Summary of the technology
The subject of the offer are unique sequences of single-stranded DNA aptamers with high affinity to any molecule (peptide, protein or DNA derivative) containing His-Tag and Arg-Tag and their application in biotechnology in analysis of His-Tagged and Arg-Tagged molecules.
New and innovative aspects
The development of modern molecular biology, biotechnology and medicine is dependent on availability of high-quality protein products. They are often produced in prokaryotic or eukaryotic expression systems. Recombinant proteins are usually fused with a short oligopeptide tag to provide fast and easy purification procedure based on the affinity chromatography technique.
Prokaryotic and eukaryotic expression systems for the production of recombinant proteins are used on a large scale in scientific research. The genetically engineered fusion tags, e.g. histidine tag (His-Tag) are used for fast and efficient purification of the desired recombinant proteins. The high affinity of His-Tag to the certain metal ions (cobalt, copper, nickel or zinc) enables the application of chromatographic media with the above mentioned ions for the purification or immobilization of His-Tagged proteins. So far there is not known Arginine Tag (Arg-Tag), which may be used for fast and efficient purification of the desired recombinant proteins
His-Tag and Arg-Tag recombinant protein can be detected in western blotting using anti-His mono- and polyclonal antibodies. Short His-Tag and Arg-Tag can be used to investigate protein-protein interactions in techniques such as pull down assays, immunoprecpitation, co-immunoprecipitation or Far-Western blot analysis.
DNA aptamers are single-stranded DNA oligonucleotides of approximately 4-100 nucleotides that exhibit a high affinity and specificity to their corresponding ligand. An optimal binding between target molecule and selected aptamer is possible due to the rich secondary and tertiary structures of the latter one.
Main advantages of its use
The proposed inventions have three major objectives the development and characterization of innovative DNA aptamers which might be widely used in recombinant protein chromatography. The developed aptamers are characterized by high specificity and affinity to peptide tags composed of amino acids that may interact with DNA (histidine, arginine). Aptamers are fully biochemically characterized e.g., the minimum DNA sequence region necessary for the binding of the tested tags are determined, the dissociation constant of the aptamer/tag complex are analyzed, the conditions of tag elution from the aptamer are tested. In addition, the stability of the aptamers are tested by determining how many protein purification cycles there may be without significant loss of aptamer activity.
The subject of the offer are unique sequences of single-stranded DNA aptamers that recognize His-Tag and their application in biotechnology in analysis of His-Tagged molecules (peptides, proteins or DNA derivatives). The innovative DNA aptamers may be widely used in recombinant protein chromatography.
The offered DNA aptamers can be used for analysis of molecules containing a His-Tag and Arg-Tag, and in particular to:
• application as a agonist for anti-histidine and anti-arginine antibodies;
• purification and detection of the target molecules containing a His-Tag and Arg-Tag. In addition to the above applications, DNA aptamers can be used as molecules with attached tags, especially fluorescent, and in particular to label the molecular targets containing His-Tag and Arg-Tag. This type of molecules can be successfully used e.g. to study the interaction between proteins using spectroscopic techniques.