Nucleic Acid Hybridization via Blocking Oligonucleotide
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Solution Overview
Problem
Current methods for hybridizing nucleic acid molecules are hindered by the formation of hairpin structures, which reduce hybridization efficiency, especially when one molecule is immobilized on a solid support, leading to slower hybridization speeds and reduced hybridization effectiveness.
Innovation Solution
A method involving a reaction mixture with a sense nucleic acid strand and an antisense nucleic acid strand, where the antisense strand is generated by hybridizing a primer oligonucleotide to a segment of the nucleic acid sequence and extending it with a nucleotide polymerase, thereby masking regions prone to hairpin formation and facilitating hybridization with a second nucleic acid molecule linked to a solid support.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If long nucleic acid single strands are used in hybridization reactions, then the hybridization coverage is improved, but the tendency to form hairpin structures increases, reducing hybridization speed and effectiveness
Solution Approach 1:
A blocking oligonucleotide is introduced as an intermediary element that hybridizes to a portion of the long nucleic acid single strand, preventing intramolecular hairpin formation while allowing the remaining regions to participate in intermolecular hybridization. The blocking oligonucleotide acts as a mediator that resolves the conflict between maintaining long strand coverage and preventing secondary structure formation.
2Stability of the object's composition
If one nucleic acid molecule is immobilized on a solid support, then hybridization stability is improved, but the formation of hairpin structures by the other strand is not prevented, reducing hybridization efficiency
Solution Approach 1:
The blocking oligonucleotide is applied in advance to the long nucleic acid single strand before hybridization with the immobilized molecule. This preliminary action prevents the formation of harmful hairpin structures by occupying complementary regions, thereby eliminating the obstacle to efficient hybridization while maintaining the stability provided by solid support immobilization.
3Quantity of substance
If circular nucleic acid molecules are used, then genetic information capacity is improved, but supercoiling stabilizes hairpin structures, significantly reducing hybridization effectiveness
Solution Approach 1:
The blocking oligonucleotide serves as a mediator that specifically targets and binds to regions of circular nucleic acid molecules that would otherwise form stable hairpin structures due to supercoiling. By introducing this intermediary element, the patent enables circular molecules to maintain their high information capacity while achieving reliable hybridization effectiveness.
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 significantly reduces the formation of hairpin structures, resulting in higher hybridization efficiency and allowing for effective hybridization even with circular nucleic acid molecules, which are particularly prone to such structures due to supercoiling, thereby enhancing the speed and effectiveness of nucleic acid hybridization processes.
Implementation Method 1
extending said primer oligonucleotide by a nucleotide polymerase to generate said antisense nucleic acid strand
Implementation Method 2
Nucleotide hybridization (in the following 'hybridization') is a bimolecular reaction where single-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules of complementary or at least partially complementary nucleotide sequences anneal to each other
Implementation Method 3
each of the single-stranded nucleic acid molecules involved in the hybridization reaction may form secondary structures such as stem-loops or hairpins, respectively, by intramolecular base pairing
Data Source
Figure 1~2B
Figure 3A~4C
Figure 5A~5B
AI summary
The present invention is directed to a method for hybridizing a nucleic acid molecule to another nucleic acid molecule and to a use of a nucleic acid molecule to reduce and/or avoid the formation of hairpin structures of a nucleic acid target molecule.