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.

Recent advancements in molecular biology and biotechnology have pushed the field of molecular diagnostics much further to benefit the society towards smart access for rapid and simplified health care. In this context, point-of-care (PoC) technologies that bring the inventions in diagnostics closer to bedside settings draw attention. This becomes all the more relevant in the case of infectious diseases which pose the major burden, in terms of mortality and economic loss, especially for third world developing countries with resource-limited settings (RLS). Moreover, emerging and re-emerging viruses, known for their rapid mutation rates, demand huge attention in terms of timely diagnosis and the need for effective treatments. Hence, appropriate and accurate tests to detect the pathogens with enhanced sensitivity and specificity would be needed to bridge the gap between bioanalytics and clinics.

This research work is an attempt to combine the tools and techniques required for the development of such efficient PoC technologies to combat infectious diseases. Among available nucleic acid-based amplification tests, padlock probing and isothermal rolling circle amplification are used to benefit from the advantages they offer for diagnostic applications, in terms of specificity, multiplexability, single molecule detection, high throughput, compatibility with various read-out platforms and inexpensive digital quantification.

In the first paper, simultaneous detection of RNA and DNA forms of adenovirus is shown to study the spatio-temporal expression patterns in both lytic and persistent infections. In situ quantification of viral DNA as well as transcripts with single cell resolution has been achieved. In the second paper, novel probe design strategy has been presented for the development of molecular assays to detect hypervariable RNA viruses. This approach becomes helpful in targeting rapidly evolving viruses by using mutation-tolerant probes for RCA. Third paper demonstrates simple RCA for rapid detection of Ebola virus in clinical samples, followed by a multiplexed detection with other re-emerging tropical viruses, namely Zika and Dengue. This study also includes a simple easy-to-operate pump-free membrane enrichment read-out, combined together with microscopy for digital quantification of the products. In the fourth paper, near point-of-care glucose sensor-based RCP detection has been proposed for Ebola virus detection. All these attempts clearly bring RCA closer to PoC settings for molecular diagnostics of virus infections.