Study On Isomerization Of A Deoxyribozyme Depend On Temperature

Deoxyribozyme (DNAzyme or DNA enzyme)or Cataltic DNA, which is discovered in recent years, is known as a kind of small molecular enzymatic DNA. It can catalyze the point pyrolysis activity of RNA molecule. Deoxyribozyme can be achieved in vitro ,because it is not exist in nature. The discovery of Deoxyribozyme is another important discovery after the dicovery of RNA ribozyme. After Breaker and Cuenoud reported the sequence and function of Deoxyribozyme ,studies on the structure and function of DZ become a hot issue in molecule engineering and new medicine development .because it has so much advantages, such as small molecular, stability of structure, high efficiency in catalyzing and easily to compose and decorate. Deoxyribozyme 10-23 has high catalytic activity and simple structure; it can neatly and devotedly identify the substrate and then cut it efficiently. The RNA cleaving deoxyribozymes provide tremendous potential application such as molecular therapy, molecular-scale computation, biological sensors and RNA analytic tools. In the past ten years, the study on structure, function and application of Deoxyribozyme becomes more and more hot .The activity center region of deoxyribozyme 10-23 is 10-23 motif which is a annular core region composed of fifteen deoxyribonucleotide. In this region, the eighth base can be replaced by T,C or A,but T has the highest activity ,and the rest sequences are highly conservative. The conservatism is necessary for the catalytic reaction of deoxyribozyme. Deoxyribozyme10-23 is extensively used in molecular therapy, molecular computer, biosensor, and RNA analysis tool. The structure of the highly conservative annular core region will influence the catalytic activity. When the conservative basic group is replaced by the Watson-Crick synapsis appears in the annular structure, the deoxyribozyme will lose activity. The study on the structure and function of DNA will help us to learn the essence of life on molecular level. And then, it is possible to find effective way to cure disease through molecular designing. In recent years, study on the reciprocity between micromolecule and macromolecule is becoming a active field . But, the study on the changes in the structure of DNA is not researched. So ,in this paper we studied the relationship between structure and activity of ribozyme by studying the stem-loop structure of the deoxyribozyme 10-23,and solved the major problems.Nowadays, the methods to study the structure of DNA includes:X-ray diffraction, NMR, UV spectrum, circular dichroism spectra (CD),IR spectrum, Raman scattering and thermoanalysis of DNA and so on. In this experiments we adopt thermoanalysis, CD and Raman spectrum to analysis the structure of stem-loop of Deoxyribozyme. In this experiment, the surface enhanced Raman scattering (SERS) which has higher sensitivity than traditional Raman scattering is used ,it can realize the detection of small dose samples, and it can detect the samples in aqueous solution without destroy the structure of the samples. SERS has a great sensitivity which can detect in unimolecule level. In aqueous solution, the detectability can reach 10-14M. It can be observed that the sample achieve the fluorescence quenching by explore the energy transformation between molecule and base in water-base or saline-base. It will avoid the disturbance of fluorescence by the sample itself or impurities. Also, we can get Raman scattering with clear fluorescence substance. Based on the superiority of surfaced enhanced Raman scattering (SERS), we choose this technique as main method to study the structure of stem-loop Deoxyribozyme SLD 6-8 which was constructed already.In this experiment, first, we used differential scanning calorimetry (DSC) to analyze the SLD6-8. We found a kind of isomerism that there are two different conformations, the structure of SLD6-8 changed when we change the temperature. And the thermal denaturation temperatures of the two structures are 45℃and 60℃. Along with the changing of temperature of the sample and the extending of soaking time, we can get preferential conformation of higher purity at the designated temperature. In order to learn the insomerism more intuitively, basisd on the minimum free energy, we choose RNAfold to calculate the existence forms of the conformations at different temperatures. We found there are some great differences between the two structures: the conformation which formed at 10℃has more stem-loop structures and it is more complex. By detecting the cutting efficiency through the method of polyacrylamide gel electrophoresis (PAGE) autoradiograph under the physiological condition, we found that the enzyme activities in both two structures are similar. The reasons are as follows: (1) the effective length of substrate specificity arm of the ribozyme and substrate is limited; just few bases are enough to combine with substrate; cutting could be achieved after the stem-loop structure was divided. (2) the catalytic core of the SLD can be shortened; the catalytic reaction can be achieved depends on ten core regions of the catalytic activity of base. (3) the low temperature conformation is a dynamic balance of itself which will be destroyed when it close to substrate to form the Watson-crick current; the stem-loop structure will be broken and combined with the substrate to catalyze the process of the reaction. At last, we approved the existence of the two different structures of SLD6-8 which was treated with different temperatures by using the means include the circular dichroism spectra and the surface enhanced Raman scattering (SERS). In this paper, we studied the isomerization of SLD and initially discuss the relationship between stem-loop and deoxyribozyme catalytic activity. We solved the problem that the traditional Raman can not test the signal of low consistency nucleic acid simples in aqueous solution. This breakthrough have a great significance in studying the relationship between the structure and the function of ribozyme.