Characterization of a catalytically efficient acidic RNA-cleaving deoxyribozyme

Abstract

We previously demonstrated—through the isolation of RNA-cleaving deoxyribozymes by in vitro selection that are catalytically active in highly acidic solutions—that DNA, despite its chemical simplicity, could perform catalysis under challenging chemical conditions [Liu,Z., Mei,S.H., Brennan,J.D. and Li,Y. (2003) J. Am. Chem. Soc. 125, 7539–7545]. One remarkable DNA molecule therefrom is pH4DZ1, a self-cleaving deoxyribozyme that exhibits a kobs of ∼1 min−1 at pH 3.8. In this study, we carried out a series of experiments to examine the sequence and catalytic properties of this acidic deoxyribozyme. Extensive nucleotide truncation experiments indicated that pH4DZ1 was a considerably large deoxyribozyme, requiring ∼80 out of the original 123 nt for the optimal catalytic activity. A reselection experiment identified ten absolutely conserved nucleotides that are distributed in three catalytically crucial sequence elements. In addition, a trans deoxyribozyme was successfully designed. Comparison of the observed rate constant of pH4DZ1 with experimentally determined rate constant for the uncatalyzed reaction revealed that pH4DZ1 achieved a rate enhancement of ∼106-fold. This study provides valuable information about this low-pH-functional deoxyribozyme and paves way for further structural and mechanistic characterization of this unique catalytic DNA.

In vitro selection of RNA-cleaving DNAzymes for bacterial detection

“…We term this type of DNAzyme "RNA-cleaving fluorogenic DNAzyme", or simply "RFD". Over the past decade, we have isolated many RFDs and examined their catalytic and signaling properties [38,39,[67][68][69][81][82][83][84][85][86][87][88][89][90]. More recently, we began to develop RFDs that that can be activated in the presence of a specific bacterium, such as Escherichia coli and Clostridium difficile [72,80,82,[91][92][93][94][95][96].…”

A Catalytic DNA Probe with Stem-loop Motif for Human T47D Breast Cancer Cells

“…29 Prior to selection, three different ESM samples were collected: ESM from the T47D cell line (ESM-TD), ESM from the MCF-10A cell line, and the ESM from the HeLa cell line. The ESM samples were prepared by removing cells after 36 h of culture in Opti-MEM ® .…”

“…Although DNAzymes that directly detect the ESM of a given living cell have been applied to identification of pathogenic bacteria, [25][26][27][28] their use in detecting eukaryotic cancer cells has not yet been reported. Detection of eukaryotic ESM is more of a challenge because eukaryotic cells have much slower growth rates and lower ESM concentrations than do prokaryotic cells.…”

Detection of Bacteria Using Fluorogenic DNAzymes

“…1) and investigated the use of RFDs as analytical tools. [17][18][19][20][21][22][23][24][25][26][27][28][29] RFDs catalyze the cleavage of a DNA-RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by a fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the uncleaved substrate minimal fluorescence.…”

“…FS1 was obtained from Keck Oligo Synthesis Facilities at Yale University, deprotected and purified by gel electrophoresis following a previously established protocol. [17][18][19][20][21][22][23][24] EC1, SS1 and LT1 were purchased from Integrated DNA Technologies and purified by gel electrophoresis. vortexing and place the tubes in -20 °C freezer for 30 min.…”