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iLocks: a novel tool for RNA assays with improved specificity
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Mats Nilsson)ORCID iD: 0000-0002-2706-8705
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 [en]
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: urn:nbn:se:su:diva-147734ISBN: 978-91-7797-043-9 (print)ISBN: 978-91-7797-044-6 (electronic)OAI: oai:DiVA.org:su-147734DiVA: diva2:1148511
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
List of papers
1. Simultaneous Single-Cell In Situ Analysis of Human Adenovirus Type 5 DNA and mRNA Expression Patterns in Lytic and Persistent Infection
Open this publication in new window or tab >>Simultaneous Single-Cell In Situ Analysis of Human Adenovirus Type 5 DNA and mRNA Expression Patterns in Lytic and Persistent Infection
Show others...
2017 (English)In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 91, no 11, UNSP e00166-17Article in journal (Refereed) Published
Abstract [en]

An efficient adenovirus infection results in high-level accumulation of viral DNA and mRNAs in the infected cell population. However, the average viral DNA and mRNA content in a heterogeneous cell population does not necessarily reflect the same abundance in individual cells. Here, we describe a novel padlock probe-based rolling-circle amplification technique that enables simultaneous detection and analysis of human adenovirus type 5 (HAdV-5) genomic DNA and virus-encoded mRNAs in individual infected cells. We demonstrate that the method is applicable for detection and quantification of HAdV-5 DNA and mRNAs in short-term infections in human epithelial cells and in long-term infections in human B lymphocytes. Single-cell evaluation of these infections revealed high heterogeneity and unique cell subpopulations defined by differential viral DNA content and mRNA expression. Further, our single-cell analysis shows that the specific expression pattern of viral E1A 13S and 12S mRNA splice variants is linked to HAdV-5 DNA content in the individual cells. Furthermore, we show that expression of a mature form of the HAdV-5 histone-like protein VII affects virus genome detection in HAdV-5-infected cells. Collectively, padlock probes combined with rolling-circle amplification should be a welcome addition to the method repertoire for the characterization of the molecular details of the HAdV life cycle in individual infected cells. IMPORTANCE Human adenoviruses (HAdVs) have been extensively used as model systems to study various aspects of eukaryotic gene expression and genome organization. The vast majority of the HAdV studies are based on standard experimental procedures carried out using heterogeneous cell populations, where data averaging often masks biological differences. As every cell is unique, characteristics and efficiency of an HAdV infection can vary from cell to cell. Therefore, the analysis of HAdV gene expression and genome organization would benefit from a method that permits analysis of individual infected cells in the heterogeneous cell population. Here, we show that the padlock probe-based rolling-circle amplification method can be used to study concurrent viral DNA accumulation and mRNA expression patterns in individual HAdV-5-infected cells. Hence, this versatile method can be applied to detect the extent of infection and virus gene expression changes in different HAdV-5 infections.

Keyword
adenovirus, persistent infection, lytic infection, rolling-circle amplification, single-cell analysis
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-144695 (URN)10.1128/JVI.00166-17 (DOI)000402166500007 ()
Available from: 2017-07-21 Created: 2017-07-21 Last updated: 2017-10-11Bibliographically approved
2. Fidelity of RNA templated end-joining by chlorella virus DNA ligase and a novel iLock assay with improved direct RNA detection accuracy
Open this publication in new window or tab >>Fidelity of RNA templated end-joining by chlorella virus DNA ligase and a novel iLock assay with improved direct RNA detection accuracy
2017 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 45, no 18, e161Article in journal (Refereed) Published
Abstract [en]

Ligation-based nucleic acid detection methods are primarily limited to DNA, since they exhibit poor performance on RNA. This is attributed to reduced end-joining efficiency and/or fidelity of ligases. Interestingly, chlorella virus DNA ligase (PBCV-1 DNA ligase) has recently been shown to possess high RNA-templated DNA end-joining activity; however, its fidelity has not yet been systematically evaluated. Herein, we characterized PBCV-1 ligase for its RNA-templated end-joining fidelity at single base mismatches in 3′ and 5′ DNA probe termini and found an overall limited end-joining fidelity. To improve the specificity in PBCV-1 ligase-driven RNA detection assays, we utilized structure-specific 5′ exonucleolytic activity of Thermus aquaticus DNA polymerase, used in the invader assay. In the iLock (invader padLock) probe assay, padlock probe molecules are activated prior ligation thus the base at the probe ligation junction is read twice in order to aid successful DNA ligation: first, during structure-specific invader cleavage and then during sequence-specific DNA ligation. We report two distinct iLock probe activation mechanisms and systematically evaluate the assay specificity, including single nucleotide polymorphisms on RNA, mRNA and miRNA. We show significant increase in PBCV-1 ligation fidelity in the iLock probe assay configuration for RNA detection.

Keyword
Invader, PBCV-1, padlock probes, RCA, SplintR
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-147727 (URN)10.1093/nar/gkx708 (DOI)000413107400005 ()
Available from: 2017-10-10 Created: 2017-10-10 Last updated: 2017-11-13Bibliographically approved
3. Detection of miRNAs using chimeric DNA/RNA iLock probes utilizing novel activity of PBCV-1 DNA ligase: RNA-templated ligation of ssRNA
Open this publication in new window or tab >>Detection of miRNAs using chimeric DNA/RNA iLock probes utilizing novel activity of PBCV-1 DNA ligase: RNA-templated ligation of ssRNA
(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
Chimeric oligonucleotides, Invader, iLock, PBCV DNA ligase 1, T4RNA ligase 2
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-147731 (URN)
Available from: 2017-10-10 Created: 2017-10-10 Last updated: 2017-10-11Bibliographically approved
4. Reverse-transcriptase activity of Phi29 DNA polymerase
Open this publication in new window or tab >>Reverse-transcriptase activity of Phi29 DNA polymerase
(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
Chimeric oligonucleotides, PBCV-1 DNA ligase 1, T4RNA ligase 2, F29 DNA polymerase, reverse- transcriptase
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-147733 (URN)
Available from: 2017-10-10 Created: 2017-10-10 Last updated: 2017-10-11Bibliographically approved

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