Nucleic Acid Reporter Barcodes for Low-Background Multiplex Sequencing

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Solution Overview

Problem

Existing multiplex proximity assays suffer from high background signal due to random interactions between unbound proximity probes, making it difficult to accurately distinguish true positive signals from false positives, and performance varies between different hybridization sites.

Innovation Solution

A pool of nucleic acid reporter molecules with shared hybridization sites and unique ID sequences is used, allowing for the differentiation of true positive signals by detecting paired barcode sequences and minimizing the need for a separate negative control reaction.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If proximity assays are performed with multiple probes to detect different analytes, then multiplex detection capability is improved, but background signal increases due to random interactions between unbound probes

Engineering Contradiction:

Improvemultiplex detection capabilityVSAvoidbackground signal

Core Design Contradiction:

Adaptability or versatilityVSObject-generated harmful factors

Solution Approach 1:

The probe population is segmented into multiple probe pairs, each targeting a specific analyte. Each probe pair consists of two probes that only interact when bound to the same analyte, segmenting the detection pathways to minimize random interactions between unrelated probes and reduce background signal.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

A splint oligonucleotide acts as an intermediary that bridges the nucleic acid domains of two probes only when they are in proximity due to analyte binding. This intermediary mechanism ensures that signal generation requires both probes to be correctly positioned by the analyte, reducing false positive interactions.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Measurement precision

If separate negative control reactions are performed to determine background levels, then measurement accuracy is improved, but assay complexity and time consumption increase

Engineering Contradiction:

Improvebackground determination accuracyVSAvoidassay complexity

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The negative control function is merged into the main assay by including all probe pairs in a single reaction mixture. The background signal is determined in-situ from the same reaction, eliminating the need for separate control reactions and reducing assay complexity while maintaining measurement accuracy.

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The assay system self-determines background levels by analyzing the distribution of signal intensities across multiple probe pairs within the same reaction. The system automatically identifies background signals from probe pairs that should not interact, eliminating the need for external control procedures.

Inventive Principle:

Principle #25Self-service

3Reliability

If unique hybridization sites are assigned to each probe pair to prevent cross-reactivity, then specificity is improved, but the number of required sequences increases

Engineering Contradiction:

Improveprobe pair specificityVSAvoidnumber of sequences

Core Design Contradiction:

ReliabilityVSQuantity of substance

Solution Approach 1:

A universal set of hybridization sites is designed that can serve multiple probe pairs. Each probe pair shares the same hybridization sites, allowing these sites to perform multiple functions across different analyte detections. This universal design maintains specificity through the splint-mediated proximity requirement while reducing the total number of unique sequences needed.

Inventive Principle:

Principle #6Universality (Multi-functionality)

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 reduces background signal interference, provides a more accurate determination of false positive levels, and simplifies the assay by eliminating the need for a separate negative control, thereby enhancing the accuracy of multiplex detection.

Implementation Method 1

The nucleic acid domains of the proximity probes comprise complementary 'hybridisation sites', which hybridise to one another

Methodology Applied

Scientific EffectHybridization:

Data Source

PatentUS12540355B1Nucleic acid reporter molecules for massively parallel DNA sequencing

Publication Date: 2026.02.03 OLINK PROTEOMICS AB

AI summary

A pool of multiple nucleic acid reporter molecule species are for use in a massively parallel DNA sequencing method. All members of an individual reporter molecule species have identical nucleic acid sequences. The members of each reporter molecule species comprise, in order from 3′-end to 5′-end: (i) a first sequencing adapter, (ii) a first identification (ID) sequence, (iii) a first hybridisation sequence, or a first hybridisation sequence and a second hybridisation sequence, (iv) a second ID sequence, and (v) a second sequencing adapter. The combination of the first ID sequence and the second ID sequence are unique to the members of an individual reporter molecule species. The first hybridisation sequence is or the first hybridisation sequence and the second hybridisation sequence are, respectively, shared between a plurality of different reporter molecule species, and the sequencing adapters are shared between all reporter molecule species.