Studies On The Media Regulation And Enzymetic Amplification Of DNA Hybridization Signal

DNA sequence determines the characteristics of DNA molecule. The change of DNA sequence affects not only the coding for a specific polypeptide, but also the coding for RNA molecules such as ribosomal RNA and transfer RNA, thus resulting in individual differences in human being. About 90% of human DNA polymorphism is single nucleotide polymorphism (SNP). SNP is single base pair position at which different sequence alternatives (alleles) exist in normal individuals in some population(s) and has close relationship with genetic diseases and drug sensitivity. Therefore, it is very important to detect SNP in clinical diagnosis and therapy.Most DNA assays are based on DNA hybridization reacted in aqueous solution or solid-supported system. Compared with solid-supported DNA hybridization, homogeneous reactions are fitter for automation because no separation or purification is needed after the allele discrimination reaction. However, Most of the homogeneous SNP typing methods need the label of fluorephore or radioactive isotope, which makes them expensive, special-apparatus-depending and time-consuming, thereby often beyond the reach of clinical laboratory.The thesis aims to develop fast and easy SNP assays in homogeneous solution which can be used routinely in disease diagnosis and therapy in clinics.1. Using a Cyanine Dye to Transmit DNA Hybridization Signal to Visible AbsorbanceIntercalating fluorescent dyes have been used to transmit dsDNA signal because of their high affinity for and large fluorescence enhancement upon binding dsDNA, but special optical apparatus is needed. The cyanine dyes are also able to recognize dsDNA structures by intercalation, exterior stacking, major groove binding, and minor groove binding. With the specific binding of cyanine dyes, DNA hybridization signal can be transmitted into visible absorbance, thus realizing easy and fast assay using ordinary spectrophotometer.A kind of cyanine dye,3,3'-diethylthiatricarbocyanine iodide (DTTC) was used to transmit the DNA hybridization signal to visible absorbance of the cyanine dye based on the specific binding interaction of duplex DNA and DTTC. The result indicated that, when binding with DNA duplex, the cyanine dye produced two absorbing peaks, in which the absorbance peak at 760nm resulted from intercalation binding mode, while the other absorbance peak at 675 nm due to groove binding mode. The absorbance at 760nm and its stability were influenced by the length of the hybrids and the experimental temperature. When the temperature was set at 25℃and 24-mer oligonucleotide was used as the probe, target DNA with concentration being as low as 1μg·mL-1 could be detected, thus realizing a more sensitive assay with the sensitivity being two orders lower than that of DNA at 260nm. Under appropriate conditions, the cyanine dye could be used not only to detect target single stranded DNA, but also to recognize SNP in target DNA fragments. Compared with other target DNA or SNP-typing assays, the present assay had advantages in simplicity and rapidity, and had an applicable potential in clinical diagnosis.2. Monitoring DNA Hybridization by UV spectra in AOT reverse micellesDNA hybridization could be monitored by measuring UV absorbance at 260 nm. But when DNA hybridized in aqueous solution at a high concentration, the absorbance would be too high to be detected. While DNA hybridized at a low concentration, the change of the absorbance at 260 nm could not be detected either.Reverse micelles, which are formulated by surfactant molecules in organic solvents, provide nanostructural water pools which can dissolve DNA at rather high concentration but the whole concentration is still low. Reverse micelles decrease DNA hybridization rate and the stability of dsDNA, thus amplify the differences between fully matched and partially matched DNA targets. Goto et al reported a 20-mer DNA hybridization in reverse micelles, but results showed the assay was time consuming.Using a 24-mer oligonucleotide as a model, we studied the influence of AOT reverse micelles on DNA hybridization rate and the methods to amplify the signal differences among different complementary DNA fragments. In order to increase DNA hybridization rate in reverse micelles, we separated the two phases by freezing the reverse micelles.After the freeze of AOT reverse micelles, DNA remained in organic solvent, but the bulk of water decreased deeply. Because fully complementary dsDNA folded more closely than those partially complementary dsDNAs, after the freezing, the water content in fully complementary dsDNA system was much lower than those partially complementary dsDNAs. Thus by measuring water content, a mutation in target DNA fragment could also be recognized.3. Transmit DNA Hybridization Signal to Catalytic Activity of an EnzymeTo develop a feasibly low cost SNP assay in a homogeneous solution, we designed a DNA probe, which had a thrombin-inhibiting aptamer domain and a stem-and-loop structure (like a molecular beacon). In this way, the hybridization signal produced at the loop could be switched directly to the signal of thrombin activity. The stem-and-loop structure was carefully designed so that under appropriate hybridization conditions, different target DNAs with sequences either fully or partially complementary to the loop sequence resulted in different degrees of the damage of the aptamer domain on the probe. The conformational changes of the probe after its hybridization with target DNAs were confirmed by CD spectra and FRET studies. Under selected conditions, different target DNAs resulted in obvious differences in thrombin activity toward fibrinogen, thereby, realizing an easy discrimination of SNPs using routinely clinical fibrinogen clotting assay. Compared with other SNP-typing assays, the present assay has advantages in simplicity and rapidity, and has potential application in clinical diagnosis.4 Binding effects of aptamers on thrombinOn the surface of thrombin, there exist two positively charged binding sites-exosite I and exosite II. The two exosites facilitate thrombin to bind various negatively charged substrates or ligands so as to regulate the different role of thrombin. Many reports have been released on the recognition mechanism of thrombin for its natural substrates or ligands through the two exosites, but there is a controversy on the allostery of thrombin during the exositeⅠand/or exositeⅡbinding(s).Crystallographic study on thrombin-thrombomodulin complex indicated no change of the conformation of thrombin's active site. Study on thrombin-fibrinogen crystal did not provide any information about the allosteric effect of fibrinogen on thrombin. However, some studies showed that the binding of TM fragment, hirudin fragment or other ligands to exositeⅠand exositeⅡproduced allosteric changes. ExositesⅠandⅡwere reported to be closely linked allosterically, i.e., binding of a ligand to one exosite resulted in nearly total loss of affinity of the other exosite for ligands, but other studies supported the independence of the interactions. The contradictory results may be due to the following facts:Firstly, thrombin and its macromolecular substrates or ligands lost flexibility in crystal, so the slight allosteric change of thrombin may be undetectable. Secondly, the binding ligands used were usually from thrombin's natural substrates, inhibitors, or the fragments of the substrates or their derivatives. Their binding to one exosite may affect the binding of other ligands to another exosite.For thrombin, two kinds of aptamers are obtained. One binds to exositeⅠ, the other binds to exositeⅡ. Because the two aptamers are non-protein, soluble, and specific for binding exositeⅠorⅡ, the conformation of thrombin with exosites empty or occupied by one or two aptamer(s) can be observed in aqueous solution using CD spectra and intrinsic fluorescence without any modifications or marks of thrombin and/or the aptamers. In addition, the aptamers have no direct linkage with the active site of thrombin. The use of small chromogenic substrate, (3-Ala-Gly-Arg-p-nitroanilide diacetate, which is cleaved by thrombin at a slow rate, for the determination of the hydrolysis activity of thrombin also facilitated the present study, making the phenomenon straightforward and obvious.CD and intrinsic fluorescence spectra indicated that after binding with aptamers the secondary structure of thrombin seemed unchanged, but the whole conformation of thrombin changed. The binding of aptamers on thrombin also made the catalytic activity of thrombin toward the chromogenic substrate (β-Ala-Gly-Arg-p-nitroanilide diacetate) increased. The present study indicated that the allostery of the two exosites seemed to be independent.5 Sensitive thrombin detection based on the interaction between aptamers and thrombinBased on the interaction between aptamers and thrombin, an attempt was made to develop a nanoporous gold (NPG)-based electrochemical aptasensor for thrombin detection. The substrate electrode NPG was in situ fabricated by a facile one-step square wave potential pulse (SWPP) treatment. The treatment involved repeated gold oxidation-reduction and intensive H2 bubbles evolution. After 100 min treatment, the active surface area of Au increased greatly (34 times). The electrochemical aptasensor was fabricated using a layer-by-layer assembling strategy. A "sandwich" structure was formed via thrombin connecting the aptamer-modified NPG and the aptamer-modified gold nanoparticles (GNPs). The AuNPs was modified with two kinds of single strand DNA (ssDNA). One was aptamer of thrombin, but the other was not, reducing the crossreaction between thrombin and its aptamer on the same GNP. The electrochemical signal produced by the [Ru(NH3)6]3+ bound to ssDNA via electrostatic interaction was measured by chronocoulometry. Due to the amplification effects of both NPG and GNPs, this novel NPG-based aptasensor could detect thrombin quantitatively in the range of 0.01-22nmol/L with a detection limit as low as 30 fmol/L. The present aptasensor also exhibited excellent selectivity, stability and reusability.