Single Streptavidin Research Articles

Measuring conductance switching in single proteins using quantum tunneling.

Interpreting the electrical signatures of single proteins in electronic junctions has facilitated a better understanding of the intrinsic properties of proteins that are fundamental to chemical and biological processes. Often, this information is not accessible using ensemble and even single-molecule approaches. In addition, the fabrication of nanoscale single-protein junctions remains challenging as they often require sophisticated methods. We report on the fabrication of tunneling probes, direct measurement, and active control (switching) of single-protein conductance with an external field in solution. The probes allowed us to bridge a single streptavidin molecule to two independently addressable, biotin-terminated electrodes and measure single-protein tunneling response over long periods. We show that charge transport through the protein has multiple conductive pathways that depend on the magnitude of the applied bias. These findings open the door for the reliable fabrication of protein-based junctions and can enable their use in future protein-embedded bioelectronics applications.

Biotin-streptavidin-guided two-step pretargeting approach using PLGA for molecular ultrasound imaging and chemotherapy for ovarian cancer.

BackgroundOvarian cancer seriously threatens the lives and health of women, and early diagnosis and treatment are still challenging. Pre-targeting is a promising strategy to improve the treatment efficacy of ovarian cancer and the results of ultrasound imaging.PurposeTo explore the effects of a pre-targeting strategy using streptavidin (SA) and paclitaxel (PTX)-loaded phase-shifting poly lactic-co-glycolic acid (PLGA) nanoparticles with perfluoro-n-pentane (PTX-PLGA-SA/PFPs) on the treatment and ultrasound imaging of ovarian cancer.MethodsPTX-PLGA/PFPs were prepared with a single emulsion (O/W) solvent evaporation method and SA was attached using carbodiimide. The encapsulation efficiency of PTX and the release characteristics were assessed with high performance liquid chromatography. The phase-change characteristics of the PTX-PLGA-SA/PFPs were investigated. The anti-carcinoembryonic antigen (CEA) antibody (Ab) was covalently attached to PTX-PLGA/PFPs via carbodiimide to create PTX-PLGA-Ab/PFPs. The targeting efficiency of the nanoparticles and the viability of ovarian cancer SKOV3 cells were evaluated in each group using a microscope, flow cytometry, and cell counting kit 8 assays.ResultsTHE PTX-PLGA-SA/PFPs were spheres with a size of 383.0 ± 75.59 nm. The encapsulation efficiency and loading capability of the nanoparticles for PTX were 71.56 ± 6.51% and 6.57 ± 0.61%, respectively. PTX was burst-released up to 70% in 2–3 d. When irradiated at 7.5 W for 3 min, the PTX-PLGA-SA/PFPs visibly enhanced the ultrasonography images (P < 0.05). At temperatures of 45°C and 60°C the nanoparticles phase-shifted into micro-bubbles and the sizes increased. The binding efficiencies of SA and Ab to the PTX-PLGA/PFPs were 97.16 ± 1.20% and 92.74 ± 5.75%, respectively. Pre-targeting resulted in a high binding efficacy and killing effect on SKOV3 cells (P < 0.05).ConclusionsThe two-step pre-targeting process can significantly enhance the targeting ability of PTX-loaded PLGA nanoparticles for ovarian cancer cells and substantially improve the therapeutic efficacy. This technique provides a new method for ultrasonic imaging and precise chemotherapy for ovarian cancer.

Surface charge density measurement of a single protein molecule with a controlled orientation by AFM

The spatial distribution of functional groups causes a charge distribution that often has a close relationship with its biofunctions. To understand them of the protein molecules, measurements of the charge distribution under physiological conditions are desired. Atomic force microscopy (AFM) has been utilized to measure the surface charge density by measuring the electric double layer (EDL) force caused by the overlap of the EDLs on the surfaces of the AFM tip and the biomolecule. Here, we demonstrated the surface charge density measurement of a single streptavidin (SA) protein molecule by the three-dimensional force mapping method based on frequency modulation AFM (FM-AFM). The SA has a strong affinity to biotin because of the electrostatic interactions between the molecules. Therefore, the surface charge density measurements of the biotin-binding sites and other surface areas of the molecule have been anticipated. However, the surface charge density of the surfaces other than the biotin-binding side has never been measured. We demonstrate the surface charge density measurement of the top surface of the single SA molecule, which is perpendicular to the biotin-binding sides, with a controlled orientation using DNA origami as a template by FM-AFM in an electrolyte solution. The surface charge density of the top surface of the SA molecule was estimated by fitting the experimental force curves to the Derjaguin-Landau-Verwey-Overbeck theory. We found that the surface charge density of the top surface of the SA molecule is comparable to those reported earlier for the biotin-binding sides of the molecule. We expect that, by using the DNA origami technology, one can control the orientation of a biomolecule attached to the substrate and measure the surface charge density of the specific surface areas of the biomolecule to obtain information that will help us to understand the relationship between their structures and functions.

Analysis of Protein-DNA Interactions Using Surface Plasmon Resonance and a ReDCaT Chip.

The recognition of specific DNA sequences by proteins is crucial to fundamental biological processes such as DNA replication, transcription, and gene regulation. The technique of surface plasmon resonance (SPR) is ideally suited for the measurement of these interactions because it is quantitative, simple to implement, reproducible, can be automated, and requires very little sample. This typically involves the direct capture of biotinylated DNA to a streptavidin (SA) chip before flowing over the protein of interest and monitoring the interaction. However, once the DNA has been immobilized on the chip, it cannot be removed without damaging the chip surface. Moreover, if the protein-DNA interaction is strong, then it may not be possible to remove the protein from the DNA without damaging the chip surface. Given that the chips are costly, this will limit the number of samples that can be tested. Therefore, we have developed a Reusable DNA Capture Technology, or ReDCaT chip, that enables a single streptavidin chip to be used multiple times making the technique simple, quick, and cost effective. The general steps to prepare the ReDCaT chip, run a simple binding experiment, and analysis of data will be described in detail. Some additional applications will also be introduced.

Placement of Single Proteins within the SERS Hot Spots of Self-Assembled Silver Nanolenses.

This study demonstrates the bottom-up synthesis of silver nanolenses. A robust coating protocol enabled the functionalization of differently sized silver nanoparticles with DNA single strands of orthogonal sequence. Coated particles 10 nm, 20 nm, and 60 nm in diameter were self-assembled by DNA origami scaffolds to form silver nanolenses. Single molecules of the protein streptavidin were selectively placed in the gap of highest electric field enhancement. Streptavidin labelled with alkyne groups served as model analyte in surface-enhanced Raman scattering (SERS) experiments. By correlated Raman mapping and atomic force microscopy, SERS signals of the alkyne labels of a single streptavidin molecule, from a single silver nanolens, were detected. The discrete, self-similar aggregates of solid silver nanoparticles are promising for plasmonic applications.

Direct Observation of Single Biopolymer Folding and Unfolding Process by Solid-State Nanopore

Biomolecular conformation and their transition play a crucial role in various in vivo or in vitro system. The most of the practical techniques for resolving the secondary structures of biomolecules could provide quite precise structural information for their solid-state or steady state, even at atomic resolution. For example, Cryo-EM determines high-resolution structures for the frozen-hydrated specimens of biomolecules. polymers, but it is still challenging to resolve the dynamic process of multiple functional conformational states for biomolecules at single-molecular scale. Here, we direct observed DNA folding and unfolding process in real-time by using sub-5 nm solid-state nanopores. In our experiments, a single-stranded DNA adhered to single monovalent streptavidin could be reversibly trapped in a solid-state nanopore. Then, the fluctuations of the blockade current could be recorded, which reveals the dynamic structural transitions among DNA secondary structures. For example, after trapping the cytosine-rich DNA strains in slightly alkaline solution, the formation of multiple unstable and semi-folded i-motif structures could be observed. More important, well time-resolved transitions between these structures could be obtained. When using slightly acidic solution, the stable structures with stable blockade current could be found. With this new approach, we can directly observe the dynamic conformational change of biomolecules at single-molecular scale, which would be of great help for resolving single molecule interactions, designing single-molecule machine and understanding the working process of biomolecular in biological system.

Ultrasensitive Label-Free Nanosensing and High-Speed Tracking of Single Proteins.

Label-free detection, analysis, and rapid tracking of nanoparticles is crucial for future ultrasensitive sensing applications, ranging from understanding of biological interactions to the study of size-dependent classical-quantum transitions. Yet optical techniques to distinguish nanoparticles directly among their background remain challenging. Here we present amplified interferometric scattering microscopy (a-iSCAT) as a new all-optical method capable of detecting individual nanoparticles as small as 15 kDa proteins that is equivalent to half a GFP. By balancing scattering and reflection amplitudes the interference contrast of the nanoparticle signal is amplified 1 to 2 orders of magnitude. Beyond high sensitivity, a-iSCAT allows high-speed image acquisition exceeding several hundreds of frames-per-second. We showcase the performance of our approach by detecting single Streptavidin binding events and by tracking single Ferritin proteins at 400 frames-per-second with 12 nm localization precision over seconds. Moreover, due to its extremely simple experimental realization, this advancement finally enables a cheap and routine implementation of label-free all-optical single nanoparticle detection platforms with sensitivity operating at the single protein level.

Cell labeling and proximity dependent biotinylation with engineered monomeric streptavidin

Because streptavidin is a homotetramer, it can bind multiple biotinylated ligands and cause target aggregation. To allow biotin detection without clustering, we previously engineered monomeric streptavidin (mSA) that is structurally similar to a single streptavidin subunit. Introducing the S25H mutation near the binding site increases the biotin dissociation half-life t1/2 to 83 minutes. The slowly dissociating mutant, mSA2, is useful in imaging studies because it allows stable labeling of biotinylated targets. We show that mSA2 conjugated with Alexa 488 binds biotinylated receptors on HEK293 with high specificity, and bound mSA2–Alexa488 does not dissociate significantly during an imaging study lasting 50 minutes. As a structural monomer, mSA2 can be fused to other proteins to create bifunctional molecules. We tested the use of mSA2 in proximity dependent biotinylation, in which mSA2 is fused to a peptide or a protein that binds a protein of interest (POI) and is used to recruit photoactivatable biotin (PA-biotin) to the target molecule. Once the resulting cluster of interacting proteins is subjected to UV-initiated distance-dependent biotinylation, subsequent affinity purification of biotinylated proteins on streptavidin beads can identify protein molecules that interact with POI. In addition to proteins that directly interact with the mSA2 fusion, mSA2 also induces biotinylation of other proteins that are associated through a series of noncovalent interactions. We show that mSA2 fused to an antibody recognition domain can be recruited to the kinase Erk-2 using a commercially available antibody and induce biotinylation of a known Erk-2 substrate, GST-Elk-1. Therefore, mSA2 can be used to implement proximity dependent biotinylation and detect transient enzyme-substrate interactions.

Probing and mapping the binding sites on streptavidin imprinted polymer surface

Molecular imprinting is an effective technique for preparing recognition sites which act as synthetic receptors on polymeric surfaces. Herein, we synthesized MIP surfaces with specific binding sites for streptavidin and characterized them at nanoscale by using two different atomic force microscopy (AFM) techniques. While the single molecule force spectroscopy (SMFS) reveals the unbinding kinetics between streptavidin molecule and binding sites, simultaneous topography and recognition imaging (TREC) was employed, for the first time, to directly map the binding sites on streptavidin imprinted polymers. Streptavidin modified AFM cantilever showed specific unbinding events with an unbinding force around 300pN and the binding probability was calculated as 35.2% at a given loading rate. In order to prove the specificity of the interaction, free streptavidin molecules were added to AFM liquid cell and the binding probability was significantly decreased to 7.6%. Moreover, the recognition maps show that the smallest recognition site with a diameter of around ~21nm which corresponds to a single streptavidin molecule binding site. We believe that the potential of combining SMFS and TREC opens new possibilities for the characterization of MIP surfaces with single molecule resolution under physiological conditions.

Breaking the Single-Molecule Limit in Biological Imaging

Chemically-selective imaging of biomolecules using labels is central to biological imaging. Although advances that allow resolving single fluorescently-labeled molecules with nanoscale resolution have been introduced, it is still a major challenge to extend the power of chemically-selective imaging to investigate single biomolecules with sub-molecular chemical and structural detail. To break this barrier, we have developed a chemo-mechanical labeling strategy employing single-stranded DNA to label target sites on a biomolecule and a nanomechanical readout mechanism based on atomic force microscopy to locate these labels. In analogy with fluorescence microscopy, the chemo-mechanical labeling strategy allows generating multi-color images wherein the sequence of DNA encodes color information. We demonstrate the basic utility of this approach by imaging the locations of biotins bound to a single streptavidin molecule.Figure 1. Chemically selective imaging within single biomolecules. a, Illustration of the imaging method employing single-stranded DNAs as chemo-mechanical labels and T-shaped cantilevers that allow locating the labels with sub-nanometer precision. b, Schematic of an application of this method to the imaging of biotin molecules bound to a single streptavidin. c, A 3-D topographical image of a single streptavidin, in which two black spots show the detected locations of biotins.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Single particle detection in CMOS compatible photonic crystal nanobeam cavities

We report the label-free detection of single particles using photonic crystal nanobeam cavities fabricated in silicon-on-insulator platform, and embedded inside microfluidic channels fabricated in poly-dimethylsiloxane (PDMS). Our system operates in the telecommunication wavelength band, thus leveraging the widely available, robust and tunable telecom laser sources. Using this approach, we demonstrated the detection of polystyrene nanoparticles with dimensions down to 12.5nm in radius. Furthermore, binding events of a single streptavidin molecule have been observed.

Nanoscale organization of proteins via block copolymer lithography and non-covalent bioconjugation

Thin films of cylinder-forming biotinylated poly(ethylene glycol)-polystyrene (PEG-b-PS) block copolymers were studied as a means to produce protein patterns. The orientation of the PEG cylinders depended on the end group functionality as well as on the preparation conditions. In the case of perpendicular cylinders, immobilization of single streptavidin molecules could be achieved. This immobilization was controlled by varying the amount of biotin in the films by mixing with non-functional PEG-b-PS.

Preparation of one- to four-branch silver nanostructures of various sizes by metallization of hybrid DNA–protein assemblies

We exploit the versatility of DNA–protein assemblies to generate branched metal nanostructures, referred to as nanoshurikens, of various sizes and degrees of branching. Branched silver nanostructures are prepared by metallization of star-shaped DNA–protein templates composed of monobiotinylated DNA molecules surrounding a single streptavidin protein core. DNA–protein templates are prepared by direct assembly that results in a mixture of 1-, 2-, 3- and 4-branch structures that can be separated by gel electrophoresis. A one-pot, bulk metallization is then performed in situ by successive addition of silver nitrate and sodium borohydride. This results in branched metal nanostructures with one to four branches of a well-defined length that is about 3-fold shorter than that of the template. We show that it is possible to tune two structural parameters: (i) the degree of branching by varying the concentration of streptavidin and (ii) the branch length (from 21 ± 5 nm to 107 ± 22 nm) by using biotinylated DNA molecules of different sizes.

In situ monitoring of single molecule binding reactions with time-lapse atomic force microscopy on functionalized DNA origami

Individual biomolecular binding events were recorded in situ by combining time-lapse atomic force microscopy and DNA origami. Single streptavidin molecules bound to specifically biotinyated DNA origami were simply counted as a function of time to obtain a direct measure of the binding rate.

Single‐molecule imaging of protein adsorption mechanisms to surfaces

Protein-surface interactions cause the desirable effect of controlled protein adsorption onto biodevices as well as the undesirable effect of protein fouling. The key to controlling protein-surface adsorptions is to identify and quantify the main adsorption mechanisms: adsorptions that occur (1) while depositing a protein solution onto dry surfaces and (2) after the deposition onto wet surfaces. Bulk measurements cannot reveal the dynamic protein adsorption pathways and thus cannot differentiate between the two adsorption mechanisms. We imaged the interactions of single streptavidin molecules with hydrophobic fused-silica surfaces in real-time. We observed both adsorbed proteins on surfaces and diffusing proteins near surfaces and analyzed their adsorption kinetics. Our analysis shows that the protein solution deposition process is the primary mechanism of streptavidin adsorption onto surfaces at the subnanomolar to nanomolar protein concentrations. Furthermore, we found that hydrophilic fused-silica surfaces can prevent the adsorption of streptavidin molecules.

Design and Synthesis of Bis-Biotin-Containing Reagents for Applications Utilizing Monoclonal Antibody-Based Pretargeting Systems with Streptavidin Mutants

Previous studies have shown that pretargeting protocols, using cancer-targeting fusion proteins, composed of 4 anti-CD20 single chain Fv (scFv) fragments and streptavidin (scFv(4)-SAv), followed by a biotinylated dendrimeric N-acetyl-galactosamine blood clearing agent (CA), 1, then a radiolabeled DOTA-biotin derivative (a monobiotin), 3a, can provide effective therapy for lymphoma xenografts in mouse models. A shortcoming in this pretargeting system is that endogenous biotin may affect its efficacy in patients. To circumvent this potential problem, we investigated a pretargeting system that employs anti-CD20 scFv(4)-SAv mutant fusion proteins with radioiodinated bis-biotin derivatives. With that combination of reagents, good localization of the radiolabel to lymphoma tumor xenografts was obtained in the presence of endogenous biotin. However, the blood clearance reagents employed in the studies were ineffective, resulting in abnormally high levels of radioactivity in other tissues. Thus, in the present investigation a bis-biotin-trigalactose blood clearance reagent, 2, was designed, synthesized, and evaluated in vivo. Additionally, another DOTA-biotin derivative (a bis-biotin), 4a, was designed and synthesized, such that radiometals (e.g., (111)In, (90)Y, (177)Lu) could be used in the pretargeting protocols employing scFv(4)-SAv mutant fusion proteins. Studies in mice demonstrated that the CA 2 was more effective than CA 1 at removing [(125)I]scFv(4)-SAv-S45A mutant fusion proteins from blood. Another in vivo study compared tumor targeting and normal tissue concentrations of the new reagents (2 and [(111)In]4b) with standard reagents (1 and [(111)In]3b) used in pretargeting protocols. The study showed that lymphoma xenografts could be targeted in the presence of endogenous biotin when anti-CD20 fusion proteins containing SAv mutants (scFv(4)-SAv-S45A or scFv(4)-SAv-Y43A) were employed in combination with CA 2 and [(111)In]4b. Importantly, normal tissue concentrations of [(111)In]4b were similar to those obtained using the standard reagents (1 and [(111)In]3b), except that the blood and liver concentrations were slightly higher with the new reagents. While the reasons for the higher blood and liver concentrations are unknown, the differences in the galactose structures of the clearance agents 1 and 2 may play a role.

Asymmetric Secondary and Tertiary Streptavidin/DNA Complexes Selectively Formed in a Nanometer-Scale DNA Well

Rectangular DNA motifs, which each have a built-in cavity in the middle, were prepared by bundling nine DNA helices into a U-shaped tile first and then assembling two of them together. The cavity was modified with two or three biotin molecules and was used as a nanometer-sized well to capture a single streptavidin (SA) tetramer size-selectively. Selective, one-step formation of asymmetric secondary or tertiary SA/DNA complexes via bidentate or tridentate capture have been shown by analyses of the complexes with denaturing polyacrylamide gel electrophoresis. When biotinylated DNA was simply added to the solution of SA, only symmetric SA/DNA complexes were formed as a mixture of primary to quaternary complexes, depending on the amount of DNA. However, when the DNA motif was preformed prior to SA addition, the asymmetric secondary complex was preferentially formed. The yield of the asymmetric secondary complex was ca. 50% when biotins were tethered to the two-turn well (6.8 nm wide) via 2.3 nm linkers, whereas the yield of the mixture of symmetric and asymmetric secondary complexes was only 7% for the reaction in bulk solution under the corresponding conditions. The larger the size of the well, the lower the yield became. Selective formation of asymmetric tertiary complex was also successful. Treatment of a SA tetramer captured in a well with a biotinylated, long dsDNA has shown that the captured SA still preserves its binding ability to additional biotinylated target.

Different Types of Lateral Diffusion Measurements Reveal that Unlike HA, Dc-SIGN is Immobilized in Microdomains

Hemagglutinin (HA), from influenza virus, and DC-SIGN, a dendritic cell C-type lectin that binds many pathogens including viruses, bacteria, and fungi, form microdomains on the plasma membrane. We investigated the dynamics of these proteins using Scanning FCS (S-FCS), defined valency quantum dot-based Single Particle Tracking (SPT) and Fluorescence Recovery After Photobleaching (FRAP). Using FRAP we verified that HA has a large mobile fraction (∼80%) that is characterized by a diffusion coefficient of ∼0.09 um2/sec and that it exchanges with the surround (Ellens et al. (1990) Biochemistry,29(41): 9697-9707). By contrast, neither DC-SIGN or Δ35-DC-SIGN, a DC-SIGN mutant missing the cytoplasmic tail, recovered after photobleaching, even after many minutes, indicating that these molecules do not exchange significantly with the surround. In order to determine the dynamics of molecules within the domains, we utilized a confocal line S-FCS method that permits the autocorrelation function between the same pixel in successive linescans (acquired in the form of a carpet or kymograph) to be calculated on the ms timescale. HA lateral motion within its microdomain is characterized by a diffusion coefficient of ∼0.10 um2/sec, similar to that measured outside the domain by FRAP. On the other hand, DC-SIGN and Δ35-DC-SIGN do not diffuse within the domain. We are currently using quantum dots that have been conjugated to a single streptavidin in order to examine lateral dynamics within these domains. Such nanoparticles will obviate a consistent interpretative limitation for gold and quantum dot SPT; namely, that particle valency cannot be specified with absolute certainty for the particle whose motion is tracked. Further characterization of these surprisingly stable DC-SIGN domains that are important for pathogen entry into dendritic cells is in progress.Supported by NIH GM 41402 (KJ &NLT)

Probing Intranuclear Environments at the Single-Molecule Level

Genome activity and nuclear metabolism clearly depend on accessibility, but it is not known whether and to what extent nuclear structures limit the mobility and access of individual molecules. We used fluorescently labeled streptavidin with a nuclear localization signal as an average-sized, inert protein to probe the nuclear environment. The protein was injected into the cytoplasm of mouse cells, and single molecules were tracked in the nucleus with high-speed fluorescence microscopy. We analyzed and compared the mobility of single streptavidin molecules in structurally and functionally distinct nuclear compartments of living cells. Our results indicated that all nuclear subcompartments were easily and similarly accessible for such an average-sized protein, and even condensed heterochromatin neither excluded single molecules nor impeded their passage. The only significant difference was a higher frequency of transient trappings in heterochromatin, which lasted only tens of milliseconds. The streptavidin molecules, however, did not accumulate in heterochromatin, suggesting comparatively less free volume. Interestingly, the nucleolus seemed to exclude streptavidin, as it did many other nuclear proteins, when visualized by conventional fluorescence microscopy. The tracking of single molecules, nonetheless, showed no evidence for repulsion at the border but relatively unimpeded passage through the nucleolus. These results clearly show that single-molecule tracking can provide novel insights into mobility of proteins in the nucleus that cannot be obtained by conventional fluorescence microscopy. Our results suggest that nuclear processes may not be regulated at the level of physical accessibility but rather by local concentration of reactants and availability of binding sites.

Implementation of alternating excitation schemes in a biochip-reader for quasi-simultaneous multi-color single-molecule detection

We report here the development of a device for single-molecule biochip readout using fast alternating excitation. The technology extends standard imaging cytometry by offering additional color channels in excitation. To enable the study of mobile objects, e.g. actively transported vesicles in living cells or freely diffusing lipids in a lipid bilayer, the frequency of the illumination pulses was chosen high enough to virtually freeze the motion of the biomolecules, as they are shifted through the illuminated area. The synchronization of sample illumination, scanning and line-camera readout yield two quasi-simultaneously recorded images covering the same sample region. Diffraction-limited resolution and high localization precision for point-light sources down to ∼10 nm was shown by scanning immobilized 100 nm fluorescence latex beads. Ultra-sensitivity was demonstrated by imaging single fluorescent streptavidin molecules diffusing in a fluid lipid bilayer. Two-color streptavidin labeled with Cy3 and Cy5 could be easily identified in the two respective excitation channels; high accordance in the dye positions confirms the applicability for colocalization studies of moving objects. Finally, scans of antibody–receptor interactions in large populations of live cells illustrate the feasibility of this method for biochip application.