Development Of Functional Dna Based Sensors And Investigations Into Their Mechanism

Author : Eric L. Null
Publisher :
ISBN 13 :
Total Pages : pages
Book Rating : 4.:/5 (774 download)

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Download or read book Functional Dna: Biochemical/biophysical Characterization & Sensing Applications written by Eric L. Null and published by . This book was released on 2011 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: In 1990 the Szostak and Gold groups independently discovered that short pieces of RNA can bind to small molecule or biological targets. In 1994 the Joyce group showed that DNA, long thought to be solely for information storage, was capable of catalysis. Naturally occurring ribozymes were discovered in the 1980s by the Cech group. Since then aptamers, which bind targets, and ribozymes or deoxyribozymes, which are catalytically active, have become known collectively as 0́−functional nucleic acids.0́+ The common theme of the works presented herein involves manipulating functional nucleic acids to further increase our understanding of their fundamental properties and to also develop applications for these molecules. Chapter 1 is an introduction to the works presented herein. Chapter 2 discusses conversion of aptamers into sensors for the determination of enantiomeric ratio, leading to a rapid method of detection with high selectivity and portability. Chapters 3, 4, and 5 are tied together through the common thread of the 8-17 DNAzyme and involve biochemical and biophysical characterization of the 8-17 DNAzyme as well as characterization of a novel red Pb2+ species formed upon cleavage of a modified 8-17 DNAzyme. Determination of the enantiomeric ratio is important as many monetarily and functionally valuable molecules are chiral, such as pharmaceuticals and chiral catalysts. For example, Xopenex,® a single enantiomer form of albuterol, has higher efficacy than the racemic mixture. There are currently multiple methods for determining the enantiomeric ratio, all of which work well, with their own particular caveats. Separations-based methods, using a variety of detectors, may require 30 minute runs and often require solvents. NMR and fluorescence-based methods are rapid, though the development of a chiral reporter requires many iterative cycles of design and synthesis. Herein, we use the power of aptamers generated by in vitro selection to design a fluorescence-based system capable of detecting 0.1% L-arginine in a solution of D-arginine in five minutes. The 8-17 DNAzyme is a RNA-cleaving DNAzyme which is active with divalent metal ions, showing the highest activity with Pb2+. The 8-17 DNAzyme has been isolated multiple times by different groups and has been the subject of many studies 0́3 both fundamental and practical. Mutational studies by the Peracchi and Lu groups have shown that certain bases in the DNAzyme are highly conserved, though the metal ion binding site is still unknown. FRET studies by the Lu group have shown that a folding step is necessary before catalysis with either Zn2+ or Mg2+, though Pb2+ does not require a folding step, leading to the postulation of a pre-arranged binding site. FRET, however, is a low-resolution technique and does not provide information on local folding, or rather changes in the conformation of the active site upon metal ion binding. Herein we show that the 8-17 DNAzyme is prearranged for Pb2+ as minimal changes in the 1H NMR spectrum are seen upon Pb2+ titration, supporting a true 0́−lock-and-key0́+ mode of catalysis. Addition of Zn2+ or Mg2+, both of which induce global folding, results in significant changes in the 1H NMR spectrum. These changes are correlated with cleavage activity, indicating local folding accompanies activity. Additionally, we show that mutation of the catalytically essential G0́ØT wobble pair to a G-C base pair results in perturbation of structure as well as reduced Zn2+ and Pb2+ interaction. Chapter 4 discusses very exciting results, demonstrating localization of Pb2+ on the backbone of the 8-17 DNAzyme leading us much closer to understanding the Pb2+ binding site and reinforcing the importance of the conserved residues in maintaining the hydrogen bonding network, rather than serving directly as ligands. Metal ion interactions with the backbone were determined through phosphorothioate mutations. A phosphorothioate is an isostructural mutation consisting of a non-bridging backbone oxygen mutated to sulfur. Metal ion affinity changes upon this mutation based on Hard Soft Acid Base (HSAB) Theory. Activity with a 0́−hard0́+ metal ion such as Mg2+ will be lost if metal ion binding at the phosphorothioate-mutated site is catalytically important, as a hard metal ion has much lower affinity for sulfur than oxygen. Activity assays were performed which showed that several highly-conserved positions are catalytically important for Pb2+ binding, demonstrating interaction of Pb2+ with the backbone for the first time. These results were confirmed via 31P NMR. A phosphorothioate mutation shifts the backbone peak over 50 ppm downfield and metal ion interaction results in a change in the chemical shift. Metal ion titrations were performed and monitored by 31P NMR which showed a larger change in chemical shift upon metal ion binding to catalytically important backbone residues than control residues. Chapter 5 leads directly out of Chapter 4 in that a phosphorothioate mutation at the cleavage site led to a color change upon the addition of Pb2+, resulting in a novel red Pb2+-DNA species that is assigned to a Pb2+-20́ø,30́ø-cyclic phosphorothioate interaction. Cleavage products were characterized by gel-based and instrumental methods which showed that a phosphorothioate mutation at the cleavage site did not result in a change in cleavage mechanism or cleavage behavior (pH dependence, etc.). Small molecule models confirmed that Pb2+ interacted with a pendant phosphorothioate, a 30́ø,50́ø-cyclic phosphorothioate, and a 20́ø,30́ø-cyclic phosphorothioate, though only the 20́ø,30́ø-cyclic phosphorothioate-Pb2+ interaction resulted in a species visible to the naked eye. Hg2+ was also shown to generate a colored species, and the mutation was extended to a phosphorodithioate (where both non-bridging oxygen atoms are substituted) and other DNAzyme systems. This system may be useful for detection of cyclic phosphate where a phosphorothioate mutation can be chemically introduced at the cleavage site of an RNA-cleaving DNAzyme or ribozyme. This system is also the first known soluble red Pb2+ species and is highly specific for 20́ø,30́ø- over 30́ø,50́ø-cyclic phosphorothioates in terms of response.