Amita Nand

Emerging technology of in situ cell free expression protein microarrays

Recently, in situ protein microarrays have been developed for large scale analysis and high throughput studies of proteins. In situ protein microarrays produce proteins directly on the solid surface from pre-arrayed DNA or RNA. The advances in in situ protein microarrays are exemplified by the ease of cDNA cloning and cell free protein expression. These technologies can evaluate, validate and monitor protein in a cost effective manner and address the issue of a high quality protein supply to use in the array. Here we review the importance of recently employed methods: PISA (protein in situ array), DAPA (DNA array to protein array), NAPPA (nucleic acid programmable protein array) and TUSTER microarrays and the role of these methods in proteomics.

Detection of embryonic stem cell lysate biomarkers by surface plasmon resonance with reduced nonspecific adsorption

Surface plasmon resonance imaging (SPRi) has emerged as a versatile biosensor to detect a wide range of biomolecular interactions with divergent potential applications. However, the use of this advanced-level technology for stem cell lysate study is still not much explored. Cell lysates are significant biological analytes used for disease diagnostics and proteomic studies, but their complex nature limits their use as an analyte for SPRi biosensors. Here, we review the problems associated with the use of SPRi for stem cell lysate study and examine the role of surface chemistry, running buffer, and blocking solution in order to minimize nonspecific adsorption (NSA). We detect the expression of Oct4, Sox2, Nanog, Rex1, and Lin28 biomarkers present in mouse embryonic stem cell (mESC) lysate against their corresponding antibodies immobilized on the sensor surface with reduced NSA. The current study shows that the conjunction of SPRi and microarray can be used as a label-free, high-throughput, and rapid technique for detection of biomarkers and their relative abundance in stem cell lysate study.

Glycoprotein profiling of stem cells using lectin microarray based on surface plasmon resonance imaging.

Lectin microarrays have emerged as a novel platform for glycan analysis during recent years. Here, we have combined surface plasmon resonance imaging (SPRi) with the lectin microarray for rapid and label-free profiling of stem cells. In this direction, 40 lectins from seven different glyco-binding motifs and three different cell lines-mouse embryonic stem cells (mESCs), mouse-induced pluripotent stem cells (miPSCs), and mouse embryonic fibroblast stem cells (MEFs)-were used. Pluripotent mouse stem cells were clearly distinguished from non-pluripotent stem cells. Eight lectins-DBA, MAL, PHA_E, PHA_L, EEL, AAL, PNA, and SNA-generated maximal value to define pluripotency of mouse stem cells in our experiments. The discriminant function based on lectin reactivities was highly accurate for the determination of stem cell pluripotency. These results suggested that glycomic analysis of stem cells leads to a novel comprehensive approach for quality control in cell-based therapy and regenerative medicine.

In situ protein microarrays capable of real-time kinetics analysis based on surface plasmon resonance imaging.

In recent years, in situ protein synthesis microarray technologies have enabled protein microarrays to be created on demand just before they are needed. In this paper, we utilized the TUS-TER immobilization technology to allow label-free detection with real-time kinetics of protein-protein interactions using surface plasmon resonance imaging (SPRi). We constructed an expression-ready plasmid DNA with a C-terminal TUS fusion tag to directionally immobilize the in situ synthesized recombinant proteins onto the surface of the biosensor. The expression plasmid was immobilized on the polyethylene imine-modified gold surface, which was then coupled with a cell-free expression system on the flow cell of the SPRi instrument. The expressed TUS fusion proteins bind on the surface via the immobilized TER DNA sequence with high affinity (∼3-7×10(-13)M). The expression and immobilization of the recombinant in situ expressed proteins were confirmed by probing with specific antibodies. The present study shows a new low cost method for in situ protein expression microarrays that has the potential to study the kinetics of protein-protein interactions. These protein microarrays can be created on demand without the problems of stability associated with protein arrays used in the drug discovery and biomarker discovery fields.

High-throughput measurement of drug–cyclodextrin kinetic rate constants by a small molecule microarray using surface plasmon resonance imaging

Applications of small molecule microarrays (SMMs) has been extensively studied but have been limited to the screening of small molecule inhibitors. Here for the first time, we conjugated SMMs with label free surface plasmon resonance imaging (SPRi) for measurement of kinetic parameters for drug–cyclodextrins interactions in high-throughput manner. A collection of insoluble drugs was immobilized onto biosensor surface using photo-cross-linked technique to form SMMs. A highly sensitive and recently reported surface chemistry based on surface initiated polymerization chemistry was used for SMMs fabrication. In total, 38 insoluble drugs were evaluated for their interaction profile and kinetic rate constants against 5 different types of cyclodextrins (CDs) including, α-CD, β-CD, γ-CD, 2-hydroxylpropyl β-CD (HP-β-CD) and sulphobutyl-ether-β-CD (SBE-β-CD). For the supramolecular drug–CD interaction kinetics, the response magnitude and detailed kinetic parameters were calculated and presented in the article. The presented method described a label free and high-throughput technique for real time measurement of kinetic constants for drug–CDs interactions which will assist the selection and use of different CDs in number of different applications.

Universal screening platform using three-dimensional small molecule microarray based on surface plasmon resonance imaging

Although much progress has been made in the field of small molecule microarrays in the past decades, its potential has been limited by a lack of efficient methodology. Herein we report a potent methodology for drug screening on a three-dimensional (3D) surface using a carbene based photo-cross-linking reaction. The simultaneous display of a large number of small molecules on a single polymer chain in various orientations allows for the retention of their activity. The presented method was tested using high throughput surface plasmon resonance (SPRi) with the immunosuppressive drugs rapamycin and FK506. We showed that rapamycin and FK506 immobilized on the 3D surface, not the conventional 2D surface, bound to the FKBP with high affinity. Using FKBP-binding ligands and FKBP mutants with altered mutual binding affinities, we observed a strong correlation between the relative binding affinities determined by SPRi and those previously reported. In addition, other important parameters including blocking, washing, robustness and surface reproducibility were also validated. Some well known kinase inhibitors of p38α, JNK and EKR2 proteins were also used to extend the applications of the method. All together, these results suggested that the newly developed 3D small molecule microarray in conjunction with SPRi can be a powerful platform for high throughput drug screening.

Echinomycin, a Potential Binder of FKBP12, Shows Minor Effect on Calcineurin Activity

Echinomycin, a member of the quinoxaline family of antibiotics, is known to be a small-molecule inhibitor of hypoxia inducible factor–1 (HIF-1) DNA binding activity. Recently, it has been shown to suppress mammalian target of rapamycin (mTOR) signaling and growth in leukemia cell lines. In this study, we investigated whether echinomycin interacts with the FKBP12 protein. Molecular docking was used, and the predicted binding energy was −10.61 kcal/mol. Moreover, surface plasmon resonance imaging and fluorescence quenching techniques were used to validate this interaction. Echinomycin binds to FKBP12 with a strong binding affinity comparable with rapamycin. Furthermore, the echinomycin-FKBP12 complex has been shown to affect calcineurin activity when tested in a calcineurin phosphatase inhibition assay. All of these studies have shown that echinomycin may have a double impact on HIF signaling by direct inhibition and through mTOR.