Affinity Tag Nucleic Acid Compositions for Rapid Hybrid Capture
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Solution Overview
Problem
Current methods for isolating and detecting nucleic acids and proteins are limited by slow kinetics, low capacity, and high costs, particularly in the use of affinity tags like histidine tags, which are not efficiently utilized for signal detection or analyte isolation.
Innovation Solution
A composition comprising a nucleic acid with an affinity binding pair attached to one or more nucleotides through a phosphate or sugar, including metal binding peptides and peptide affinity groups, allowing for specific hybridization and capture on a solid matrix, enabling efficient isolation and detection of nucleic acids and proteins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If hybrid capture methods are used to isolate nucleic acids with selected sequences, then sequence-specific isolation is improved, but hybridization kinetics become slower
Solution Approach 1:
The probe is divided into two functional segments: a hybridization domain (nucleic acid sequence) for specific binding and an affinity tag domain (non-nucleic acid moiety) for rapid capture. This segmentation allows each domain to optimize its function independently, resolving the contradiction between specific isolation and fast kinetics.
Solution Approach 2:
The affinity tag acts as an intermediary between the hybridized probe and the solid support. Instead of relying on slow mixed-phase hybridization for capture, the affinity tag provides a rapid, high-affinity binding interface that accelerates the isolation process while maintaining sequence specificity.
2Manufacturing precision
If biotinylated primers are used with strepavidin-coated microtitre plates, then nucleic acid capture is improved, but capacity is reduced due to large bulky proteins on solid matrix
Solution Approach 1:
The invention extracts the capture function from large bulky proteins (strepavidin) and replaces it with smaller affinity tags (metal binding peptides, aptamers, or other non-nucleic acid moieties) that can be attached to the probe. This reduction in tag size increases the number of tags that can be accommodated on the solid support, thereby increasing overall capture capacity.
Solution Approach 2:
The invention changes the physical parameter of the affinity tag from large protein molecules to smaller molecules or peptides. This parameter change allows higher density packing of tags on the solid support surface, directly increasing the quantity of nucleic acid that can be captured simultaneously.
3Manufacturing precision
If histidine tags are used for protein purification, then isolation of proteins is improved, but binding kinetics and capacity are limited
Solution Approach 1:
The invention changes the affinity tag from histidine peptides (which have moderate affinity for chelated metals) to affinity tags with higher binding constants, such as engineered protein domains or high-affinity peptide sequences. This parameter change in binding affinity directly improves both kinetics and capacity.
Solution Approach 2:
The invention uses composite affinity tags that combine multiple binding motifs or use multi-domain proteins as tags, creating a composite structure with enhanced overall affinity and capacity compared to simple histidine tags, thereby improving productivity.
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 enhances the efficiency of nucleic acid and protein isolation and detection by improving binding kinetics and capacity, reducing costs, and expanding the range of applicable sequences, while allowing for flexible attachment of affinity peptides to proteins and antibodies.
Implementation Method 1
metal binding peptides and peptide affinity groups, allowing for specific hybridization and capture on a solid matrix
Implementation Method 2
allowing for specific hybridization and capture on a solid matrix
Data Source
AI summary
The present invention concerns compositions and processes that use affinity tags for isolating, and detecting or quantifying analytes, including nucleic acids, proteins and polypeptides. Compositions include nucleic acid compositions and protein compositions with affinity binding pairs, including metal binding peptides and immobilized metals, or peptide affinity groups.