I:Construction Of Protein Microarray Platform And TORCH Antigen Microarray II: A Movel MicroRNA Profiling Microarray III:Conformation And Unfolding Of Leucyl-tRNA Synthetase Of Aquifex Aeolicus

Part I: Construction of protein microarray platformand TORCH antigen microarrayProtein microarray is an important tool of proteomics research. It is made byarraying a large scale of trace amounts of protein onto activated glass slides withrobot in an addressable manner. It implements analyzation of protein compounds insample with a miniaturized, rapid, high throughput and parellel manner. Inconstruction of protein microarray platform, the present study involves making of alow cost biochip arrayer based on computer-controlled plotter, developing a methodof making surface-activated glass slides substrate, developing a colorimetric proteinmicroarray detection method. These present an effective solution for common lab tomake and use protein microarray without expensive instruments. As a demonstrationof protein microarray application, a TORCH antigen chip was developed for diagnosisof prenatal infections that cause congenital anomalies. The antigen chip can detectconcentrations of antibodies to 8 antigens in one serum simultaneously, candifferentiate kinds of positive and negative sera, and has good reproducibility.Part II: A novel microRNA profiling microarraymicroRNA (miRNA) is a large class of small noncoding RNA that was foundvery important in biological function recently. Profiling of miRNA expression wasmostly depend on Northern blot, it was work and time consuming. Based on the abovemicroarray technology platform, a novel miRNA expression profiling microarray wasdeveloped using the diol group of 3' terminal of miRNA and quantum dot which is anovel fluorescent probe with very good fluorescence properties. miRNAs wereoxidized with periodate into dialdehyde form and biotinylated withbiotin-X-hydrazide before hybridization with the oligonucleotide microarray andsubsequently detected with streptavidin-conjugated quantum dots. The detection limitof this microarray for miRNA was 0.4 fmol, and the detection dynamic range spannedabout 2 orders of magnitude. A model microarray to profile 11 miRNAs from leaf androot of rice (Oryzasativa L. ssp. indica) seedlings was made. The analysis results ofthe miRNAs had a good reproducibility and were consistent with the northern blotresult. To avoid using high-cost detection equipment, colorimetric detection, a methodbased on nanogold probe coupled with silver enhancement, was also successfullyintroduced into miRNA profiling microarray detection.Part III: Conformation and unfolding of leucyl-tRNA synthetaseof Aquifex aeolicusWe studied the conformation and unfolding of leucyl-tRNA synthetase(αβ-LeuRS) from hyperthermophilic bacteria Aquifex aeolicus induced byguanidinium hydrochloride (GuaHCl) and high hydrostatic pressure, monitored withfluorescence spectroscopy and 4-th derivative UV absorption spectroscopy.Tryptophan residues in αβ-LeuRS and β-subunit were both found to be in a relativelyhydrophilic microenvironment. They both have two hydrophobic bis-ANS bindingsite. Thermodynamic experiments showed that the β-subunit could be fully unfoldedby 3-4 M of GuaHCl. Unfolding dynamics showed that unfolding of β-subunitinduced by GuaHCl was a fast tristate process, while the unfolding process ofαβ-LeuRS was very slow and complicate. The β-subunit was found to be fullyunfolded induced by pressure of 260 MPa in presence of 2 M of GuaHCl. The volumechange was found to be decreasing with temperature increasing. It was found thatNaCl, glycerol and sucrose significantly stabilize the protein from unfolding and thestabilization is associated not only with an increase in the free energy but also with adecrease in volume change.