Probe Silencing With Blocker Oligonucleotides for Spatial Analysis
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Solution Overview
Problem
Existing spatial analysis methods fail to provide information on the position of single cells within a tissue sample and indiscriminately detect analytes, including those not of interest, leading to poor performance and reduced efficiency in determining analyte location and abundance.
Innovation Solution
The method involves designing synthetic antisense molecules to selectively silence probes used in templated ligation processes, using blocker oligonucleotides to block hybridization with undesired analytes, thereby improving the efficiency of spatial analysis by selectively reducing or excluding overexpressed analytes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If capture probes are used to detect analytes in spatial analysis, then analyte detection is achieved, but off-target analytes are also detected indiscriminately leading to poor performance
Solution Approach 1:
The patent applies preliminary anti-action by introducing blocker oligonucleotides that are complementary to probes targeting highly expressed genes (such as SERF1A) before the templated ligation process. These blockers hybridize to the probes in advance, preventing them from binding to their target analytes during the detection process. This pre-blocking strategy selectively silences probes that would otherwise cause off-target detection and sequencing saturation, thereby improving measurement precision without sacrificing overall detection capability
Solution Approach 2:
The blocker oligonucleotides serve as intermediaries between the probe set and the highly expressed analytes. Rather than directly removing the problematic probes or analytes, the blockers mediate the interaction by binding to probes and preventing their engagement with target analytes. This intermediary approach allows selective silencing of specific probes while maintaining the integrity of the overall templated ligation system and preserving spatial information
2Adaptability or versatility
If templated ligation probes are used to achieve coverage of entire transcriptome, then comprehensive analyte detection is achieved, but highly expressed analytes cause sequencing saturation and reduce efficiency
Solution Approach 1:
The patent applies partial action by selectively silencing only the subset of probes that target highly expressed genes, rather than removing all probes or reducing overall detection sensitivity. The blocker oligonucleotides are designed to specifically target probes for genes like SERF1A that are known to be highly expressed, allowing the majority of the transcriptome to remain detectable while preventing sequencing saturation from a small subset of overrepresented transcripts
Solution Approach 2:
The invention changes the parameters of the detection system by introducing blocker oligonucleotides that alter the effective concentration and binding affinity of specific probes. By adjusting the blocker concentration and sequence specificity, the system dynamically controls which probes are silenced, enabling optimization of transcriptome coverage while preventing sequencing saturation from highly expressed analytes
3Measurement precision
If probes targeting highly expressed genes are used, then detection sensitivity for those genes is improved, but sequencing saturation increases by 50% or more
Solution Approach 1:
The blocker oligonucleotides are introduced in advance to hybridize with probes targeting highly expressed genes, preventing these probes from binding to their abundant targets during the templated ligation process. This preliminary blocking action maintains the ability to detect these genes at their native expression levels without allowing them to dominate the sequencing output, thereby reducing sequencing saturation while preserving detection sensitivity
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 number of genes detected, sensitivity, and spatial diversity by 5-100% and reduces sequencing saturation levels by 50% or more, compared to methods without blocker oligonucleotides, thereby improving the accuracy and efficiency of spatial transcriptomic analysis.
Implementation Method 1
hybridizing the first blocker oligonucleotide to the first probe or the second probe, thereby blocking the first probe or the second probe from hybridizing to the first analyte or the second analyte
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.