RNA aptamers as sensors for the miR-200 family of microRNAs

Kersten, Corinna (2026): RNA aptamers as sensors for the miR-200 family of microRNAs. Dissertation, LMU München: Faculty of Chemistry and Pharmacy

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs that play an important role in post-transcriptional gene regulation and have emerged as important biomarkers for many diseases, including cancer. Among them, the miR-200 family stands out due to its important role in epithelial-to-mesenchymal transition (EMT), a key process in the formation of cancer metastasis. In this thesis, an RNA-based biosensor was developed to enable the direct, amplification-free detection of miR-200c, a miR-200 family member that plays an important role in the development of disease. The sensor designs tested were based on the fluorescence light up aptamer Broccoli and consist of a toehold-like hairpin or a strand displacement design approach or a combination of both, resulting in three major design approaches: Hairpin (HP), Strand displacement (SD) and Hairpin strand displacement (HPSD). Among them, the HPSD aptamer demonstrated superior performance regarding sensitivity, signal intensity and specificity. The HPSD aptamer enables real-time, in vitro detection of miR-200c in a simple assay format with a detection limit of 5-10 nM. Importantly, it can discriminate between the mature miR-200c and its precursors (pre- and pri-miR-200c), allowing the investigation of miRNA biogenesis. Additionally, the aptamer can distinguish miR-200c from miR-200a, but not miR-200c from miR-200b, highlighting its high selectivity towards group I miR-200 family members. HPSD was successfully used to detect endogenous miR-200c in small RNA isolates from Caco-2 cells, showing its value as a diagnostic detection tool. Apart from this, the design can easily be adapted to detect miR-200a, instead of miR-200c, proving its versatility as a diagnostic tool. In addition to the successful in vitro characterization of the HPSD aptamer, the goal was to image miR-200c in live cells. However, despite intense optimizations, specific aptamer fluorescence was not consistently observed in miR-200c expressing cells. Further analysis showed that the performance of the aptamer is highly dependent on the MgCl2 concentration and that it is thermally instable at physiological temperatures, which might explain the challenges in live-cell imaging. In parallel, another fluorescent aptamer, named RhoBast, was successfully turned into a guanine-responsive sensor, by exchanging parts of the aptamer with a guanine riboswitch. However, RhoBast could not be turned into a miR-200c sensor, mainly due to either too stable off or on-states. In summary, in this thesis, a novel miR-200c sensor, termed HPSD aptamer, was developed enabling specific and sensitive detection of miR-200c. Due to its in vitro performance and design flexibility it is a promising tool for developing miRNA sensors for diagnostic or research applications. The HPSD aptamer-based assay for miR-200c detection is faster, more cost-effective, and more user-friendly, compared to other gold-standard miRNA detection methods, such as qPCR, Northern blotting, microarrays or small RNA sequencing, making it suitable for point-of care or other diagnostic tests.

• Item Type: Theses (Dissertation, LMU Munich)
• Subjects: 500 Natural sciences and mathematics; 500 Natural sciences and mathematics > 540 Chemistry and allied sciences
• Faculties: Faculty of Chemistry and Pharmacy
• Language: English
• Date of oral examination: 19. June 2026
• 1. Referee: Schneider, Sabine
• Signature of the printed copy: 0001/UMC 32216