Efficient Purification of Cowpea Chlorotic Mottle Virus by a Novel Peptide Aptamer.

The cowpea chlorotic mottle virus (CCMV) is a plant virus explored as a nanotechnological platform. The robust self-assembly mechanism of its capsid protein allows for drug encapsulation and targeted delivery. Additionally, the capsid nanoparticle can be used as a programmable platform to display different molecular moieties. In view of future applications, efficient production and purification of plant viruses are key steps. In established protocols, the need for ultracentrifugation is a significant limitation due to cost, difficult scalability, and safety issues. In addition, the purity of the final virus isolate often remains unclear. Here, an advanced protocol for the purification of the CCMV from infected plant tissue was developed, focusing on efficiency, economy, and final purity. The protocol involves precipitation with PEG 8000, followed by affinity extraction using a novel peptide aptamer. The efficiency of the protocol was validated using size exclusion chromatography, MALDI-TOF mass spectrometry, reversed-phase HPLC, and sandwich immunoassay. Furthermore, it was demonstrated that the final eluate of the affinity column is of exceptional purity (98.4%) determined by HPLC and detection at 220 nm. The scale-up of our proposed method seems to be straightforward, which opens the way to the large-scale production of such nanomaterials. This highly improved protocol may facilitate the use and implementation of plant viruses as nanotechnological platforms for in vitro and in vivo applications.

Aptahistochemistry in diagnostic pathology: technical scrutiny and feasibility.

Antibodies have been the workhorse for diagnostic immunohistochemistry to specifically interrogate the expression of certain protein to aid in histopathological diagnosis. This review introduces another dimension of histochemistry that employs aptamers as the core tool, the so-called aptahistochemistry. Aptamers are an emerging class of molecular recognition elements that could recapitulate the roles of antibodies. The many advantageous properties of aptamers suited for this diagnostic platform are scrutinized. An in-depth discussion on the technical aspects of aptahistochemistry is provided with close step-by-step comparison to the more familiarized immunohistochemical procedures, namely functionalization of the aptamer as a probe, antigen retrieval, optimization with emphasis on incubation parameters and visualization methods. This review offers rationales to overcome the anticipated challenges in transition from immunohistochemistry to aptahistochemistry, which is deemed feasible for an average diagnostic pathology laboratory.

Complementarity of SOMAscan to LC-MS/MS and RNA-seq for quantitative profiling of human embryonic and mesenchymal stem cells.

Dynamic range limitations are challenging to proteomics, particularly in clinical samples. Affinity proteomics partially overcomes this, yet suffers from dependence on reagent quality. SOMAscan, an aptamer-based platform for over 1000 proteins, avoids that issue using nucleic acid binders. Targets include low expressed proteins not easily accessible by other approaches. Here we report on the potential of SOMAscan for the study of differently sourced mesenchymal stem cells (MSC) in comparison to LC-MS/MS and RNA sequencing. While targeting fewer analytes, SOMAscan displays high precision and dynamic range coverage, allowing quantification of proteins not measured by the other platforms. Expression between cell types (ESC and MSC) was compared across techniques and uncovered the expected large differences. Sourcing was investigated by comparing subtypes: bone marrow-derived, standard in clinical studies, and ESC-derived MSC, thought to hold similar potential but devoid of inter-donor variability and proliferating faster in vitro. We confirmed subtype-equivalency, as well as vesicle and extracellular matrix related processes in MSC. In contrast, the proliferative nature of ESC was captured less by SOMAscan, where nuclear proteins are underrepresented. The complementary of SOMAscan allowed the comprehensive exploration of CD markers and signaling molecules, not readily accessible otherwise and offering unprecedented potential in subtype characterization. SIGNIFICANCE: Mesenchymal stem cells (MSC) represent promising stem cell-derived therapeutics as indicated by their application in >500 clinical trials currently registered with the NIH. Tissue-derived MSC require invasive harvesting and imply donor-to-donor differences, to which embryonic stem cell (ESC)-derived MSC may provide an alternative and thus warrant thorough characterization. In continuation of our previous study where we compared in depth embryonic stem cells (ESC) and MSC from two sources (bone marrow and ESC-derived), we included the aptamer-based SOMAscan assay, complementing LC-MS/MS and RNA-seq data. Furthermore, SOMAscan, a targeted proteomics platform developed for analyzing clinical samples, has been benchmarked against established analytical platforms (LC-MS/MS and RNA-seq) using stem cell comparisons as a model.

Protocol: MYTHBUSTERS: a universal procedure for sample preparation for mass spectrometry.

Improvements in proteomic strategies from the development of new and robust separation and identification techniques have led to broad applications of proteomics to solve numerous biological questions. For all analyses, sample quality is unquestionably a critical factor; therefore protein extraction is of outmost importance. The ideal extraction method should provide reproducible spectra of the most comprehensive repertoire of proteins, while minimizing sample loss and degradation. It is already known that to capture the whole proteome is an unenforceable task. Many protein extraction protocols have been described, yet there is no "one perfect procedure" taking into account the vast diversity of biological and physical properties of proteins, including their charge, size, hydrophobicity, interactions and sub-cellular localization. The research presented here reflects the main obstacle occurring in proteomic experimental design; i.e. the lack of reproducibility as a result of alterations in protein extraction methods. We have performed a series of experiments, aimed towards identification of the aptamer-binding partners in cancerous cells. Aptamers are chemically synthesized, short, single-stranded nucleic acids with a strictly defined three-dimensional structure, which allows them to interact with a target molecule with high affinity. The low immunogenicity and cellular- targeting properties of aptamers might facilitate design of suitable drugs with low side-effects. Aptamers can be used for identification of molecules associated with a pathogenic state of a cell. Aptamers can be considered as a powerful tool, since they possess unique properties to benefit cancer diagnosis, prevention and treatment. We have used different types of protein extraction methods prior to analyses of complex biological samples by mass spectrometry, based on slight changes of homogenization buffers, and have observed the changes in the identified compounds. These results should prove to be very useful for future proteomic studies and the design of studies in terms of sample preparation, especially sample homogenization and protein extraction.

Aptamer-Phage Reporters for Ultrasensitive Lateral Flow Assays.

We introduce the modification of bacteriophage particles with aptamers for use as bioanalytical reporters, and demonstrate the use of these particles in ultrasensitive lateral flow assays. M13 phage displaying an in vivo biotinylatable peptide (AviTag) genetically fused to the phage tail protein pIII were used as reporter particle scaffolds, with biotinylated aptamers attached via avidin-biotin linkages, and horseradish peroxidase (HRP) reporter enzymes covalently attached to the pVIII coat protein. These modified viral nanoparticles were used in immunochromatographic sandwich assays for the direct detection of IgE and of the penicillin-binding protein from Staphylococcus aureus (PBP2a). We also developed an additional lateral flow assay for IgE, in which the analyte is sandwiched between immobilized anti-IgE antibodies and aptamer-bearing reporter phage modified with HRP. The limit of detection of this LFA was 0.13 ng/mL IgE, ∼100 times lower than those of previously reported IgE assays.

In vitro selection of a photo-responsive peptide aptamer using ribosome display.

A photo-responsive peptide aptamer against microbeads immobilized streptavidin was isolated using in vitro selection combined with photo-manipulation. This is the first example of the introduction of a peptide aptamer in the photo-control of dynamic molecular recognition.

Hemin/G-quadruplexes as DNAzymes for the fluorescent detection of DNA, aptamer-thrombin complexes, and probing the activity of glucose oxidase.

Hemin/G-quadruplex catalyzes the H(2)O(2)-mediated oxidation of Amplex Red to the fluorescent product resorufin. This process is implemented to develop hairpin nucleic acid structures for the detection of DNA, to probe the catalytic activity of glucose oxidase, to use the thrombin-aptamer complex as a catalytic readout structure, and to quantitatively analyze telomere chain composition.

Development of interdigitated arrays coated with functional polyaniline/MWCNT for electrochemical biodetection: application for human papilloma virus.

In this study, polyaniline-multiwalled carbon nanotube film (PANi-MWCNT) has been polymerized on interdigitated platinum electrode arrays (IDA), fabricated by MEMS technology for the detection of human papillomavirus (HPV) infection, using immobilized peptide aptamers as affinity capture reagent. Label-free, electrochemical detection of the specific immune reaction between antigen peptide aptamer HPV-16-L1 (with a molecular weight of 1825 Da), the most common genotype in cytological normal women worldwide, and its specific antibody of HPV-16 (which is much bigger with molecular weight of ca. 150 kDa) on multifunctional PANi-MWCNT based arrays was reported. The most significant advantage of this technique consists of reagentless and multiple detection of antigen-antibody complex formation on well conducting IDA interface of PANi-MWCNT, without intermediate steps or any labeling reagents, as normally required in the previous works.

Application of 3,4,9,10-perylenetetracarboxylic diimide microfibers as a fluorescent sensing platform for biomolecular detection.

In this paper, we report on the use of 3,4,9,10-perylenetetracarboxylic diimide microfibers (PDIMs) as an effective fluorescent sensing platform for DNA detection for the first time. This sensing system exhibits a detection limit as low as 15 nmol L(-1) and has a high selectivity down to single-base mismatch. The general concept used in this approach is based on adsorption of fluorescently labeled single-stranded DNA (ssDNA) probe by PDIM due to the strong π-π stacking between unpaired DNA bases and PDIM. As a result, the fluorophore is brought into close proximity of PDIM, leading to substantial fluorescence quenching. In the presence of the target, the specific hybridization of the probe with its complementary DNA sequence generates a double stranded DNA (dsDNA) which detaches from PDIM, leading to fluorescence recovery. Its generality of this sensing platform for protein detection is also demonstrated.

Development of peptide aptamer microarrays for detection of HPV16 oncoproteins in cell extracts.

Protein microarrays represent an emerging technology that promises to facilitate high-throughput proteomics. The major goal of this technology is to employ peptides, full-length proteins, antibodies, and small molecules to simultaneously screen thousands of targets for potential protein-protein interactions or modifications of the proteome. This article describes the performance of a set of peptide aptamers specific for the human papillomavirus (HPV) type 16 oncoproteins E6 and E7 in a microarray format. E6 and E7 peptide aptamer microarrays were probed with fluorescence-labeled lysates generated from HPV-infected cervical keratinocytes expressing both E6 and E7 oncoproteins. Peptide aptamer microarrays are shown to detect low levels of E6 and E7 proteins. Peptide aptamers specific for cellular proteins included on these microarrays suggested that expression of CDK2, CDK4, and BCL-6 may be affected by HPV infection and genome integration. We conclude that peptide aptamer microarrays represent a promising tool for proteomics and may be of value in biological and clinical investigations of cervical carcinogenesis.

Fluorescence resonance energy transfer between an anionic conjugated polymer and a dye-labeled lysozyme aptamer for specific lysozyme detection.

Surface charge switch of a 6-carboxyfluorescein (FAM) labeled lysozyme aptamer from negative to positive upon lysozyme binding triggers fluorescence resonance energy transfer (FRET) between an anionic conjugated polymer and the aptamer, which provides a convenient method for lysozyme detection in biological media with high sensitivity and selectivity.

Au nanoparticles grafted sandwich platform used amplified small molecule electrochemical aptasensor.

We report a sensitively amplified electrochemical aptasensor using adenosine triphosphate (ATP) as a model. ATP is a multifunctional nucleotide that is most important as a "molecular currency" of intracellular energy transfer. In the sensing process, duplexes consisting of partly complementary strand (PCS1), ATP aptamer (ABA) and another partly complementary strand (PCS2) were immobilized onto Au electrode through the 5'-HS on the PCS1. Meanwhile, PCS2 was grafted with the Au nanoparticles (AuNPs) to amplify the detection signals. In the absence of ATP, probe methylene blue (MB) bound to the DNA duplexes and also bound to guanine bases specifically to produce a strong differential pulse voltammetry (DPV) signal. But when ATP exists, the ABA-PCS2 or ABA-PCS1 part duplexes might be destroyed, which decreased the amount of MB on the electrode and led to obviously decreased DPV signal. This phenomenon can be used to detect ATP and get a very sensitive detection limit low to 0.1nM, and the detection range could extend up to 10(-7)M. Compared to the sensing platform without PCS2 grafted AuNPs, amplified function of this sensing system was also evidently proved. Therefore, such PCS1-ABA-PCS2/AuNPs sensing system could provide a promising signal-amplified model for aptamer-based small-molecules detection.

Au-nanoparticles as an electrochemical sensing platform for aptamer-thrombin interaction.

A novel electrochemical method for the detection of bioaffinity interactions based on a gold-nanoparticles sensing platform and on the usage of stripping voltammetry technique was developed. The oxidation of gold surface (resulted in gold oxide formation) upon polarization served as a basis for analytical response. As a model, thrombin-thrombin binding aptamer couple was chosen. The aptamer was immobilized on a screen-printed electrode modified with gold-nanoparticles by avidin-biotin technology. Cathodic peak area was found proportional to thrombin quantity specifically adsorbed onto electrode surface. Sigmoid calibration curve as is typical for immunoassay was obtained, with thrombin detection limit of 10(-9)M. Linear range corresponds from 10(-8) to 10(-5)M thrombin concentration or 2 x 10(-14) to 2 x 10(-11)mol/electrode (R=0.996). Binding of thrombin to an aptamer has also been detected using the ferricyanide/ferrocyanide redox couple as electrochemical indicator.

Molecular characterization of Sin3 PAH-domain interactor specificity and identification of PAH partners.

Sin3 is the central component of a multisubunit co-repressor complex. A number of DNA-binding proteins are targeted by the Sin3 complex to chromatin through association with its paired amphipathic helix (PAH) domains. Here, we performed a yeast two-hybrid screening using a peptide aptamer library and identified peptides that interact with either PAH1 or PAH2. Analysis of PAH2 interacting peptides uncovered motifs similar to previously characterized PAH2 interacting proteins, Mad, Ume6 and kruppel-like members, while analysis of PAH1 interacting peptides revealed an LXXLL motif. In addition, a tandem affinity purification (TAP)-tagging approach of Sin3b resulted in the isolation of known and novel interactors amongst which neural retina leucine (NRL) zipper. Strikingly, one of the identified PAH2 interacting peptide showed strong resemblance to the NRL region amino acids 125-150. Direct association between PAH2 and NRL was shown and NRL(125-150) mediated transcriptional repression in reporter assays. Finally, we reveal that PAH1 and PAH2 amino acids 7, 14 and 39 shown previously to be important for Mad-PAH2 interaction, also play an important role in the specificity of interaction between PAH1, PAH2 and identified aptamers. Our results provide novel insights into the molecular determinant of the specificity of PAH1 and PAH2 for their interacting partners.

Ultra high-speed sorting.

BACKGROUND: Cell sorting has a history dating back approximately 40 years. The main limitation has been that, although flow cytometry is a science, cell sorting has been an art during most of this time. Recent advances in assisting technologies have helped to decrease the amount of expertise necessary to perform sorting. METHODS: Droplet-based sorting is based on a controlled disturbance of a jet stream dependent on surface tension. Sorting yield and purity are highly dependent on stable jet break-off position. System pressures and orifice diameters dictate the number of droplets per second, which is the sort rate limiting step because modern electronics can more than handle the higher cell signal processing rates. RESULTS: Cell sorting still requires considerable expertise. Complex multicolor sorting also requires new and more sophisticated sort decisions, especially when cell subpopulations are rare and need to be extracted from background. High-speed sorting continues to pose major problems in terms of biosafety due to the aerosols generated. CONCLUSIONS: Cell sorting has become more stable and predictable and requires less expertise to operate. However, the problems of aerosol containment continue to make droplet-based cell sorting problematical. Fluid physics and cell viability restraints pose practical limits for high-speed sorting that have almost been reached. Over the next 5 years there may be advances in fluidic switching sorting in lab-on-a-chip microfluidic systems that could not only solve the aerosol and viability problems but also make ultra high-speed sorting possible and practical through massively parallel and exponential staging microfluidic architectures.

Screening of a glutamic acid-binding aptamer from arginine-modified DNA library.

A binder that is specific for a target will be applicable to detection of a variety of molecules such as bioactive substances, environmental contaminants and so on. Nucleic acid aptamers obtained by in vitro selection method are interesting and promising binders. To enhance the ability of nucleic acid aptamers, we prepared a combinatorial modified DNA library containing arginyl uracil base by using arginyl dUTP, then attempted screening of glutamic acid-binding aptamers from the library by in vitro selection method.