Spectroscopic study of restriction deoxyribozyme: Structural and activity survey

The Cu dependent restriction deoxyribozyme is the unique example of known deoxyribozymes. The uniqueness of this deoxyribozyme is originated from specific cleaving of single stranded DNA and formation of triple helix DNA structure which is necessary for substrate recognition and binding. The most established method for measuring the kinetic parameters of deoxyribozyme is based on use of radiolabeled substrates which have several difficulties. In this study we present accurate, fast and inexpensive methods for kinetic study of the deoxyribozyme which is based on extrinsic fluorescence and UV-visible spectroscopy techniques. As mentioned above, DNA triple helix formation is necessary for substrate identification and also enzyme activity. Circular dichroism spetropolarimetery is used for structural study of enzyme. Analysis of spectrum results from this technique indicates structural changes which is a direct evidence for the triple helix formation in enzyme-substrate complex. Extrinsic fluorescence experiment is based on high affinity of SYBR gold to double stranded DNA compared to single stranded DNA. Enzyme activity can be measured by SYBR gold fluorescence emission upon addition of cofactor to the enzyme-substrate complex. Continuous hyperchromicity assay method which is based on UV-visible spectroscopy was used for measuring of enzyme activity by hyperchromicity of the enzyme-substrate complex after addition of cofactor. Comparison of the results show that the continuous hyperchromicity assay is more accurate than the extrinsic fluorescence method, because of this method is based on intrinsic physicochemical properties of DNA without interference of external factors.