Nanoparticle self-assembly on a DNA-scaffold written by single-molecule cut-and-paste

Self-assembly guided by molecular recognition has in the past been employed to assemble nanoparticle superstructures like hypercrystals or nanoparticle molecules. An alternative approach, the direct molecule-by-molecule assembly of nanoscale superstructures, was demonstrated recently. Here we present a hybrid approach where we first assemble a pattern of binding sites one-by-one at a surface and then allow different nanoparticles to attach by self-assembly. For this approach, biotin bearing DNA oligomers were picked up from a depot using a cDNA strand bound to an AFM tip. These units were deposited in the target area by hybridization, forming a recognition pattern on this surface. Fluorescent semiconductor nanoparticles conjugated with streptavidin were allowed to assemble on this scaffold and to form the final nanoparticle superstructures.     

Signaling aptamer/protein binding by a molecular light switch complex

A novel method of signaling aptamer/protein binding for aptamer-based protein detection has been developed using a molecular light switch complex, [Ru(phen)2(dppz)]2+. The method takes advantage of the sensitive luminescence signal change of [Ru(phen)2(dppz)]2+ intercalating to the aptamer upon protein/aptamer binding. A 37-nt DNA aptamer against immunoglobulin E (IgE) was first tested as a model system. The luminescence of the [Ru(phen)2(dppz)]2+/IgE aptamer decreased with the increase of IgE. By monitoring the luminescence change, we were able to detect the binding events between the aptamer and IgE for IgE quantitation in homogeneous solutions as well as in serum. The assay was highly selective and sensitive with a detection limit of 100 pM for IgE. This new method is very simple and without the need for the covalent coupling of fluorophores to aptamers. The generalizability of the method was demonstrated by the direct detection of two other proteins, oncoprotein platelet derived growth factor-BB (PDGF-BB) using its DNA aptamer and alpha-thrombin using its RNA aptamer. This new approach is expected to promote the exploitation of aptamer-based biosensors for protein assays in biochemical and biomedical studies.     

In situ single-molecule detection of antibody-antigen binding by tapping-mode atomic force microscopy

We performed in situ detection of specific and nonspecific binding during immunoreaction on surfaces at the same location before and after analyte was injected using tapping-mode atomic force microscopy (TM-AFM) in liquid and demonstrated the ability of TM-AFM to monitor the occurrence of single-molecule binding events and to distinguish nonspecific from specific binding by examining topographical change. Two antigen/antibody pairs were investigated: chorionic gonadotropin (hCG)/mouse monoclonal anti-hCG and goat IgG (anti-intact hCG)/ mouse monoclonal anti-goat IgG. Antibody (or antigen) molecules were covalently immobilized on uniform mixed self-assembled monolayers (SAMs) terminated with carboxylic acid and hydroxyl groups. Mixed SAMs allow the control of the density of immobilized antibody (or antigen) on surfaces to achieve the detection of individual antigens, antibodies, and antigen/antibody complexes. This in situ TM-AFM-based detection method allows the single-molecule detection of antigen/antibody binding under near-physiological environment and the distinction of nonspecific from specific binding. It could be extended into a microarray.     

Direct optical detection of viral nucleoprotein binding to an anti-influenza aptamer

We have demonstrated label-free optical detection of viral nucleoprotein binding to a polyvalent anti-influenza aptamer by monitoring the surface-enhanced Raman (SERS) spectra of the aptamer-nucleoprotein complex. The SERS spectra demonstrated that selective binding of the aptamer-nucleoprotein complex could be differentiated from that of the aptamer alone based solely on the direct spectral signature for the aptamer-nucleoprotein complex. Multivariate statistical methods, including principal components analysis, hierarchical clustering, and partial least squares, were used to confirm statistically significant differences between the spectra of the aptamer-nucleoprotein complex and the spectra of the unbound aptamer. Two separate negative controls were used to evaluate the specificity of binding of the viral nucleoproteins to this aptamer. In both cases, no spectral changes were observed that showed protein binding to the control surfaces, indicating a high degree of specificity for the binding of influenza viral nucleoproteins only to the influenza-specific aptamer. Statistical analysis of the spectra supports this interpretation. AFM images demonstrate morphological changes consistent with formation of the influenza aptamer-nucleoprotein complex. These results provide the first evidence for the use of aptamer-modified SERS substrates as diagnostic tools for influenza virus detection in a complex biological matrix.     

Highly parallel single-molecule amplification approach based on agarose droplet polymerase chain reaction for efficient and cost-effective aptamer selection

We have developed a novel method for efficiently screening affinity ligands (aptamers) from a complex single-stranded DNA (ssDNA) library by employing single-molecule emulsion polymerase chain reaction (PCR) based on the agarose droplet microfluidic technology. In a typical systematic evolution of ligands by exponential enrichment (SELEX) process, the enriched library is sequenced first, and tens to hundreds of aptamer candidates are analyzed via a bioinformatic approach. Possible candidates are then chemically synthesized, and their binding affinities are measured individually. Such a process is time-consuming, labor-intensive, inefficient, and expensive. To address these problems, we have developed a highly efficient single-molecule approach for aptamer screening using our agarose droplet microfluidic technology. Statistically diluted ssDNA of the pre-enriched library evolved through conventional SELEX against cancer biomarker Shp2 protein was encapsulated into individual uniform agarose droplets for droplet PCR to generate clonal agarose beads. The binding capacity of amplified ssDNA from each clonal bead was then screened via high-throughput fluorescence cytometry. DNA clones with high binding capacity and low K(d) were chosen as the aptamer and can be directly used for downstream biomedical applications. We have identified an ssDNA aptamer that selectively recognizes Shp2 with a K(d) of 24.9 nM. Compared to a conventional sequencing-chemical synthesis-screening work flow, our approach avoids large-scale DNA sequencing and expensive, time-consuming DNA synthesis of large populations of DNA candidates. The agarose droplet microfluidic approach is thus highly efficient and cost-effective for molecular evolution approaches and will find wide application in molecular evolution technologies, including mRNA display, phage display, and so on.     

Direct binding of a redox protein for single-molecule electron transfer measurements

An electron transfer protein is engineered with two thiol groups introduced at different positions in the molecular structure to allow robust binding to two gold electrodes. Atomic force microscopy and scanning tunneling microscopy single-molecule studies show that the engineered proteins: (1) bind to a gold electrode in defined orientation dictated by the thiol-pair utilised, and (2) have a higher conductance than the wild-type proteins indicating a more efficient electron transmission due to the strong gold-thiol contacts.     

瓜环与四苯基卟啉锌的功能组装

采用紫外-可见吸收光谱(UV-Vis)、红外光谱(IR)、扫描电镜(SEM)技术探讨了瓜环Q[7,8]与具有光动力疗效的光敏剂四苯基卟啉锌(Zn TPP)分子之间的组装作用。结果表明,Q[7,8]与Zn TPP均形成以1∶1为主的组装物;溶液p H值在5.0~11.0范围内,组装体较稳定;主-客体间的相互作用都能够自发进行,组装过程是放热反应,以熵驱动为主;与Zn TPP相比,组装体的荧光量子产率并没有明显下降;Zn TPP与Q[7,8]组装后,溶解度得到极大的提高。     

Single-stranded DNA binding protein-assisted fluorescence polarization aptamer assay for detection of small molecules

Here, we describe a new fluorescence polarization aptamer assay (FPAA) strategy which is based on the use of the single-stranded DNA binding (SSB) protein from Escherichia coli as a strong FP signal enhancer tool. This approach relied on the unique ability of the SSB protein to bind the nucleic acid aptamer in its free state but not in its target-bound folded one. Such a feature was exploited by using the antiadenosine (Ade)-DNA aptamer (Apt-A) as a model functional nucleic acid. Two fluorophores (fluorescein and Texas Red) were introduced into different sites of Apt-A to design a dozen fluorescent tracers. In the absence of the Ade target, the binding of the labeled aptamers to SSB governed a very high fluorescence anisotropy increase (in the 0.130-0.200 range) as the consequence of (i) the large global diffusion difference between the free and SSB-bound tracers and (ii) the restricted movement of the dye in the SSB-bound state. When the analyte was introduced into the reaction system, the formation of the folded tertiary structure of the Ade-Apt-A complex triggered the release of the labeled nucleic acids from the protein, leading to a strong decrease in the fluorescence anisotropy. The key factors involved in the fluorescence anisotropy change were considered through the development of a competitive displacement model, and the optimal tracer candidate was selected for the Ade assay under buffer and realistic (diluted human serum) conditions. The SSB-assisted principle was found to operate also with another aptamer system, i.e., the antiargininamide DNA aptamer, and a different biosensing configuration, i.e., the sandwich-like design, suggesting the broad usefulness of the present approach. This sensing platform allowed generation of a fluorescence anisotropy signal for aptamer probes which did not operate under the direct format and greatly improved the assay response relative to that of the most previously reported small target FPAA.     

Selection strategy to generate aptamer pairs that bind to distinct sites on protein targets

Many analytical techniques benefit greatly from the use of affinity reagent pairs, wherein each reagent recognizes a discrete binding site on a target. For example, antibody pairs have been widely used to dramatically increase the specificity of enzyme linked immunosorbent assays (ELISA). Nucleic acid-based aptamers offer many advantageous features relative to protein-based affinity reagents, including well-established chemical synthesis, thermostability, and low production cost. However, the generation of suitable aptamer pairs has posed a significant challenge, and few such pairs have been reported to date. To address this important challenge, we present multivalent aptamer isolation systematic evolution of ligands by exponential enrichment (MAI-SELEX), a technique designed for the efficient selection of aptamer pairs. In contrast to conventional selection methods, our method utilizes two selection modules to generate separate aptamer pools that recognize distinct binding sites on a single target. Using MAI-SELEX, we have isolated two groups of 2'-fluoro-modified RNA aptamers that specifically recognize the αV or β3 subunits of integrin αVβ3. These aptamers exhibit low nanomolar affinities for their targets, with minimal cross-reactivity to other closely related integrin homologues. Moreover, we show that these aptamer pairs do not interfere with each other's binding and effectively detect the target even in complex mixtures such as undiluted serum.     

Evaluating the binding affinities of NF-kappaB protein to the single-nucleotide mismatch DNA binding sites by using double-stranded DNA microarray

Protein-DNA sequence-specific interaction plays an essential role in many biological processes. Here we immobilized a series of double-stranded DNA probes on an agarose coated slide to investigate the binding affinity of NF-kappaB p50 homodimer to the single-nucleotide mismatches (G<-->A or T<-->C) of the 10 base pair (bp) protein binding sites. The results demonstrated that the nucleotides at different positions contribute differently to the p50p50/DNA binding interaction. Within the 10 bp binding sites, the 5tG or 6cA mismatch has less effect on the protein-DNA binding affinity. Even the 5tG mismatch may have the ability to enhance the protein-DNA interaction (5t/w = 1.07). On the other hand, the 7cA or 10tG mismatch blocked the protein-DNA interaction more significantly than other six single-nucleotide mismatches. (7c/W = 0.37, 10t/W = 0.35). It also indicated that the duplex DNA probes immobilized on the agarose-coated surface were apt to be recognized by DNA-binding proteins, and this method would provide a reliable method for exploring the binding affinities of DNA-binding proteins with a larger number of DNA targets.     

Effect of linker structure on surface density of aptamer monolayers and their corresponding protein binding efficiency

A systematic study is reported on the effect of linker size and its chemical composition toward ligand binding to a surface-immobilized aptamer, measured using surface plasmon resonance. The results, using thrombin as the model system, showed that as the number of thymidine (T) units in the linker increases from 0 to 20 in four separate increments (T(0), T(5), T(10), T(20)), the surface density of the aptamer decreased linearly from approximately 25 to 12 pmol x cm(-2). The decrease in aptamer surface density occurred due to the increased size of the linker molecules. In addition, thrombin binding capacity was shown to increase as the linker length increased from 0 to 5 thymidine nucleotides and then decreased as the number of thymidine residues increased to 20 due to a balance between two different effects. The initial increase was due to increased access of thrombin to the aptamer as the aptamer was moved away from the surface. For linkers greater in length than T(5), the overall decrease in binding capacity was primarily due to a decrease in the surface density. Incorporation of a hexa(ethylene glycol) moiety into the linker did not affect the surface density but increased the amount of thrombin bound. In addition, the attachment of the linker at the 3'- versus the 5'-end of the aptamer resulted in increased aptamer surface density. However, monolayers formed with equal surface densities showed similar amounts of thrombin binding irrespective of the point of attachment.     

Kondo effect by controlled cleavage of a single-molecule contact

Conductance measurements of a molecular wire, contacted between an epitaxial molecule-metal bond and the tip of a scanning tunnelling microscope, are reported. Controlled retraction of the tip gradually de-hybridizes the molecule from the metal substrate. This tunes the wire into the?Kondo regime in which the renormalized molecular transport orbital serves as a spin impurity at half-filling and the Kondo resonance opens up an additional transport channel. Numerical renormalization group simulations suggest this type of behaviour to be generic for a common class of metal-molecule bonds. The results demonstrate a new approach to single-molecule experiments with atomic-scale contact control and prepare the way for the ab?initio simulation of many-body transport through single-molecule junctions.     

Polymer nanochannels fabricated by thermomechanical deformation for single-molecule analysis

A simple method for fabricating nanoscale channels based on thermomechanical deformation of rigid polymer substrates is demonstrated. Polycarbonate preforms containing microchannels with cross-sectional dimensions on the order of tens of micrometers are controllably deformed to produce submicrometer dimensions. The reduced channel dimensions are achieved by heating the preform while applying a uniaxial tensile force to reduce channel cross sections through the Poisson effect. Nanochannels with circular or elliptical cross sections are defined by varying the channel position and preform geometry prior to deformation. Arrays of parallel nanochannels with critical dimensions down to 400 nm are described. Using the fabrication method, a nanochannel network is fabricated for the detection of single protein molecules via confocal fluorescence microscopy. The chip includes a detection channel with cross-sectional dimensions approaching the confocal volume dimensions of the detection optics and a larger adjacent reference channel used to optimize focusing. Detection of fluorescently labeled bovine serum albumin at 15 and 150 nM concentrations is presented, demonstrating the ability to perform single-molecule fluorescence measurements in polycarbonate chips using visible wavelengths for excitation and detection.     

Exploration of possible binding sites of nanoparticles on protein by cross-linking chemistry coupled with mass spectrometry

For the first time, the possible binding site of nanoparticles on protein was revealed by cross-linking chemistry coupled with mass spectrometry. The peptides located very close to the poly(acrylic acid) (PAA)-coated Fe(3)O(4) nanoparticles (NPs) during interaction with human serum albumin (HSA) were cross-linked to the surface of NPs. Following protease digestion, the attached peptides were cleaved off the particle surface and identified by matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS). The peptides were found to be part of the so-called drug binding site 2 of HSA; and the competitive binding to HSA between the corresponding drug, ibuprofen, and the NPs was observed. Our results demonstrated that cross-linking chemistry coupled with MS was a quick and simple method for locating the possible binding sites of NPs on protein. Information on NP-protein binding interface will benefit the study of how the interactions are governed by the physicochemical properties of NPs, for guiding the design of functional bionano constructs. It can also help to predict the biological consequence of protein adsorption on NPs, for obtaining more knowledge on nanotoxicity.     

Electrophoretic quantitation of nucleic acids without amplification by single-molecule imaging

We have developed a novel high-performance quantitative assay for unamplified nucleic acids that is based on single-molecule imaging. The apparatus is a simple but highly sensitive single-molecule detection system that uses a normal CCD camera instead of an image-intensified CCD camera. After the DNA molecules in a sample were labeled with YOYO-1, they were induced to migrate electrophoretically in a polymer solution and imaged. No chemical or biochemical amplification was required. Direct quantitation of the sample by counting molecules was possible, because the number counted over the measurement period was directly proportional to the concentration of DNA molecules in the sample. Nonspecifically labeled impurities that would degrade the sensitivity of the assay were successfully reduced and discriminated from the DNA molecules by differences in electrophoretic mobility. By using beta-actin DNA (838 bp) as a model sample, we demonstrate that this protocol was fast (10-min measurement period), highly sensitive (limit of quantitation: approximately 10(3) copies/sample, or 3 x 10(-16) M), quantitative, and covered a wide linear dynamic range (approximately 10(4)). This high-performance assay promises to be a powerful technology for the quantitation of specific varieties of mRNA in the study of gene functions and diseases and in the clinical detection of mutant cells.     

Lithographically directed assembly of one-dimensional DNA nanostructures via bivalent binding interactions

In order to exploit the outstanding physical properties of one-dimensional (1D) nanostructures such as carbon nanotubes and semiconducting nanowires and nanorods in future technological applications, it will be necessary to organize them on surfaces with precise control over both position and orientation. Here, we use a 1D rigid DNA motif as a model for studying directed assembly at the molecular scale to lithographically patterned nanodot anchors. By matching the inter-nanodot spacing to the length of the DNA nanostructure, we are able to achieve nearly 100% placement yield. By varying the length of single-stranded DNA linkers bound covalently to the nanodots, we are able to study the binding selectivity as a function of the strength of the binding interactions. We analyze the binding in terms of a thermodynamic model which provides insight into the bivalent nature of the binding, a scheme that has general applicability for the controlled assembly of a broad range of functional nanostructures.      

Protein labeling enhances aptamer selection by methods of kinetic capillary electrophoresis

Methods of kinetic capillary electrophoresis (KCE) facilitate highly efficient selection of DNA aptamers for protein targets. The inability to detect native proteins at low concentrations in capillary electrophoresis creates, however, a significant obstacle for many important protein targets. Here we suggest that protein labeling with new Chromeo dyes can help to overcome this obstacle. By labeling a number of proteins with Chromeo P503, we show that the labeling procedure enables accurate detection of proteins in CE without significantly affecting their electrophoretic mobility or their ability to bind DNA. Moreover, Chromeo P503 does not appear to label the amino-groups of buffer components to a significant extent, making the labeling procedure compatible with a large number of selection and run buffers. Fluorescent labeling of protein targets with Chromeo dyes empowers selection of aptamers by KCE methods and promises to increase the rate at which aptamers for new targets are being developed and introduced in various applications.     

Identifying the SPARC binding sites on collagen I and procollagen I by atomic force microscopy

SPARC (secreted protein acidic and rich in cysteine) is a matricellular protein associated with the extracellular matrix (ECM). It has been found that the production of collagen I is a requisite for the association of SPARC with ECM, and studies with SPARC-null mice indicate that SPARC plays a role in modifying the structure of collagen fibers. It is not known, however, whether SPARC interacts with the collagen I precursor, procollagen I. In this study, the binding of SPARC to collagen I and procollagen I was verified by surface plasmon resonance. The SPARC-binding sites on collagen I and procollagen I were identified by directly visualizing their complexes using tapping-mode atomic force microscopy (TM-AFM). The characteristic chain end feature in collagen I is not readily detected by AFM, so unambiguous location of the binding sites relative to the C- or N-termini is difficult. In contrast, procollagen I, with its large globular C-propeptide, permits easy identification of the C-terminus. Histograms were constructed and compared based on the distances of the bound SPARC to the C-terminus of procollagen I and to the closest end of collagen I. There is a broad distribution of SPARC binding sites on procollagen I with the most preferred binding region located approximately 1/3 from the C-terminus. Characterization of the SPARC-binding sites on collagen I and procollagen I provides useful information for further understanding of the functional implications of their interactions.     

Rapid measurement of single-molecule conductance

A new device for measuring the conductance values of single-molecule junctions which are covalently bound to two electrodes is presented. The system works by repeatedly bringing two electrodes into and out of contact in a solution of molecules while measuring the current between the two electrodes during withdrawal. When molecules connect the two electrodes, steps occur in the current transient, and a statistical analysis provides the most probable conductance value for a single-molecule junction. This system provides an order of magnitude increase in speed over previous devices used for single-molecule conductance measurements, and the applicability of this tool is demonstrated in array based measurements as well as in biologically relevant samples where the conductances of single amino acid residues are measured.