Helicase SELEX Aptamer Selection via Enzymatic Activity

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Solution Overview

Problem

Current methods for selecting aptamers and riboswitches, such as SELEX, are limited by the need for target immobilization, often select binding sites away from the enzyme active site, and tend to favor rigid structures over flexible ones, which are not suitable for biosensor applications.

Innovation Solution

The Helicase SELEX process, which involves functional selection based on enzymatic activity, enriches nucleic acid motifs that promote helicase activity in the presence of an activating compound, allowing for the selection of aptamers and switches that modulate helicase activity specifically.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If traditional SELEX method is used for selecting aptamers, then binding affinity to target is improved, but the selected aptamers bind away from the enzyme active site and lack functional relevance

Engineering Contradiction:

Improvebinding affinityVSAvoidfunctional relevance

Core Design Contradiction:

Measurement precisionVSReliability

Solution Approach 1:

The patent inverts the traditional SELEX approach by using functional selection instead of affinity selection. Instead of selecting aptamers based on their binding affinity to the target protein, the method selects aptamers based on their ability to modulate helicase activity. The selection process uses helicase activity as a readout to identify aptamers that functionally interact with the target, thereby ensuring both binding affinity and functional relevance simultaneously.

Inventive Principle:

Principle #13The other way round (Inversion)

Solution Approach 2:

The patent employs a disposable reporter construct system where the helicase activity is coupled to a reporter gene expression. This allows for easy selection and disposal of the reporter plasmids after use, enabling high-throughput functional screening without the need for complex, reusable selection systems. The reporter constructs are designed to be discarded after a single selection cycle, simplifying the overall process.

Inventive Principle:

Principle #27Cheap short-living objects (Disposable)

2Device complexity

If traditional SELEX method is used, then selection process is simplified, but the selected aptamers have rigid structures unsuitable for biosensor applications

Engineering Contradiction:

Improveselection process complexityVSAvoidstructural flexibility

Core Design Contradiction:

Device complexityVSAdaptability or versatility

Solution Approach 1:

The patent changes the selection parameter from binding affinity to functional activity. By using helicase activity modulation as the selection criterion, the method naturally selects for aptamers with appropriate structural flexibility and dynamic properties needed for biosensor applications. This parameter change allows the selection process to indirectly optimize for structural characteristics suitable for functional use without explicitly controlling them.

Inventive Principle:

Principle #35Parameter changes

3Ease of manufacture

If target immobilization is required for SELEX, then selection can be performed, but the choice of possible targets is limited

Engineering Contradiction:

Improveselection feasibilityVSAvoidtarget choice flexibility

Core Design Contradiction:

Ease of manufactureVSAdaptability or versatility

Solution Approach 1:

The patent replaces the mechanical immobilization system with a functional readout system. Instead of requiring physical immobilization of the target protein on a surface, the method uses helicase activity as a measurable functional output. This substitution allows selection to be performed in solution phase with freely diffusing molecules, greatly expanding the choice of possible targets while maintaining selection feasibility.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

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 process enables the development of aptamers and switches that effectively modulate helicase activity in response to specific inducers, providing a more targeted and flexible solution for biosensor applications.

Implementation Method 1

Incubation of said library with said helicase in appropriate conditions for the dissociation of certain duplex constructs by the helicase, resulting in release of the aptamers substrates of the helicase

Methodology Applied

Scientific EffectEnzymatic unwinding: Enzyme

Data Source

PatentUS20230374492A1Process for selection of aptamers, riboswitches and desoxyriboswitches

Publication Date: 2023.11.23 CENT NAT DE LA RECH SCI (C N R S)

AI summary

The invention relates to a process for selecting aptamers substrates of one helicase enzyme, comprising the implementation of a helicase SELEX process comprising several cycles, wherein each cycle comprises the following steps: a) Providing a library of nucleic acid duplex constructs comprising one nucleic acid strand containing a random sequence of 10 to 100 nucleotides framed by fixed sequences at each end; b) Incubation of said library with said helicase in appropriate conditions for the dissociation of certain duplex constructs by the helicase, resulting in release of the aptamers substrates of the helicase; c) Isolation and amplification of said aptamers substrates of the helicase; d) Creation of a novel library of nucleic acid duplex constructs enriched in duplex constructs comprising aptamers substrates of the helicase.