediary enables simultaneous characterization of multiple targets through a unified sequence-based readout, simplifying the workflow compared to maintaining separate detection systems for different probe types
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 simplifies sample preparation, reduces complexity and cost, and enhances sensitivity and multiplexing capability, enabling the identification of EV subpopulations and their molecular cargo for potential therapeutic targets and diagnostics.
Implementation Method 1
immunoprobing target vesicles with the DNA-tagged antibodies
Implementation Method 2
adding the DNA-tags for nucleic acids, emulsifying the solution into droplets, adding PCR reagents
Implementation Method 3
A method and system that uses unique molecular identifiers (UMIs) on oligonucleotide labels introduced into the solution with single vesicles
Data Source
AI summary
According to various embodiments, a system and method for characterizing protein and nucleic acid content of a plurality of individual particles. The method includes encapsulating individual particles into compartments also containing analyte specific binding complements with oligonucleotide tags comprising a unique molecular identifier sequence, a sequence to identify the analyte specific binding complement, and a homology domain sequence. Allowing the oligonucleotide tags to hybridize on homology domain to form initial tag pairs, amplifying the tag pairs, using an enzyme to cut at the homology domain, allowing tags to re-hybridize, pooling the compartments, and sequencing. Finally, predicting co-encapsulated analytes by computational identification of clusters based on more frequently found oligonucleotide tag pairs.