Monovalent Streptavidin that Senses Oligonucleotides

Taylor, Steven K.; Wang, Jingxian; Kostic, Natasa; Stojanović, Milan N.

doi:10.1002/anie.201209948

Cited by 7 publications

(11 citation statements)

References 27 publications

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“…From the projection shown in Figure A, we can see three bound biotin molecules clearly, which indicates that the binding sites can be reached by a bi- or trivalent ligand simultaneously. Taylor et al have demonstrated that trisbiotinylated oligonucleotides with a length of >20 nucleotides could occupy three of streptavidin’s four biotin-binding sites . In the crystal structure, the distances between biotin molecules are measured in a range from 19 to 35 Å (Figure B).…”

Section: Resultsmentioning

confidence: 99%

“…Taylor et al have demonstrated that trisbiotinylated oligonucleotides with a length of >20 nucleotides could occupy three of streptavidin's four biotin-binding sites. 33 In the crystal structure, the distances between biotin molecules are measured in a range from 19 to 35 Å (Figure 1B). Accordingly, we proposed a three-arm structure composed of a rigid core 3,5-bis(phenylethynyl)aniline to which three flexible oligo[ethylene glycol] (OEG) chains are connected (Figure 1C).…”

Section: ■ Results and Discussionmentioning

confidence: 99%

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A Y-Shaped Three-Arm Structure for Probing Bivalent Interactions between Protein Receptor–Ligand Using AFM and SPR

et al. 2018

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The goal of this research was to develop linkage chemistry for the study of bivalent interactions between a receptor and its ligand using atomic force microscopy (AFM) and surface plasmon resonance (SPR). We conceived a three-arm structure composed of flexible chains connected to a large rigid core with orthogonal functional groups at their ends for formation and attachment (or immobilization) of bivalent ligands. To demonstrate the principle, we chose the well-known biotin-streptavidin interaction as a model system. On the basis of a crystal structure of the biotin-streptavidin complex, we designed and synthesized a bisbiotin ligand to have a Y shape with two biotin motifs on its arms for binding and a functional group on its stem for immobilization or attachment, referred to as y-bisbiotin. First, we found that the y-bisbiotin ligand stabilized the streptavidin more than its monobiotin counterpart did in solution, which indicates that the bivalent interaction was synergistic. The y-bisbiotin was attached to AFM tips through a click reaction for the force measurement experiments, which showed that unbinding the bisbiotin from streptavidin needed twice the force of unbinding a monobiotin. For the SPR study, we added a ω-thiolated alkyl chain to y-bisbiotin for its incorporation into a monolayer. The SPR data indicated that the streptavidin dissociated from a mixed monolayer bearing y-bisbiotin much slower than from the one bearing monobiotin. This work demonstrates unique chemistry for the study of bivalent interactions using AFM and SPR.

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Section: Resultsmentioning

confidence: 99%

Section: ■ Results and Discussionmentioning

confidence: 99%

A Y-Shaped Three-Arm Structure for Probing Bivalent Interactions between Protein Receptor–Ligand Using AFM and SPR

et al. 2018

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“…The streptavidin–biotin system is widely used in chemistry, biology, biotechnology, and medicine 37 . For example, it has been used in live cell imaging, 38 the identification of the proteins that undergo posttranslational modification(PTM), 39 the identification of RNA‐binding proteins, 40 the detection of in vivo protein–DNA interactions, 41 drug delivery for cancer and gene therapy, 42 and the development of novel sensors 43 . Here we show that Biotin‐DTA is sensitive enough to detect endogenous telomerase activity in tumor cells.…”

Section: Discussionmentioning

confidence: 99%

Nonradioactive direct telomerase activity detection using biotin‐labeled primers

Wang

Jin

et al. 2021

Clinical Laboratory Analysis

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Background Telomerase is a ribonucleoprotein enzyme responsible for maintenance of telomere length which expressed in more than 85% of cancer cells but undetectable in most normal tissue cells. Therefore, telomerase serves as a diagnostic marker of cancers. Two commonly used telomerase activity detection methods, the telomerase repeated amplification protocol (TRAP) and the direct telomerase assay (DTA), have disadvantages that mainly arise from reliance on PCR amplification or the use of an isotope. A safe, low‐cost and reliable telomerase activity detection method is still lacking. Method We modified DTA method using biotin‐labeled primers (Biotin‐DTA) and optimized the method by adjusting cell culture temperature and KCl concentration. The sensitivity of the method was confirmed to detect endogenous telomerase activity. The reliability was verified by detection of telomerase activity of published telomerase regulators. The stability was confirmed by comparing the method with TRAP method. Results Cells cultured in 32°C and KCl concentration at 200 mM or 250 mM resulted in robust Biotin‐DTA signal. Endogenous telomerase activity can be detected, which suggested an similar sensitivity as DTA using radioactive isotope markers. Knockdown of telomerase assembly regulator PES1 and DKC1 efficiently reduced telomerase activity. Compared with TRAP method, Biotin‐DTA assay offers greater signal stability over a range of analyte protein amounts. Conclusion Biotin‐labeled, PCR‐free, and nonradioactive direct telomerase assay is a promising new method for the easy, low‐cost, and quantitative detection of telomerase activity.

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“…A tetrameric but monovalent streptavidin with a single biotin binding site was developed as an alternative approach . Although this monovalent streptavidin is very effective for biotin labeling without cross‐linking, a truly monomeric (strept)avidin protein is still highly desired for minimal perturbation of biotin‐labeled targets and even more diverse applications of avidin proteins . For example, monomeric avidin can be genetically fused with other proteins of interest without oligomerization, and this fusion will also allow the creation of new forms of avidin proteins with different spatial organization and valency of the biotin binding sites.…”

Section: Figurementioning

confidence: 99%

A Rhizavidin Monomer with Nearly Multimeric Avidin‐Like Binding Stability Against Biotin Conjugates

et al. 2016

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Developing a monomeric form of an avidin-like protein with highly stable biotin binding properties has been a major challenge in biotin-avidin linking technology. Here we report a monomeric avidin-like protein-enhanced monoavidin-with off-rates almost comparable to those of multimeric avidin proteins against various biotin conjugates. Enhanced monoavidin (eMA) was developed from naturally dimeric rhizavidin by optimally maintaining protein rigidity during monomerization and additionally shielding the bound biotin by diverse engineering of the surface residues. eMA allowed the monovalent and nonperturbing labeling of head-group-biotinylated lipids in bilayer membranes. In addition, we fabricated an unprecedented 24-meric avidin probe by fusing eMA to a multimeric cage protein. The 24-meric avidin and eMA were utilized to demonstrate how artificial clustering of cell-surface proteins greatly enhances the internalization rates of assembled proteins on live cells.

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Monovalent Streptavidin that Senses Oligonucleotides

Cited by 7 publications

References 27 publications

A Y-Shaped Three-Arm Structure for Probing Bivalent Interactions between Protein Receptor–Ligand Using AFM and SPR

A Y-Shaped Three-Arm Structure for Probing Bivalent Interactions between Protein Receptor–Ligand Using AFM and SPR

Nonradioactive direct telomerase activity detection using biotin‐labeled primers

A Rhizavidin Monomer with Nearly Multimeric Avidin‐Like Binding Stability Against Biotin Conjugates

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