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Quantitative Microplate Assay for Real-Time Nuclease Kinetics
Stockholm University, Faculty of Science, Department of Neurochemistry.
Stockholm University, Faculty of Science, Department of Neurochemistry.
Number of Authors: 2
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.

Place, publisher, year, edition, pages
2016. Vol. 11, no 4, e0154099
National Category
Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-130869DOI: 10.1371/journal.pone.0154099ISI: 000374898500143PubMedID: 27101307OAI: oai:DiVA.org:su-130869DiVA: diva2:934015
Available from: 2016-06-07 Created: 2016-06-07 Last updated: 2016-08-11Bibliographically approved
In thesis
1. Gene therapy tools: oligonucleotides and peptides
Open this publication in new window or tab >>Gene therapy tools: oligonucleotides and peptides
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Genetic mutations can cause a wide range of diseases, e.g. cancer. Gene therapy has the potential to alleviate or even cure these diseases. One of the many gene therapies developed so far is RNA-cleaving deoxyribozymes, short DNA oligonucleotides that specifically bind to and cleave RNA. Since the development of these synthetic catalytic oligonucleotides, the main way of determining their cleavage kinetics has been through the use of a laborious and error prone gel assay to quantify substrate and product at different time-points. We have developed two new methods for this purpose. The first one includes a fluorescent intercalating dye, PicoGreen, which has an increased fluorescence upon binding double-stranded oligonucleotides; during the course of the reaction the fluorescence intensity will decrease as the RNA is cleaved and dissociates from the deoxyribozyme. A second method was developed based on the common denominator of all nucleases, each cleavage event exposes a single phosphate of the oligonucleotide phosphate backbone; the exposed phosphate can simultaneously be released by a phosphatase and directly quantified by a fluorescent phosphate sensor. This method allows for multiple turnover kinetics of diverse types of nucleases, including deoxyribozymes and protein nucleases.

The main challenge of gene therapy is often the delivery into the cell. To bypass cellular defenses researchers have used a vast number of methods; one of these are cell-penetrating peptides which can be either covalently coupled to or non-covalently complexed with a cargo to deliver it into a cell. To further evolve cell-penetrating peptides and understand how they work we developed an assay to be able to quickly screen different conditions in a high-throughput manner. A luciferase up- and downregulation experiment was used together with a reduction of the experimental time by 1 day, upscaling from 24- to 96-well plates and the cost was reduced by 95% compared to commercially available assays. In the last paper we evaluated if cell-penetrating peptides could be used to improve the uptake of an LNA oligonucleotide mimic of GRN163L, a telomerase-inhibiting oligonucleotide. The combination of cell-penetrating peptides and our mimic oligonucleotide lead to an IC50 more than 20 times lower than that of GRN163L.

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University, 2016. 66 p.
Keyword
Gene therapy, oligonucleotide, peptide, RNA-cleaving deoxyribozyme, deoxyribozyme, DNAzyme, cell-penetrating peptide, CPP, enzyme, enzyme kinetics, kinetic assay, assay, telomerase, telomerase inhibitor, imetelstat, GRN163L
National Category
Biochemistry and Molecular Biology Other Chemistry Topics
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-132271 (URN)978-91-7649-460-8 (ISBN)
Public defence
2016-09-30, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2016-09-07 Created: 2016-08-04 Last updated: 2016-08-24Bibliographically approved
2. Kinetic assays for RNA-cleaving deoxyribozymes and other nucleases
Open this publication in new window or tab >>Kinetic assays for RNA-cleaving deoxyribozymes and other nucleases
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:nbn:se:su:diva-126537 (URN)978-91-7649-323-6 (ISBN)
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

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