Nuclease-Deficient Argonaute Barcode Detection for Large Panels
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Solution Overview
Problem
Existing methods for detecting barcode sequences in biological samples face challenges in designing distinct barcode subunits with high affinity for correct barcode-binding probes and low affinity for off-target binding, especially as the number of distinct barcode subunits increases and subunit length decreases.
Innovation Solution
Utilizing nuclease-deficient Argonaute proteins in complex with barcode-binding probes to enhance sequence-specific binding, ensuring exact complementarity within the seed region for precise barcode detection, thereby improving specificity and efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If the number of distinct barcode subunits is increased to detect more analytes, then the panel size increases, but the affinity for correct binding decreases and off-target binding increases
Solution Approach 1:
The patent introduces Argonaute proteins as intermediary molecules that bind to barcode subunits with high specificity through guide RNA-complementarity. This intermediary binding mechanism allows for increased panel size while maintaining high binding specificity, as the Argonaute-protein complex acts as a selective mediator between the barcode probe and detection system
Solution Approach 2:
The patent changes the binding parameters by using Argonaute proteins with engineered binding affinities and specificities. By modifying the protein's interaction parameters with different barcode subunits, the system can accommodate larger panels while maintaining reliable discrimination between correct and off-target binding through optimized binding kinetics
2Adaptability or versatility
If the length of barcode subunits is decreased to increase the number of distinct barcodes, then the diversity of barcodes increases, but the binding affinity and specificity decrease
Solution Approach 1:
The Argonaute protein serves as an intermediary that compensates for the reduced length of barcode subunits. The protein's structured binding interface and guide RNA provide additional specificity elements, allowing short barcode subunits to maintain high binding precision through the cooperative binding mechanism of the Argonaute complex
Solution Approach 2:
The patent creates a composite detection system combining Argonaute proteins, guide RNAs, and shortened barcode subunits. This composite structure leverages the strengths of each component: the protein's high affinity, the RNA's specificity, and the short barcode's diversity, achieving both high barcode diversity and maintained binding precision
3Device complexity
If conventional probe binding is used without Argonaute proteins, then the system is simpler, but the specificity and affinity for barcode detection are insufficient
Solution Approach 1:
The Argonaute protein is introduced as a specialized intermediary that provides the necessary specificity and affinity for reliable barcode detection. While this increases system complexity, the protein's natural binding mechanisms and guide RNA interactions create a highly specific detection system that overcomes the limitations of simple probe binding
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
The method provides increased specificity and affinity for barcode detection, reducing off-target binding and enabling the decoding of a large number of barcode probes with improved binding stability and kinetics.
Implementation Method 1
the guide nucleic acid directs Argonaute binding to a target sequence complementary to the guide nucleic acid or a portion thereof (e.g., complementary to a seed sequence of the guide nucleic acid) with high sensitivity and specificity
Implementation Method 2
each barcode-binding probe is in a complex with a nuclease-deficient Argonaute protein and each barcode-binding probe comprises a barcode-binding domain that binds to a sequence of the barcode subunit
Data Source
AI summary
The present disclosure relates in some aspects to methods for analyzing target nucleic acids and their spatial locations in a biological sample using Argonaute proteins. In some aspects, a barcode probe library comprising a plurality of probes each comprising a plurality of barcode subunits that identifies a target analyte is detected in situ in the sample. Also provided are compositions and kits for use in accordance with the methods.