Small molecule inhibitor for more precise biotin/streptavidin tagging affords improved live cell imagingTechnology #cu13286
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Streptavidin is one of the most widely used tagging and labeling schemes in biomedical research. Though for many applications streptavidin is ideal, certain methods, such as protein quantification, are complicated or impossible due to the fact that the streptavidin tetramer binds up to four distinct biotin molecules at a given time. Additionally, biotinylated proteins can be cross-linked by labeled streptavidin, which has a confounding effect in live cell imaging since the proteins are artificially coupled. To circumvent this issue, this technology presents a monovalent streptavidin prepared using a small molecule inhibitor that blocks three of the four biotin-binding sites. The inhibitor consists of three biotin moieties joined via a customizable oligonucleotide backbone. Due to covalent linkage, all three biotin moieties are highly likely to be bound by the same streptavidin molecule. Thus, the inhibitor serves to convert normal streptavidin into monovalent streptavidin with a very simple modification process.
Use of inhibitor eliminates reliance on costly and time-consuming genetic manipulation and remains compatible with all commercially available streptavidin derivatives
Approaches towards producing monovalent streptavidin generally involve modifying the protein through mutation. This is of limited use since the resulting streptavidin either has lower affinity for biotin or must be prepared through a painstaking process that will complicate and lengthen experimental procedures. This technology’s inhibitor approach is quicker than genetic manipulation and can be readily adapted to almost every protocol without modification. Commercially available streptavidin derivatives can be converted to monovalent forms simply by adding the inhibitor. Furthermore, because the inhibitor contains a customizable oligonucleotide backbone, the presence of a complementary oligonucleotide causes one of the biotin moieties to become unbound from the streptavidin. This allows conditional switching from mono- to divalent streptavidin depending on the presence and concentration of an RNA or DNA target, and allows for complex and precise labeling schemes.
The inhibitor and resulting monovalent streptavidin have been tested with a range of chromatographic and spectrophotometric assays.
- Improves biotin labeling for live-cell imaging by blocking crosslinking
- Creates simple one-to-one biotin/streptavidin complex which simplifies analysis
- Allows biotin labeling to be used for direct counting quantification
- Improvement of a biotin immobilization strategy
- Sensitive detection of RNA or DNA
- Block strategy can also be used to make smaller inhibitor that produces divalent streptavidin
- Significantly faster and cheaper method to produce monovalent streptavidin than other strategies
- Highly compatible with nearly all commercially-available labeled or modified streptavidin
- Improves spatial resolution allowing usage in super resolution imaging methods
- Oligonucleotide backbone can be fully customized
- Interactions with oligonucleotide backbone act as conditional switch that can free up one binding site
- Inhibitor itself can be tagged or labeled, if desired
Patent Pending (WO/2013/036826
Tech Ventures Reference: IR 2927