Discrimination of Alternative Spliced Isoforms by Real-Time PCR Using Locked Nucleic Acid (LNA) Substituted Primer

Determination of quantitative expression levels of alternatively spliced isoforms provides an important approach to the understanding of the functional significance of each isoform. Real-time PCR using exon junction overlapping primers has been shown to allow specific detection of each isoform. However, this design often suffers from severe cross amplification of sequences with high homology at the exon junctions. We used human GFRα2b as a model to evaluate the specificity of primers substituted with locked nucleic acids (LNAs). We demonstrate here that single LNA substitutions at different positions of 3’ terminus could improve the discrimination of the primers against GFRα2a template, a highly homologous isoform. While LNA substitutions of GFRα2b primer at the residues possessing different sequences as GFRα2a has limited improvement in specificity, two consecutive LNA substitutions preceding the different sequences has dramatically improved the discrimination by greater than 100,000-fold compared to the non-substituted primer. Thus, LNA when substituted at certain residues can allow the discrimination of highly homologous sequences.Singapore-MIT Alliance (SMA

Study of GDNF-Family Receptor Alpha 2 And Inhibitory Activity of GDNF-Family Receptor Alpha 2b (GFRα2b) Isoform

The glial cell-line derived neurotrophic factor (GDNF) and neurturin (NTN) belong to a structurally related family of neurotrophic factors. NTN exerts its effect through a multi-component receptor system consisting of the GDNF family receptor alpha 2 (GFRα2), proto-oncogene RET and/or NCAM. GFRα2 is spliced into at least three isoforms, GFRα2a, GFRα2b and GFRα2c. The present study investigated the expression and functional differences of GFRα2 isoforms. These receptor isoforms are differentially expressed in specific human brain regions. Using Neuro2A model, GDNF and NTN promote neurite outgrowth via GFRα2a and GFRα2c, but not GFRα2b. These GFRα2 isoforms regulate different early response genes when stimulated with GDNF and NTN. Interestingly, using co-expression models, GFRα2b inhibits ligand induced neurites outgrowth of GFRα2a and GFRα2c, and also the related receptor, GFRα1a. More intriguingly, ligands activated GFRα2b was also able to attenuate neurite extension induced by an unrelated stimulation using retinoic acid. MAPK activation induced by GDNF was not attenuated by GFRα2b in a co-expression model, while the early response genes expression profile (up-regulation of FosB) was similar to that induced by GFRα2b alone. This study suggest that GFRα2b is not merely a dominant negative isoform, but signals through a yet to be determined mechanism to antagonize and inhibit neuritogenesis. Together, these data suggest a new paradigm for the regulation of growth factor signaling and neurite outgrowth via an inhibitory splice variant of the receptor.Singapore-MIT Alliance (SMA

Single Stranded DNA Induced Assembly of Gold Nanoparticles

The binding affinity of single stranded DNA (ssDNA) for gold nanoparticle surface is studied in this work. The data indicate that the strength of interaction between ssDNA and Au particle surface is closely related to the particle size, with smaller particles (5 nm) producing the most pronounced effects. From these experimental findings, a single stranded DNA (ssDNA) based method to assimilate 13 and 5 nm gold nanoparticles was developed, and verified by transmission electron microscopy (TEM).Singapore-MIT Alliance (SMA

Multivariate Perturbation of a Growth Factor-Cytokine Signalling Network Reveals Complex Systemic Responses in Glioblastoma Cells

Glioblastoma cells can evade TRAIL-induced apoptosis through various strategies involving the growth factor-activated MEK–MAPK/ERK and PI3K–Akt/PKB pro-survival signalling cascades. Although these signalling cascades have been studied extensively, our understanding of how they interact and participate in modulating apoptosis as part of a dynamic cell-wide network of signalling proteins is limited by traditional univariate experimental paradigms. Here, we study three human glioblastoma cell lines with differential response to TRAIL-induced apoptosis: LN229 (resistant), T98G, and A172 (both susceptible). We show that differential TRAIL susceptibility in these cell lines is unrelated to expression levels of agonist (DR4 and DR5) or antagonist (DcR1, DcR2, and OPG) receptors for TRAIL and thus TRAIL-induced apoptosis in these cell lines is modulated at the intracellular signalling level. Serum, comprising multiple factors that regulate cellular activity, enhances TRAIL resistance in T98G but not LN229 and A172 cell lines. This protective effect against TRAIL-induced apoptosis is recapitulated by the prototypical survival factor PDGF in T98G cells. Univariate inhibition of cell survival signalling cascades with MEK inhibitor U0126 and PI3K inhibitor LY294002 sensitized T98G cells to TRAIL but did not abrogate PDGF-mediated protection. However, further perturbation with inhibitors in a combinatorial and multivariate manner reveal synergistic effects and complex systemic responses which may be a basis for uncovering novel insights into the regulation of TRAIL-induced apoptosis.Singapore-MIT Alliance (SMA

Experimental design-aided systematic pathway optimization of glucose uptake and deoxyxylulose phosphate pathway for improved amorphadiene production

Artemisinin is a potent antimalarial drug; however, it suffers from unstable and insufficient supply from plant source. Here, we established a novel multivariate-modular approach based on experimental design for systematic pathway optimization that succeeded in improving the production of amorphadiene (AD), the precursor of artemisinin, in Escherichia coli. It was initially found that the AD production was limited by the imbalance of glyceraldehyde 3-phosphate (GAP) and pyruvate (PYR), the two precursors of the 1-deoxy-d-xylulose-5-phosphate (DXP) pathway. Furthermore, it was identified that GAP and PYR could be balanced by replacing the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) with the ATP-dependent galactose permease and glucose kinase system (GGS) and this resulted in fivefold increase in AD titer (11 to 60 mg/L). Subsequently, the experimental design-aided systematic pathway optimization (EDASPO) method was applied to systematically optimize the transcriptional expressions of eight critical genes in the glucose uptake and the DXP and AD synthesis pathways. These genes were classified into four modules and simultaneously controlled by T7 promoter or its variants. A regression model was generated using the four-module experimental data and predicted the optimal expression ratios among these modules, resulting in another threefold increase in AD titer (60 to 201 mg/L). This EDASPO method may be useful for the optimization of other pathways and products beyond the scope of this study.Singapore-MIT Alliance for Research and Technology (SMART

Profiling of ligand-receptor induced signalling- a novel protein chip technique

Cellular signalling pathways are the master controls of the biology of the cell, which includes cell communication, growth, death, and differentiation. The activities of these signalling proteins directly influence gene function by regulation of the signalling pathways that mediate cellular responses. Recent advanced techniques have given rise to a number of emerging tools for the analysis of cellular signalling that profile the proteome or the protein complement of the genome. However, these tools for signal profiling still face significant challenges such as sensitivity, specificity and be a high throughput method before they are widely adopted. Sensitivity issues are paramount in detecting signalling proteins that are normally in low amounts. Conventional protein chip technology promises to be a powerful tool for large scale high-throughput proteome profiling but there are still significant drawbacks. Here we report the development and application of a novel multiplexed and high-throughput platform for the quantitative profiling of activated intracellular sig nalling proteins subsequent to ligand-receptor induced signalling. This spatially addressable biochip platform will allow comprehensive mapping of interconnected signal pathways, through identification of key functional signalling proteins (ânodes’) in each pathway and quantifying their state of activity.Singapore-MIT Alliance (SMA

Correlating Gene Transfection Efficiency and the Physical Properties of Various Cationic Poly(methacrylate) Systems

Transfection efficiencies of several polymeric gene carriers were compared and correlated quantitatively to the amounts of cellular accumulation of plasmid DNA and to the expression of mRNA by quantitative real time PCR. Three cationic methacrylate polymer systems with similar chemical structure were used in this study, namely: poly(dimethylamino)ethyl methacrylate (PDMA) homopolymer, PEO-b-PDMA copolymer and PEO-b-poly(diethylamino)ethyl methacrylate (PEO-b-PDEA) copolymer. Despite their similar chemical structures, their transfection efficiencies were significantly different. PEO-b-PDEA copolymer was significantly less efficient as gene carrier compared to both PDMA and PEO-b-PDMA systems. Results from quantitative real-time polymerase chain reaction (real-time PCR), cytotoxicity and Zeta potential measurements showed correlations between the physical properties of the polymers and the efficiencies of cellular uptake of the transgene and transfections. In the case of PEO-b-PDEA system, cytotoxicity was due primarily to the excess polymers that did not participate in the DNA binding. In addition, the inability of the polymer/DNA complexes to interact with cell effectively was identified as the main barrier for high efficiency of transfection. This study demonstrated that the use of quantitative real-time PCR in combination with other physical characterization techniques can provide greater insights into the transfection barrier at different cellular levels.Singapore-MIT Alliance (SMA

Core-Shell Assisted Bimetallic Assembly of Pt and Ru Nanoparticles by DNA Hybridization

We have discovered that the current protocols to assemble Au nanoparticles based on DNA hybridization do not work well with the small metal nanoparticles (e.g. 5 nm Au, 3.6 nm Pt and 3.2 nm Ru particles). Further investigations revealed the presence of strong interaction between the oligonucleotide backbone and the surface of the small metal nanoparticles. The oligonucleotides in this case are recumbent on the particle surface and are therefore not optimally oriented for hybridization. The nonspecific adsorption of oligonucleotides on small metal nanoparticles must be overcome before DNA hybridization can be accepted as a general assembly method. Two methods have been suggested as possible solutions to this problem. One is based on the use of stabilizer molecules which compete with the oligonucleotides for adsorption on the metal nanoparticle surface. Unfortunately, the reported success of this approach in small Au nanoparticles (using K₂BSPP) and Au films (using 6-mercapto-1-hexanol) could not be extended to the assembly of Pt and Ru nanoparticles by DNA hybridization. The second approach is to simply use larger metal particles. Indeed most reports on the DNA hybridization induced assembly of Au nanoparticles have made use of relatively large particles (>10 nm), hinting at a weaker non-specific interaction between the oligonucleotides and large Au nanoparticles. However, most current methods of nanoparticle synthesis are optimized to produce metal nanoparticles only within a narrow size range. We find that core-shell nanoparticles formed by the seeded growth method may be used to artificially enlarge the size of the metal particles to reduce the nonspecific binding of oligonucleotides. We demonstrate herein a core-shell assisted growth method to assemble Pt and Ru nanoparticles by DNA hybridization. This method involves firstly synthesizing approximately 16 nm core-shell Ag-Pt and 21 nm core-shell Au-Ru nanoparticles from 9.6 nm Ag seeds and 17.2 nm Au seeds respectively by the seed-mediated growth method. The core-shell nanoparticles were then functionalized by complementary thiolated oligonucleotides followed by aging in 0.2 M PBS buffer for 6 hours. The DNA hybridization induced bimetallic assembly of Pt and Ru nanoparticles could then be carried out in 0.3 M PBS buffer for 10 hours.Singapore-MIT Alliance (SMA

Protein Microarray: "Theory" to "Real Practice"

Fueled by ever-growing genomic information and rapid developments of proteomics–the large scale analysis of proteins and mapping its functional role has become one of the most important disciplines for characterizing complex cell function. For building functional linkages between the biomolecules, and for providing insight into the mechanisms of biological processes, last decade witnessed the exploration of combinatorial and chip technology for the detection of bimolecules in a high throughput and spatially addressable fashion. Among the various techniques developed, the protein chip technology has been rapid. Recently we demonstrated a new platform called “Spacially addressable protein array” (SAPA) to profile the ligand receptor interactions. To optimize the platform, the present study investigated various parameters such as the surface chemistry and role of additives for achieving high density and high-throughput detection with minimal nonspecific protein adsorption. In summary the present poster will address some of the critical challenges in protein micro array technology and the process of fine tuning to achieve the optimum system for solving real biological problems.Singapore-MIT Alliance (SMA

Association Behavior of Biotinylated and Non-Biotinylated PolyEthylene Oxide-b-Poly(2-(Diethylamino)Ethyl Methacrylate)

Biotinylated and non-biotinylated copolymers of ethylene oxide (EO) and 2-(diethylamino)ethyl methacrylate (DEAEMA) were synthesized by the atom transfer radical polymerization technique (ATRP). The chemical compositions of the copolymers as determined by NMR are represented by PEO₁₁₃PDEAEMA₇₀ and biotin-PEO₁₀₄PDEAEMA₉₃ respectively. The aggregation behavior of these polymers in aqueous solutions at different pHs and ionic strengths was studied using a combination of potentiometric titration, dynamic light scattering (DLS), static light scattering (SLS), and transmission electron microscopy (TEM). Both PEO-b-PDEAEMA and biotin-PEO-b-PDEAEMA diblock copolymers form micelles at high pH with hydrodynamic radii (Rh) of about 19 and 23 nm, respectively. At low pH, the copolymers are dispersed as unimers in solution with Rh of about 6-7 nm. However, at a physiological salt concentration (cs) of about 0.16M NaCl and a pH of 7-8, the copolymers form large loosely packed Guassian chains, which were not present at the low cs of 0.001M NaCl. The critical micelle concentrations (CMC) and the cytotoxicity of the copolymers were investigated to determine a suitable polymer concentration range for future biological applications. Both PEO-b-PDEAEMA and biotin-PEO-b-PDEAEMA diblock copolymers possess identical CMC values of about 0.0023 mg/g, while the cytotoxicity test indicated that the copolymers are not toxic up to 0.05mg/g (> 83% cell survival at this concentration).Singapore-MIT Alliance (SMA