Modified Thymine Bases for Nucleic Acid Hybridization

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Solution Overview

Problem

Current nucleic acid technologies face challenges in achieving high specificity and sensitivity, particularly when target nucleic acids are in limited quantities, and existing nucleotide analogs do not adequately enhance hybridization stability and solubility.

Innovation Solution

Development of novel, non-naturally occurring thymidine-like modified bases that increase binding affinity and specificity for adenine or 2,6-diaminopurine bases, incorporated into polynucleotide oligomers to enhance hybridization and solubility, allowing for shorter oligomers and improved aqueous solubility.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If conventional nucleotide analogs are used to enhance hybridization stability, then binding affinity may be improved, but solubility deteriorates due to hydrophobic character of aromatic fluorophores and quencher moieties

Engineering Contradiction:

Improvehybridization stabilityVSAvoidsolubility

Core Design Contradiction:

ReliabilityVSObject-affected harmful factors

Solution Approach 1:

The patent introduces a water-soluble modified thymine base as an intermediary component that mediates between the hydrophobic fluorophore/quencher moieties and the aqueous environment. This modified base acts as a solubility-enhancing intermediary that allows the hydrophobic detection moieties to function while maintaining overall oligomer solubility in aqueous solutions

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent modifies the chemical structure of thymine by adding a hydrophilic substituent group to create a water-soluble modified thymine base. This parameter change in the molecular structure (adding hydrophilic character) directly improves the solubility parameter of the oligomer while maintaining hybridization stability

Inventive Principle:

Principle #35Parameter changes

2Reliability

If longer oligomer sequences are used to achieve sufficient binding affinity, then hybridization stability improves, but the hydrophobic effects of aromatic fluorophores and quencher moieties are exacerbated

Engineering Contradiction:

Improvebinding affinityVSAvoidhydrophobic aggregation

Core Design Contradiction:

ReliabilityVSObject-affected harmful factors

Solution Approach 1:

The water-soluble modified thymine base serves as a solubility-enhancing intermediary distributed within the oligomer sequence, counteracting the hydrophobic effects of aromatic fluorophores and quencher moieties. This allows the use of optimized oligomer lengths for binding affinity without exacerbating hydrophobic aggregation

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

By changing the solubility parameter of the oligomer through incorporation of hydrophilic modified thymine bases, the patent enables optimization of oligomer length for maximum binding affinity while maintaining solubility and preventing hydrophobic aggregation

Inventive Principle:

Principle #35Parameter changes

3Adaptability or versatility

If standard thymine bases are used in polynucleotide oligomers, then natural hybridization properties are maintained, but binding affinity and specificity are insufficient for limited quantity targets

Engineering Contradiction:

Improvenatural hybridization propertiesVSAvoidbinding affinity

Core Design Contradiction:

Adaptability or versatilityVSReliability

Solution Approach 1:

The patent applies local quality modification by specifically modifying only the thymine bases in the oligomer sequence while maintaining the overall nucleotide structure and natural hybridization properties. This localized modification enhances binding affinity and specificity without completely altering the natural hybridization behavior

Inventive Principle:

Principle #3Local quality

Solution Approach 2:

The patent changes specific chemical parameters of the thymine base (adding hydrophilic substituents) to enhance binding affinity and specificity while maintaining compatibility with natural hybridization processes, enabling detection of limited quantity targets

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

The modified bases significantly enhance the binding affinity and specificity of polynucleotide oligomers, enabling the use of shorter sequences and improving solubility, which helps in offsetting hydrophobic effects of aromatic fluorophores and quencher moieties, promoting stable hybridization and detection.

Implementation Method 1

The modified bases of the present disclosure when incorporated into polynucleotide oligomers have been discovered to surprisingly increase the binding affinity and specificity of those oligomers comprising them for hybridization with complementary sequences

Methodology Applied

Scientific EffectHybridization:

Data Source

PatentEP3126372B1Modified thymine polynucleotide oligomers and methods

Publication Date: 2019.02.06 CEPHEID INC

Figure 1A~1B
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Figure 3A~3B

AI summary

Disclosed are modified thymine bases that provide enhanced base-pairing affinity for adenine or 2,6-diaminopurine bases in polynucleotide hybridization complexes. Also disclosed are polynucleotide oligomers, polynucleotide hybridization complexes that comprise such modified thymine bases. Also disclosed are various methods of use. For example, in some embodiments, modified polynucleotide oligomers disclosed herein can be used as primers and probes for nucleic acid amplification and/or detection.