Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Detection of miRNAs using chimeric DNA/RNA iLock probes utilizing novel activity of PBCV-1 DNA ligase: RNA-templated ligation of ssRNA
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]

Accurate detection of miRNAs with complementary probes is challenging due to the short target size, and often high sequence similarity between isoforms belonging to the same miRNA family. Ligation based methods can provide powerful discrimination of subtle sequence variation among target sequences, but they have been difficult to implement for direct RNA analysis due to the sloppiness and inefficiency of most DNA ligases on RNA substrates. In this work, we have studied if RNA substitutions in padlock probes can provide higher catalytic efficiencies for PBCV-1 DNA ligase on RNA substrates. We also characterise end-joining fidelity for Chlorella virus DNA ligase (PBCV DNA ligase 1) and T4RNA ligase 2 (T4Rnl2) in RNA-templated 3'-OH RNA/5’-pDNA chimeric probe ligation. Although we observed considerable ligation efficiency improvement towards short miRNA targets for PBCV-1 ligated chimeric probes, it showed no sequence specificity towards mismatches at the ligation junction. T4Rnl2 showed some base discrimination, but not satisfactory for robust RNA sequence analysis. To increase end-joining fidelity in PBCV-1 DNA ligase catalysed direct RNA detection assays (iLock probes), we have recently introduced an alternative ligation assay design in which ligation probes first undergo sequence- specific 5’ FLAP removal in order to create ligatable substrates. We have tested various chimeric iLock probe designs where RNA substitutions were introduced at different positions in the FLAP and at the ligation junction. We defined two particular nucleotide positions in the iLock probe sequence that when substituted with RNA, significantly increased iLock probe activation and ligation. We further characterized the end-joining fidelity of PBCV-1 and T4Rnl2 catalysed iLock reactions. Both enzymes showed high ligation fidelities for single nucleotide polymorphisms on RNA and miRNA. Finally, we demonstrate a multiplexed chimeric iLock probe miRNA profiling assay using sequencing-by-ligation as readout. 

Keyword [en]
Chimeric oligonucleotides, Invader, iLock, PBCV DNA ligase 1, T4RNA ligase 2
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-147731OAI: oai:DiVA.org:su-147731DiVA: diva2:1148311
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

Open Access in DiVA

No full text

Search in DiVA

By author/editor
Krzywkowski, TomaszKühnemund, MalteNilsson, Mats
By organisation
Department of Biochemistry and BiophysicsScience for Life Laboratory (SciLifeLab)
Biological Sciences

Search outside of DiVA

GoogleGoogle Scholar

urn-nbn

Altmetric score

urn-nbn
Total: 320 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf