In vitro selection of monovalent metal ion-dependent DNAzymes

Enzymatic DNA, DNAzyme, DNA enzyme, in vitro selection, deoxyribozyme, lithium sensor, potassium sensor

McGhee, Claire Elizabeth

关键词: Enzymatic DNA, DNAzyme, DNA enzyme, in vitro selection, deoxyribozyme, lithium sensor, potassium sensor;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/101361/MCGHEE-DISSERTATION-2018.pdf?sequence=1&isAllowed=y

美国|英语

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

【 摘 要 】

DNA is no longer thought of as only the purveyor of genetic information, but also as a highly programmable and functional biopolymer. It was discovered in 1994 that some single-stranded DNA sequences can fold into complex tertiary structures to catalyze reactions, these DNA sequences are called DNAzymes, or DNA enzymes. Like with protein enzymes, the sequence of a DNAzyme dictates the folding of DNA into unique tertiary structure which ultimately programs its catalytic function. The folding of DNA is a product of a combination of Watson-Crick base-pairing and nonconventional base-pairing. One of the most common types of DNAzymes studied is the RNA-cleaving DNAzyme. RNA-cleaving DNAzymes catalyze cleavage of the phosphodiester backbone of an all-RNA oligomer, or a chimeric DNA sequence at an embedded RNA site, splitting the oligomer in two, through transesterification or hydrolysis. The study of RNA-cleaving DNAzymes have shown preferential activity with certain metal ion cofactors. The preferential catalytic activity of RNA-cleaving DNAzymes with selective metal ion cofactors, in conjunction with advances in DNA synthesis and DNA nanotechnology, have led to their application as metal ion sensors.DNAzyme-based metal ion sensors have been developed for a variety of sensing platforms including but not limited to: fluorescence, electrochemical, electrochemiluminescence, colorimetric, luminescent, surface-enhance Raman scattering, and glucose-based sensors. These sensors capitalize of the release of the shortened oligomer upon cleavage of the phosphodiester backbone, caused by a in DNA hybridization melting temperature. This physical release of the short oligomer can be detected by modifying the DNAzyme and substrate, for example with a fluorophore-quencher pair. This technology has been applied for the detection of metal ions in environmental samples, but more recent discoveries are also transitioning DNAzyme-based metal ion sensors into the biological and medical fields. One particular goal is to use the Na+-specific NaA43 DNAzyme for point-of-care or cellular sensing. While other sodium sensors do exist, the high selectivity, sensitivity, solubility, and biocompatibility of the NaA43 DNAzyme make it highly competitive in the sensor field. When coupled with the ability to apply the same general DNAzyme to multiple different sensing platforms, using DNAzymes to sense difficult to detect monovalent metal ions could help advance both point-of-care monitoring and fundamental biological understanding of these metal ions.Lithium is one of the most well-established drugs for the treatment of bipolar disorder and mania and is shown to have neuroprotective properties which attenuate the effects of traumatic brain injury. However, despite its efficacy the mechanism by which lithium works is still debated and Li+ detection strategies remain elusive. Therefore, it was determined that a Li+-specific DNAzyme with ability to be applied in cellular studies and point-of-care sensors would be beneficial.In order to identify a Li+-specific DNAzyme in vitro selection was performed. In vitro selection is a combinatorial selection technique which cultivates DNAzymes with desired activity from a pool of 1014-15 varied DNA sequences through a repetitive screening process. By tailoring in vitro selection, it can be designed to enrich a DNA pool with DNAzymes with selectively reactivity with a specified metal ion cofactor. In order to accomplish the identification of Li+-selective DNAzymes the buffers and in vitro selection procedure was modified to remove almost all labile metal ions form the selection process with the exception of Li+.After modifying the selection procedure, a DNA pool was cultivated with DNAzymes selective for Li+ over other monovalent, divalent, and trivalent metal ions. The new DNAzymes were able to detect low millimolar Li+, however, the reaction was slow, and potentially impractical for sensor applications. To increase the catalytic rate of the DNAzyme a reselection of a pool generated by partial randomization of the enzymatic core, was carried out. The resulting 20-4 DNAzyme was 7-10 times faster than the original sequence. The 20-4 DNAzyme was successfully truncated and converted into a trans-cleaving DNAzyme, without significant loss of activity. Attempts to further increase the reaction rate of the 20-4 DNAzyme by coupling the cleavage reaction with catalytic hairpin assembly were met with moderate success. Overall, the 20-4 DNAzyme shows promise towards Li+-detection for biological applications.