Spatial Nucleic Acid Analysis With 5′-End Sequence Capture

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Solution Overview

Problem

Existing spatial analysis techniques primarily capture the 3′ ends of nucleic acid analytes, neglecting valuable 5′ end proximal sequences, which contain crucial information about target analytes such as mRNA encoding immune cell receptors.

Innovation Solution

A method involving reverse transcription with a first primer complementary to the target nucleic acid, incorporation of a non-templated polynucleotide sequence, and use of a second primer with a capture sequence to hybridize the 5′ end proximal sequences to capture probes on a spatial array, allowing for the determination of the analyte's location.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If standard spatial array capture methods are used, then capture efficiency is improved, but only 3′ end sequences are captured while 5′ end sequences are lost

Engineering Contradiction:

Improvecapture efficiencyVSAvoid5′ end sequence information

Core Design Contradiction:

ProductivityVSLoss of information

Solution Approach 1:

The patent inverts the conventional capture approach by designing capture probes that bind to the 5′ end of cDNA molecules rather than the traditional 3′ end. This inversion enables sequencing reads to extend from the 5′ end through the entire transcript, capturing previously lost 5′ end sequences while maintaining capture efficiency through optimized probe design and hybridization conditions.

Inventive Principle:

Principle #13The other way round (Inversion)

Solution Approach 2:

The patent incorporates template switching oligonucleotides during the reverse transcription process that preemptively add capture sequences to the 5′ end of cDNA molecules. This preliminary action ensures that the 5′ end sequences are prepared with capture capabilities before the actual capture and sequencing steps, enabling comprehensive 5′ end sequencing without requiring additional processing steps.

Inventive Principle:

Principle #10Preliminary action

2Device complexity

If reverse transcription is performed without template switching, then process simplicity is maintained, but 5′ end capture is impossible

Engineering Contradiction:

Improveprocess simplicityVSAvoid5′ end sequence data

Core Design Contradiction:

Device complexityVSLoss of information

Solution Approach 1:

The patent introduces template switching oligonucleotides as intermediary molecules during reverse transcription. These oligos serve as mediators that transfer the template switching function from the RNA template to the cDNA product, enabling the incorporation of capture sequences at the 5′ end without requiring complex enzymatic modifications or additional reagents beyond standard reverse transcription components.

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent modifies the reverse transcription parameters by adjusting primer concentrations, extension temperatures, and cycle conditions to optimize template switching efficiency. These parameter changes enable robust 5′ end capture while maintaining process simplicity, as the modifications are made within the existing reverse transcription framework rather than requiring entirely new methodologies.

Inventive Principle:

Principle #35Parameter changes

3Quantity of substance

If sequencing reads are limited to regions near the 3′ end, then sequencing cost is reduced, but valuable 5′ end information is discarded

Engineering Contradiction:

Improvesequencing coverageVSAvoid5′ end proximal sequences

Core Design Contradiction:

Quantity of substanceVSLoss of information

Solution Approach 1:

The patent transitions from a one-dimensional sequencing approach (reading only from the 3′ end toward the 5′ end) to a two-dimensional approach by capturing and sequencing from the 5′ end toward the 3′ end. This dimensional change in sequencing direction enables comprehensive coverage of the entire transcript length, including previously inaccessible 5′ end regions, while maintaining cost-effectiveness through optimized read length and coverage strategies.

Inventive Principle:

Principle #17Another dimension (Dimensionality change)

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

Enables high-resolution spatial analysis of nucleic acid analytes by capturing and sequencing 5′ end proximal sequences, providing comprehensive data on analyte location and expression within biological samples.

Implementation Method 1

hybridizing the first primer to the target nucleic acid

Methodology Applied

Scientific EffectHybridization:

Implementation Method 2

reverse transcribed with a first primer including a sequence complementary to the target nucleic acid

Methodology Applied

Scientific EffectReverse transcription:

Implementation Method 3

incorporating a polynucleotide sequence including at least three nucleotides to the 3′ end of the extension product

Methodology Applied

Scientific EffectEnzymatic incorporation: Enzyme

Implementation Method 4

hybridizing a second primer to the polynucleotide sequence including at least three nucleotides of the extension product

Methodology Applied

Scientific EffectHybridization:

Implementation Method 5

extending the extension product using the second primer as a template, thereby incorporating a complement of the capture sequence into the extension product

Methodology Applied

Scientific EffectDNA polymerization: