Protein-Catalyzed Capture Agents.

Protein-catalyzed capture agents (PCCs) are synthetic and modular peptide-based affinity agents that are developed through the use of single-generation in situ click chemistry screens against large peptide libraries. In such screens, the target protein, or a synthetic epitope fragment of that protein, provides a template for selectively promoting the noncopper catalyzed azide-alkyne dipolar cycloaddition click reaction between either a library peptide and a known ligand or a library peptide and the synthetic epitope. The development of epitope-targeted PCCs was motivated by the desire to fully generalize pioneering work from the Sharpless and Finn groups in which in situ click screens were used to develop potent, divalent enzymatic inhibitors. In fact, a large degree of generality has now been achieved. Various PCCs have demonstrated utility for selective protein detection, as allosteric or direct inhibitors, as modulators of protein folding, and as tools for in vivo tumor imaging. We provide a historical context for PCCs and place them within the broader scope of biological and synthetic aptamers. The development of PCCs is presented as (i) Generation I PCCs, which are branched ligands engineered through an iterative, nonepitope-targeted process, and (ii) Generation II PCCs, which are typically developed from macrocyclic peptide libraries and are precisely epitope-targeted. We provide statistical comparisons of Generation II PCCs relative to monoclonal antibodies in which the protein target is the same. Finally, we discuss current challenges and future opportunities of PCCs.

Dual-target recognition sandwich assay based on core-shell magnetic mesoporous silica nanoparticles for sensitive detection of breast cancer cells.

A novel dual-target recognition sandwich strategy for selective capture and detection of MCF-7 breast cancer cells based on core-shell magnetic mesoporous silica (Fe3O4@nSiO2@mSiO2@apt) nanoparticles was developed. Fe3O4@nSiO2@mSiO2@apt nanoparticles, which were prepared by a layer-by-layer method and were used for the first time to capture cancer cells, have large surface areas, particularly accessible mesochannels, and good biocompatibility, enabling aptamers to be compactly anchored onto the surface of the core-shell magnetic nanoparticles. A mucin 1 protein (MUC1)-targeted Fe3O4@nSiO2@mSiO2@apt nanoparticle was used as an affinity magnetic isolate material to capture target MCF-7 cells selectively and to reduce interference through affinity interaction between the anti-MUC1 aptamer and the MUC1 protein over-expressed on the surface of the MCF-7 cells. Meanwhile, a folate receptor (FR)-targeted affinity fluorescent probe (FA-BSA-FITC) was developed by coupling folic acid and FITC to the surface of BSA, enabling high sensitivity, selective fluorescent labeling of FR over-expressed MCF-7 cells. A dual-target recognition sandwich assay was developed based on the MUC1-targeted magnetic nanoparticles and the FR-targeted fluorescent probes. Under optimum conditions, a quantitative assay of MCF-7 cells was achieved with a dynamic range of 102-105 cells/mL (R2 = 0.9991). This assay showed high specificity and sensitivity to the target MCF-7 cells. Finally, the proposed strategy could be extended to detect MCF-7 cells in human plasma and whole blood with a recovery range of 86.1-104.0% and a RSD range of 1.2-8.4%, respectively. This indicates that the dual-target recognition method developed in this research exhibits good selectivity, anti-interference capability, and reliability even in plasma and whole blood samples and is more suitable for complex samples than previous targeted assays. Therefore, the approach proposed here may have great potential for early breast cancer diagnosis.

Engineering and characterization of fluorogenic glycine riboswitches.

A set of 12 fluorogenic glycine riboswitches with different thermodynamic and kinetic response properties was engineered. For the design of functional riboswitches, a three-part RNA approach was applied based on the idea of linking a RNA sensor, transmitter and actuator part together. For the RNA sensor and actuator part, we used the tandem glycine aptamer structure from Bacillus subtillis, and fluorogenic aptamer Spinach, respectively. To achieve optimal signal transduction from the sensor to the actuator, a riboswitch library with variable transmitter was screened with a microfluidic large-scale integration chip. This allowed us to establish the complete thermodynamic binding profiles of the riboswitch library. Glycine dissociation constants of the 12 strong fluorescence response riboswitches varied between 99.7 and 570 µM. Furthermore, the kinetic glycine binding (k(on)), and dissociation (k(off)) rates, and corresponding energy barriers of the 10 strongest fluorescence response riboswitches were determined with the same chip platform. k(on) and k(off) were in the order of 10(-3)s(-1) and 10(-2)s(-1), respectively. Conclusively, we demonstrate that systematic screening of synthetic and natural linked RNA parts with microfluidic chip technology is an effective approach to rapidly generate fluorogenic metabolite riboswitches with a broad range of biophysical response properties.

Design of a dual aptamer-based recognition strategy for human matrix metalloproteinase 9 protein by piezoelectric biosensors.

MMP-9, human matrix metalloproteinase 9, belongs to the family of zinc-dependent peptide-bond hydrolases and is involved in the degradation of the extracellular matrix (ECM). In clinics, it is well known that elevated MMP-9 serum levels are associated with cardiovascular dysfunctions, several aspects of the physiology and pathology of the central nervous system, neuropsychiatric disorders and degenerative diseases related to brain tumors, and excitotoxic/neuroinflammatory processes. Due to the large interest of diagnostics in this protein, efforts to set up sensitive methods to detect MMP-9 for early diagnosis of a number of metabolic alterations are rapidly increasing. In this panorama, biosensors could play a key role; therefore we explored for the first time the development of an aptamer-based piezoelectric biosensor for a sensitive, label free, and real time detection of MMP-9. The detecting strategy involved two different aptamers in a sandwich-like approach able to detect down to 100 pg mL(-1) (1.2 pM) of MMP-9 as detection limit in standard solution. As proof of principle, commercial serum was investigated in terms of possible interferents, their identification and role in MMP-9 detection. The estimated detection limit for MMP-9 is about 560 pg mL(-1) (6.8 pM) in untreated serum.

Designed synthesis of aptamer-immobilized magnetic mesoporous silica/Au nanocomposites for highly selective enrichment and detection of insulin.

We designed and synthesized aptamer-immobilized magnetic mesoporous silica/Au nanocomposites (MMANs) for highly selective detection of unlabeled insulin in complex biological media using MALDI-TOF MS. The aptamer was easily anchored onto the gold nanoparticles in the mesochannels of MMANs with high capacity for highly efficient and specific enrichment of insulin. With the benefit from the size-exclusion effect of the mesoporous silica shell with a narrow pore size distribution (∼2.9 nm), insulin could be selectively detected despite interference from seven untargeted proteins with different size dimensions. This method exhibited an excellent response for insulin in the range 2-1000 ng mL(-1). Moreover, good recoveries in the detection of insulin in 20-fold diluted human serum were achieved. We anticipate that this novel method could be extended to other biomarkers of interest and potentially applied in disease diagnostics.

Application of molecular antiviral compounds: novel approach for durable resistance against geminiviruses.

Both transgenic as well as traditional breeding approaches have not been completely successful in inducting resistance against geminiviruses in crop plants. This demands the utilization of non-viral, non-plant compounds possessing antiviral characteristics as an alternate and effective strategy for developing durable resistance against geminiviruses. In recent years, several antiviral molecules have been developed for the treatment of plant virus infections. These molecular antiviral compounds target various geminiviral-DNA and -protein via interacting with them or by cleaving viral RNA fragments. Applications of these proteins such as GroEL, g5g and VirE2 have also provided a convincing evidence of resistance against geminiviruses. Taking advantage of this information, we can generate robust resistance against geminiviruses in diverse crop plants. In this context, the present review provides epigrammatic information on these antiviral compounds and their mode of action in modulating virus infection.

Inhibition of the ferric uptake regulator by peptides derived from anti-FUR peptide aptamers: coupled theoretical and experimental approaches.

The FUR protein (ferric uptake regulator) is an iron-dependent global transcriptional regulator. Specific to bacteria, FUR is an attractive antibacterial target since virulence is correlated to iron bioavailability. Recently, four anti-FUR peptide aptamers, composed of 13 amino acid variable loops inserted into a thioredoxinA scaffold, were identified, which were able to interact with Escherichia coli FUR (EcFUR), inhibit its binding to DNA and to decrease the virulence of pathogenic E. coli in a fly infection model. The first characterization of anti-FUR linear peptides (pF1 6 to 13 amino acids) derived from the variable part of the F1 anti-FUR peptide aptamer is described herein. Theoretical and experimental approaches, in original combination, were used to study interactions of these peptides with FUR in order to understand their mechanism of inhibition. After modeling EcFUR by homology, docking with Autodock was combined with molecular dynamics simulations in implicit solvent to take into account the flexibility of the partners. All calculations were cross-checked either with other programs or with experimental data. As a result, reliable structures of EcFUR and its complex with pF1 are given and an inhibition pocket formed by the groove between the two FUR subunits is proposed. The location of the pocket was validated through experimental mutation of key EcFUR residues at the site of proposed peptide interaction. Cyclisation of pF1, mimicking the peptide constraint in F1, improved inhibition. The details of the interactions between peptide and protein were analyzed and a mechanism of inhibition of these anti-FUR molecules is proposed.

A multifunctional nanomicelle for real-time targeted imaging and precise near-infrared cancer therapy.

Simultaneous targeted cancer imaging, therapy and real-time therapeutic monitoring can prevent over- or undertreatment. This work describes the design of a multifunctional nanomicelle for recognition and precise near-infrared (NIR) cancer therapy. The nanomicelle encapsulates a new pH-activatable fluorescent probe and a robust NIR photosensitizer, R16FP, and is functionalized with a newly screened cancer-specific aptamer for targeting viable cancer cells. The fluorescent probe can light up the lysosomes for real-time imaging. Upon NIR irradiation, R16FP-mediated generation of reactive oxygen species causes lysosomal destruction and subsequently trigger lysosomal cell death. Meanwhile the fluorescent probe can reflect the cellular status and in situ visualize the treatment process. This protocol can provide molecular information for precise therapy and therapeutic monitoring.

Aptamer-targeted hyperbranched polymers: towards greater specificity for tumours in vivo.

Hyperbranched polymers conjugated to a peptide-aptamer were prepared using a combination of RAFT polymerisation and click chemistry for targeting tumour cells in vivo. The polymers showed enhanced cell-uptake in vitro (compared to unconjugated polymer) while excellent specificity for solid tumours was observed in vivo using a mouse model of melanoma.

Evolution of disulfide-rich peptide aptamers using cDNA display.

Protein scaffolds containing some disulfide bonds (e.g., Knottin, Kunitz domain, etc.) are promising candidates for molecular recognition. cDNA display has been developed to screen functional disulfide-rich peptide aptamers from a vast library by promoting disulfide bond shuffling after the synthesis of peptides in a cell-free translation system. Here we present a detailed protocol for the selection of disulfide-rich peptide aptamers against interleukin 6 receptor (IL-6R) from a 35-amino acid peptide library containing 32 amino acids in the random region, which is linked to its genotype by cDNA display.

A designed cell-permeable aptamer-based corepressor peptide is highly specific for the androgen receptor and inhibits prostate cancer cell growth in a vector-free mode.

The repression of the androgen receptor (AR) activity is a major objective to inhibit prostate cancer growth. One underlying mechanism for efficient hormone therapy is based on corepressors that inactivate the AR. In line with this, castration-resistant prostate cancer is associated with malfunction or reduced corepressor action. To overcome this, the overexpression of endogenous corepressors, however, affects many other transcription factors. Therefore, an AR-specific corepressor could be of advantage. Using a yeast peptide aptamer two-hybrid screen with the full-length human AR, we identified a short amino acid-stretch that binds specifically to the human AR in yeast and in mammalian cells and not to the closely related progesterone or glucocorticoid receptors. Furthermore, fused to a silencing domain, this aptamer-based corepressor (AB-CoR) exhibits corepressor activity by inhibiting both the AR-mediated transactivation and expression of the AR target gene PSA. Furthermore, stable expression of the AB-CoR inhibits growth of human LNCaP prostate cancer cells. Moreover, we generated a cell-permeable AB-CoR by fusing a protein transduction domain to establish a vector-free transport system. Treatment of LNCaP cells with the bacterially expressed and affinity-purified cell-permeable AB-CoR peptide resulted in a significant inhibition of both AR-mediated transactivation and prostate cancer cell proliferation. Thus, generation of a novel AR-specific aptamer-based corepressor may present a vector-free inhibition of AR-dependent prostate cancer growth as a novel approach.

Antibodies are challenged.

Studies on antibody were documented as early as in 1890. They are proteins found in blood or other body fluid of vertebrates, and are used by the immune system to identify and neutralize antigens (like foreign objects, pathogens like bacteria and virus etc). Antibodies are dominating the biomedical research field especially detection, imaging and inhibition of biological target molecules, and therapeutics so far. However, recently aptamer has been seen to compete with antibodies in all the above areas. Aptamers are single stranded oligonucleotides or peptides that fold into well defined three dimensional shapes, allowing them to bind their targets with high affinity and specificity. Aptamer technology is relatively new and discovered only in 1990. Because of synthetic origin and similar function as antibodies, they are often termed as chemical antibody. Within 25 years of discovery, the first generation of aptamer drug "Macugen" is already marketed and available for public use. The Global market for aptamer was $236 million in 2010 and is expected to be valued at nearly $1.8 billion by 2014, with a growing compound annual growth rate of 67.5%. Various drugs being on the pipeline for clinical trials this emerging field of medical biotechnology is raising significant interest. This article gives an overview how aptamers are similar yet distinctly different from antibodies in terms of synthesis, handling, and applicability.

Facile detection of specific RNA-polypeptide interactions by MALDI-TOF mass spectrometry.

A simple method for the detection of specific RNA-polypeptide interactions using MALDI-TOF mass spectroscopy is described. Instead of direct observation of the RNA-polypeptide complex, we attempted the indirect observation of the binding event by focusing on the disappearance of the free polypeptide signal upon interaction with RNA. As a result, specific binding of the Rev-response element (RRE) RNA of the HIV with two RRE-binding peptide aptamers, DLA and RLA peptides, as well as the bacteriophage lambda boxB RNA with the lambda N peptide was observed. We also show that specific RNA-binding peptides can be identified from a mixture of peptides with varying RNA-binding affinity, showing that the method could be applied to high-throughput screening from simple peptide libraries. The method described in this study provides a quick and simple method for detecting specific RNA-polypeptide interactions that avoids difficulties associated with direct observation of RNA and RNA-polypeptide complexes, which may find various applications in the analysis of RNA-polypeptide interactions and in the identification of novel RNA-binding polypeptides.

Utilization of the pleiotropy of a peptidic aptamer to fabricate heterogeneous nanodot-containing multilayer nanostructures.

Peptide aptamers (=binders) against inorganic materials often show a capacity for mineralization of their target atoms; thus they are able to function both as binding molecules and as mediators for mineralization. Although the mechanisms underlying these two properties of peptide aptamers are not yet fully understood, they have been used separately to fabricate various nanostructures. Here, we present a novel method of nanofabrication, in which binding and mineralization by a peptide aptamer are alternately utilized to assemble multilayered nanostructures comprised of metal loaded cage proteins ornamented with Ti-binding peptides.