Blocker Oligonucleotides for Spatial Analysis Background Reduction

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Solution Overview

Problem

Current spatial analysis techniques fail to effectively remove non-polyadenylated RNA species, such as ribosomal and mitochondrial RNA, which compete with target analytes for hybridization, leading to increased background signals and data loss, and introduce biases in nucleic acid analysis.

Innovation Solution

The use of blocker oligonucleotides, including locked nucleic acids, that hybridize to undesirable nucleic acids, preventing them from binding to capture probes and allowing for specific and targeted binding of analytes of interest, thereby reducing background noise and sample loss.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If non-polyadenylated RNA species are not removed, then the total RNA population is preserved, but background signal increases and target analyte binding is reduced

Engineering Contradiction:

Improvetarget analyte binding specificityVSAvoidbackground signal from non-specific binding

Core Design Contradiction:

Measurement precisionVSObject-generated harmful factors

Solution Approach 1:

The patent introduces blocker oligonucleotides as intermediary molecules that specifically bind to non-polyadenylated RNA species (rRNA, mtRNA) before these RNAs can hybridize to capture probes. The blockers act as mediators that prevent harmful non-specific binding while allowing target analytes to proceed with specific capture, thereby resolving the contradiction between preserving total RNA and reducing background signal.

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent extracts and removes non-polyadenylated RNA species from the total RNA population through hybridization with blocker oligonucleotides. By selectively binding and removing these unwanted RNA species before the capture step, the system eliminates the source of background signal while maintaining the integrity of the target analyte population.

Inventive Principle:

Principle #2Taking out (Extraction)

2Loss of substance

If biotinylated probes are used to remove undesirable RNA, then RNA removal is achieved, but data bias is introduced and sample loss increases

Engineering Contradiction:

Improveundesirable RNA removalVSAvoiddata accuracy

Core Design Contradiction:

Loss of substanceVSMeasurement precision

Solution Approach 1:

The patent employs blocker oligonucleotides as disposable, single-use blocking agents that are not carried through to the sequencing step. Unlike biotinylated probes that remain in the final library, the blockers are washed away after performing their temporary function of preventing rRNA and mtRNA binding, thereby eliminating bias while maintaining removal effectiveness.

Inventive Principle:

Principle #27Cheap short-living objects (Disposable)

Solution Approach 2:

The blocker oligonucleotides serve as temporary intermediary molecules that facilitate RNA removal without becoming part of the final data set. They mediate the removal process through hybridization and are subsequently discarded, preventing any bias in the final sequencing data while achieving effective sample cleanup.

Inventive Principle:

Principle #24Intermediary (Mediator)

3Loss of substance

If multiple enzymatic steps are used for RNA removal, then RNA removal is effective, but nucleic acid integrity is compromised and protocol complexity increases

Engineering Contradiction:

Improveundesirable RNA removalVSAvoidnucleic acid integrity

Core Design Contradiction:

Loss of substanceVSReliability

Solution Approach 1:

The patent replaces multiple enzymatic steps with a purely hybridization-based mechanism using blocker oligonucleotides. Instead of using enzymes that may degrade or modify nucleic acids, the system uses complementary base pairing to selectively bind and remove unwanted RNA species, thereby maintaining nucleic acid integrity while achieving effective removal.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Solution Approach 2:

The patent extracts unwanted RNA species through a simple hybridization-based separation mechanism rather than complex enzymatic processing. By using blocker oligonucleotides that specifically bind to rRNA and mtRNA, the system achieves clean extraction without the integrity-compromising effects of multiple enzymatic steps.

Inventive Principle:

Principle #2Taking out (Extraction)

4Measurement precision

If blocker oligonucleotides with non-natural nucleic acids are used, then binding specificity is improved, but manufacturing complexity increases

Engineering Contradiction:

Improveblocker binding specificityVSAvoidblocker oligonucleotide synthesis

Core Design Contradiction:

Measurement precisionVSEase of manufacture

Solution Approach 1:

The patent incorporates non-natural nucleic acids (such as locked nucleic acids or LNA) into the blocker oligonucleotides to dramatically improve binding affinity and specificity for unwanted RNA species. These parameter changes in the molecular structure enable the blockers to outcompete target analytes for binding, resolving the contradiction between manufacturing simplicity and binding effectiveness.

Inventive Principle:

Principle #35Parameter changes

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 specificity and efficiency of analyte capture, reducing undesirable nucleic acid binding by up to 100% and increasing target analyte binding by a similar margin, while minimizing biases and sample loss, thereby improving the accuracy of spatial analysis.

Implementation Method 1

hybridizing the blocker oligonucleotide to the undesirable nucleic acid molecule

Methodology Applied

Scientific EffectHybridization:

Implementation Method 2

The non-natural nucleic acids confer the blocker oligonucleotides a high melting temperature, making them unlikely to disassociate throughout the spatial analysis workflow

Methodology Applied

Scientific EffectMelting temperature stabilization: