Abstract
Determining kinetic constants is important in the field of RNA-cleaving deoxyribozymes (DNAzymes). Using todays conventional gel assays for DNAzyme assays is time-consuming and laborious. There have been previous attempts at producing new and improved assays; however these have drawbacks such as incompatibility with structured DNAzymes, enzyme or substrate modifications and increased cost. Here we present a new method for determining single-turnover kinetics of RNA-cleaving DNAzymes in real-time and in a high-throughput fashion. The assay is based on an intercalating fluorescent dye, PicoGreen, with high specificity for double-stranded DNA and heteroduplex DNA-RNA in this case formed between the DNAzyme and the target RNA. The fluorescence decreases as substrate is converted to product and is released from the enzyme. Using a Flexstation II multimode plate reader with built in liquid handling we could automate parts of the assay. This assay gives the possibility to determine single-turnover kinetics for up to 48 DNAzymes simultaneously. As the fluorescent probe is extrinsic there is no need for enzyme or substrate modifications, making this method less costly compared to other methods. The main novelty of this assay is the possibility of using full-length mRNA as the DNAzyme target.
Highlights
RNA-cleaving DNAzymes are non-naturally occurring molecules first developed in 1994 by Breaker and Joyce through systematic evolution of ligands by exponential enrichment (SELEX) [1]
There are several drawbacks in the method such as it not being optimized for high-throughput screenings and the inability to measure kinetics of highly structured DNAzymes, minimizing the number of possible DNAzymes applicable for this assay
We identified the main obstacles as signal variation and signal strength when using ethidium bromide (EtBr) as a fluorescent intercalator together with a structured DNAzyme
Summary
RNA-cleaving DNAzymes are non-naturally occurring molecules first developed in 1994 by Breaker and Joyce through systematic evolution of ligands by exponential enrichment (SELEX) [1]. Further selection resulted in DNAzymes (‘8–17’ and ‘10–23’) capable of cleaving RNA under simulated physiological conditions [2]. DNAzymes generally comprise two binding arms reverse complementary to a target RNA and a catalytic core to induce cleavage of the target RNA, much like hairpin ribozymes, and are mainly composed of deoxyribonucleotides. The kinetic constants of a DNAzyme are determined using short synthetic substrate that mimics the target region of the mRNA. Since mRNAs are large molecules with intramolecular bonds DNAzyme binding to the target site can be sterically hindered which will not be detected when using short mimic substrates. RNAcleaving DNAzymes are generally dependent on divalent metal-ions for the cleavage of the PLOS ONE | DOI:10.1371/journal.pone.0135984 August 26, 2015
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