Blocker Oligonucleotides for Probe Silencing in Spatial Analysis
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Solution Overview
Problem
Existing methods for spatial analysis of biological samples using templated ligation fail to selectively silence probes targeting highly expressed analytes, leading to off-target detection and reduced efficiency in determining analyte location and abundance.
Innovation Solution
The use of antisense synthetic molecules, such as blocker oligonucleotides, to selectively silence probes in templated ligation processes, thereby improving the detection of specific analytes by blocking hybridization with undesired probes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If capture probes are used to detect analytes by hybridizing to poly(A) tail, then a high number of analytes can be detected, but off-target analytes are also detected reducing measurement precision
Solution Approach 1:
The probe is divided into multiple functional domains: a capture domain for specific analyte binding, a spacer region for structural separation, and a barcode sequence for identification. This segmentation allows the probe to maintain high specificity through the capture domain while enabling comprehensive detection through the barcode system.
Solution Approach 2:
A barcode sequence acts as an intermediary between the specific capture domain and the detection system. The barcode is linked to the capture domain but does not directly interact with the analyte, allowing indirect detection that maintains specificity while enabling high-throughput analysis of multiple analytes.
2Measurement precision
If probes targeting highly expressed analytes are used, then detection sensitivity for those analytes is high, but sequencing saturation occurs reducing productivity
Solution Approach 1:
The system changes the concentration parameter of probe-barcode complexes in solution, allowing highly expressed analytes to be detected at lower probe concentrations, thereby reducing sequencing saturation while maintaining detection sensitivity through optimized binding kinetics.
3Measurement precision
If blocker oligonucleotides are added to silence specific probes, then detection specificity is improved, but device complexity increases
Solution Approach 1:
Blocker oligonucleotides are designed to self-complement with specific probe sequences, allowing them to automatically silence off-target probes without requiring external control mechanisms. The blockers perform their silencing function autonomously through hybridization, reducing the need for complex regulatory systems.
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
Enhances the number of genes detected, sensitivity, and spatial diversity in spatial analysis by up to 100% and reduces sequencing saturation levels by up to 50% compared to methods without blocker oligonucleotides.
Implementation Method 1
By designing synthetic molecules that are antisense to probes to be silenced, one can selectively silence such probes and block hybridization with their target analytes
Data Source
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AI summary
Provided herein are methods for selective silencing of one or more probes used in templated ligation, or RNA-templated ligation. In some embodiments, the selective silencing is achieved using one or more blocker oligonucleotides.