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Reverse-transcriptase activity of Phi29 DNA polymerase
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).ORCID iD: 0000-0002-2706-8705
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
(English)Manuscript (preprint) (Other academic)
Abstract [en]

F29 (Phi29) DNA polymerase is an enzyme commonly used in DNA amplification methods such as rolling circle amplification (RCA) and multiple strand displacement amplification (MDA), as well as in DNA sequencing methods such as single molecule real-time (SMRT) sequencing. Here we report the ability of F29 DNA polymerase to amplify partially RNA-containing circular substrates during RCA. We found that circular substrates with single RNA substitutions support a similar amplification rate as pure DNA substrates. We observed that increasing the number of consecutive RNA substitutions in the circular templates suppress replication, and cannot be recovered by addition of M-MuLV reverse-transcriptase. In summary, this novel ability of F29 to accept RNA-containing substrates broadens the spectrum of applications for F29 mediated RCA. Applications include amplification of chimeric circular probes, such as padlock probes and molecular inversion probes. 

Keyword [en]
Chimeric oligonucleotides, PBCV-1 DNA ligase 1, T4RNA ligase 2, F29 DNA polymerase, reverse- transcriptase
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-147733OAI: oai:DiVA.org:su-147733DiVA: diva2:1148320
Available from: 2017-10-10 Created: 2017-10-10 Last updated: 2017-10-11Bibliographically approved
In thesis
1. iLocks: a novel tool for RNA assays with improved specificity
Open this publication in new window or tab >>iLocks: a novel tool for RNA assays with improved specificity
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The Central Dogma of molecular biology describes a framework for how genetic information is transferred in cells, placing RNA as a messenger between DNA and translated proteins. During the last years, interest in RNA research has grown tremendously due to the increasing understanding and recognition of the importance of RNA in regulation of gene expression, biochemical catalysis, and genome integrity surveillance. Most importantly, RNA content, unlike DNA, changes constantly, fine-tuning the cellular response to match the environmental conditions. There is a clear potential for RNA biomarkers, reflecting both the natural and pathological conditions in vivo.

Various methods have been developed to study RNA, of which the most common tools and techniques are described in this thesis. Since many of these gold standard methods are based on detecting RNA derivative (cDNA), there is a wide scope for efficient alternative tools directly targeting RNA. In Paper I, the spatiotemporal expression of human adenovirus-5 mRNA in epithelial and blood cells infected with the virus has been studied. For this, padlock probes and rolling circle amplification (RCA) were used to visualize, quantify and analyse both viral and host cell cDNAs in different infection scenarios, at single cell level. In Paper II, direct RNA detection fidelity has been evaluated using padlock probes. A novel type of probe (iLock) that is activated on RNA via invasive cleavage mechanism, prior to RCA was developed in this approach. Using iLocks, a substantial improvement of direct RNA sensing fidelity has been observed. In Paper III, RNA modifications were introduced in otherwise DNA iLock probes to enhance the probes’ efficiency on miRNAs. Using chimeric iLock probes, multiplexed differentiation of conserved miRNA family members were performed with next- generation sequencing-by-ligation readout. Efficient replication of chimeric probes used in Paper III implies that the Phi29 DNA polymerase readily accepts RNA-containing circles as amplification substrates. In Paper IV, real-time RCA monitoring for measurement of amplification rates and analysis of amplification patterns of various RNA-containing circles was achieved. Moreover, the RCA products were sequenced as a proof for the reverse-transcriptase activity of the Phi29 DNA polymerase.

This thesis effectively contributes to a better understanding of mechanisms influencing RNA detection with, but not limited to, padlock probes. It expands the available RNA analyses toolkit with novel strategies and solutions, which can be potentially adapted for RNA-focused research, in general and molecular diagnostics, in particular.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2017. 59 p.
Keyword
RNA, miRNA, non-coding RNA, padlock probes, rolling circle amplification, invader, single cell, in situ, adenovirus, virology, diagnostics
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-147734 (URN)978-91-7797-043-9 (ISBN)978-91-7797-044-6 (ISBN)
Public defence
2017-11-24, Högbomsalen, Geovetenskapens hus, Svante Arrhenius väg 12, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.

Available from: 2017-10-31 Created: 2017-10-11 Last updated: 2017-11-01Bibliographically approved

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