DNA Aptamer Binding Histidine Tags for Protein Purification
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Solution Overview
Problem
Current methods for identifying and purifying proteins with histidine tags, such as immobilized metal affinity chromatography, rely on antibodies which can be limited in specificity and availability, necessitating a more efficient and alternative approach for detecting and isolating these proteins.
Innovation Solution
Development of unique DNA aptamers with specific nucleotide sequences (e.g., 5′-GTTTGCCGGTGGGCAGGTCTAGGGTCTGCTCGGGATTGCGGAGGAACATGCGTCGCAAAC-3′) that exhibit strong affinity for histidine tags, allowing for selective purification and detection of proteins with these tags, potentially replacing traditional antibodies and enhancing protein analysis techniques.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If antibodies are used for detecting and purifying His-tag proteins, then protein detection and purification can be achieved, but the methods are limited in specificity and availability
Solution Approach 1:
The patent uses DNA aptamers as synthetic copies that replicate the binding function of antibodies against His-tags. These aptamers are generated through SELEX technology, creating artificial binding molecules that copy the antibody's ability to specifically recognize and bind to the histidine tag sequence, thereby providing an alternative with improved availability and comparable or enhanced specificity
Solution Approach 2:
The patent changes the fundamental parameter of the binding molecule from protein-based (antibodies) to nucleic acid-based (DNA aptamers). This parameter change enables the binding agent to be produced through in vitro selection and chemical synthesis rather than requiring immunogenicity and biological production, thus improving availability while maintaining or enhancing specificity through optimized aptamer sequences
2Productivity
If traditional antibody-based methods are used, then protein identification is possible, but the methods lack efficiency and require more complex procedures
Solution Approach 1:
The patent replaces the complex biological system of antibody production and purification with a streamlined in vitro DNA aptamer selection and binding process. The SELEX methodology substitutes multiple biological steps with in vitro chemical and physical selection steps, reducing procedural complexity while enhancing efficiency through direct selection of high-affinity binders
Solution Approach 2:
The patent extracts the essential binding function from the complex antibody system and isolates it into a simplified DNA aptamer system. By taking out only the critical recognition and binding capability and implementing it through nucleic acid sequences, the method achieves the same functional outcome with reduced procedural complexity and improved efficiency
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 DNA aptamers provide a robust and specific means for identifying and purifying proteins with histidine tags, enabling efficient protein detection and analysis, including the use of fluorescent labeling for monitoring protein interactions, thereby overcoming limitations of traditional antibody-based methods.
Implementation Method 1
DNA aptamers are defined as single-stranded deoxrybonucleic acid molecules of about 40-100 nucleotides in length, which have the ability to bind a ligand with high specificity and affinity
Data Source
AI summary
A DNA aptamer was obtained which has an affinity for His-tag, and contains a nucleotide sequence selected from SEQ ID No. 1 and SEQ ID No. 2, which has clear applications.