Development of functional DNA-based sensors and investigations into their mechanism

8-17 DNAzyme;catalytic beacon sensor;temperature independent lead sensor;Fluorescence Resonance Energy Transfer (FRET);phosphorothioate modifications;in vitro selection;endotoxin;Phosphorus-31 Nuclear Magnetic Resonance (31P NMR)

Nagraj, Nandini

关键词: 8-17 DNAzyme;    catalytic beacon sensor;    temperature independent lead sensor;    Fluorescence Resonance Energy Transfer (FRET);    phosphorothioate modifications;    in vitro selection;    endotoxin;    Phosphorus-31 Nuclear Magnetic Resonance (31P NMR);   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/15538/2_Nagraj_Nandini.pdf?sequence=4&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

The discovery that nucleic acids could perform functional roles in addition to being genetic materials carriers opened doors to a new paradigm in nucleic acid chemistry. Catalytic DNA molecules also known as deoxyribozymes or DNAzymes were first isolated in 1994 through an in vitro selection procedure and have since been engineered and isolated to perform various functions that include both RNA and DNA cleavage and ligation. The 8-17 DNAzyme is an RNA-cleaving DNAzyme that has shown high selectivity for Pb2+ under different selection conditions. It has been explored extensively in terms of its applications for bio-sensing as well as for exploring its mechanism from a more fundamental perspective.A critical barrier of DNA-based sensors for practical applications, such as environmental monitoring, is their highly variable sensing performance with changing temperatures, due to the reliance of sensor design on temperature-dependent hybridization. In Chapter 2, this issue has been addressed through the introduction of mismatches in the DNA hybridization arms of this Pb2+-specific 8-17 DNAzyme and these fluorescent sensors can resist temperature-dependent variations from 4 °C to 30 °C. The strategy of using mismatches to tune the temperature dependence is a novel and inexpensive method that can be applied in other nucleic acid sensors for either metal ions or other molecular targets. Currently there is no structure, (either X-ray or NMR) available for the 8-17 DNAzyme. Hence, understanding its mechanism has posed a challenge, particularly in regard to the high selectivity of Pb2+ for this DNAzyme. In Chapter 3, the systematic activity, folding and structural studies of the 8-17 DNAzyme with both monovalent and divalent metal ions has been carried out. The results obtained suggest a clear trend between the folding and activity of all the metal ions studied, the lower the activity, the lesser the folding and vice-versa. Structural studies based on CD and folding studies based on FRET have demonstrated that Pb2+ behaves in a manner that is different from other metal ions and hence it is hypothesized that the 8-17 DNAzyme may have a specific binding pocket for Pb2+.The possibility that the 8-17 DNAzyme might have a metal ion binding pocket for Pb2+ has been investigated in Chapter 4, through systematic phosphorothioate (PS) modifications on the backbone of the DNAzyme. Kinetic assays with the PS modified bases have shown that there are specific bases on the enzyme strand which are important for activity mainly in the presence of Pb2+. The activities of the identical PS modified enzymes are, however, not significantly altered in the presence of Mg2+ and Cd2+. 31P NMR has been used as an additional tool to directly visualize the backbone phosphates since a single PS modification shifts the signal of the phosphate downfield by ~50 ppm.These results, in conjunction, have led to the identification of a proposed metal ion binding site, specifically a potential Pb2+-binding site for the 8-17 DNAzyme.The starting point towards the development of a successful functional nucleic acid-based sensor is its isolation and this is done through in vitro selection. In vitro selection to isolate DNAzymes for Hg2+ and Cd2+ and the use of negative selections to overcome Pb2+ interference at various stages of selection has been described in Chapter 5. Chapter 6 describes the structure-switching strategy to isolate DNA aptamers specific for endotoxins. It is anticipated that the results obtained from the current study and future characterizations will lead to the development of functional DNA sensors for endotoxins.