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Kinetic assays for RNA-cleaving deoxyribozymes and other nucleases
Stockholm University, Faculty of Science, Department of Neurochemistry. (Ülo Langel)ORCID iD: 0000-0001-8813-1096
2016 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

In this thesis two different assays for real-time RNA-cleaving deoxyribozyme and general nuclease kinetics are presented. Previous publications on nuclease kinetic assays have been riddled with drawbacks of labeling, discontinuity, cost etc. To tackle some of the drawbacks two assays were developed; the first specifically for RNA-cleaving deoxyribozymes to allow real-time kinetic measurements independently of whether the deoxyribozyme has low or high levels of secondary structure and when cleaving a full length messenger RNA (mRNA) substrate; the second assay was developed as a means to measure kinetics of virtually any nuclease by utilizing the single ubiquitous phenomenon in nuclease cleavage, the exposure of a phosphate upon hydrolysis of the phosphate backbone.

In Paper I the assay for RNA-cleaving deoxyribozyme kinetics is presented as a development of a previously published assay. The search for a fluorescent intercalating dye with more preferential properties than ethidium bromide resulted in PicoGreen. This dye allowed the assay to be used for deoxyribozymes with low and high levels of secondary structure as well as using full length mRNA substrates.

Paper II presents the second assay of this thesis, an assay where phosphates exposed by nuclease cleavage are released from their products by phosphatases; the released inorganic phosphates are quantified in real-time by a biosensor. The assay allows for real-time kinetics without the use of labels (i.e. natural enzymes and substrates). Regardless of whether the nuclease was a protein, nucleic acid-based, an exo- or endonuclease, processive or single-target nuclease the assay suited them equally well.

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry , 2016. , 51 p.
National Category
Chemical Sciences Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-126537ISBN: 978-91-7649-323-6OAI: oai:DiVA.org:su-126537DiVA: diva2:900974
Presentation
2016-02-26, Heilbronnsalen, C458, Svante Arrhenius väg 16B, Stockholm, 14:00 (English)
Opponent
Supervisors
Available from: 2016-02-12 Created: 2016-02-05 Last updated: 2016-08-11Bibliographically approved
List of papers
1. A High-Throughput Kinetic Assay for RNA-Cleaving Deoxyribozymes
Open this publication in new window or tab >>A High-Throughput Kinetic Assay for RNA-Cleaving Deoxyribozymes
2015 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 8, e0135984Article in journal (Refereed) Published
Abstract [en]

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 multi-mode 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.

National Category
Biological Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-120911 (URN)10.1371/journal.pone.0135984 (DOI)000360069400083 ()
Available from: 2015-09-24 Created: 2015-09-18 Last updated: 2016-08-11Bibliographically approved
2. Quantitative Microplate Assay for Real-Time Nuclease Kinetics
Open this publication in new window or tab >>Quantitative Microplate Assay for Real-Time Nuclease Kinetics
2016 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 4, e0154099Article in journal (Refereed) Published
Abstract [en]

Utilizing the phenomenon of nucleases exposing oligonucleotide phosphate backbones to phosphatases we present a novel quantitative method for kinetics of nuclease catalysis. Inorganic phosphate released from nuclease products by phosphatases could be quantified in real-time by a fluorescent sensor of inorganic phosphate. Two different nucleases were employed, showing the versatility of this assay for multiple turnover label-free nuclease studies.

National Category
Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-130869 (URN)10.1371/journal.pone.0154099 (DOI)000374898500143 ()27101307 (PubMedID)
Available from: 2016-06-07 Created: 2016-06-07 Last updated: 2016-08-11Bibliographically approved

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