Precision Nucleic Acid Diagnostics using Novel Oligonucleotide Reagents and Amplification-Free Assays

Sofie Slott

Research output: Book/ReportPh.D. thesis

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Abstract

Precision in cancer diagnostics is of high interest to improve survival rates, costs and treatment guidance. Nucleic acid biomarkers are promising tools for use in precision cancer diagnostics. However, detection of nucleic acid biomarkers remains a challenge. This thesis will address some of the issues related to detection of nucleic acid biomarkers for early cancer diagnostic.
In the thesis, three strategies to optimize current amplification-free FLEET platform, established in Astakhova group, have been investigated. We applied calculations from the Peyrard-Bishop model to make insertions of LNA in high-affinity capture sequences targeting oncogenes, and predicted their melting temperatures (Tm). Selected probes were synthesized, and had their Tm measured, resulting in an accuracy of 1°C. This indicated that the PB model can be applied for future design of ultra-specific probes. Secondly, we implemented the FLEET assay in microfluidic PMMA chips using TC-tagged capture probes immobilized with UV light. We fabricated microfluidic PMMA chips and estimated LOD to be 6.4pM – 32pM using a spectrofluorometer.
The assay was tested with BRAF-probes, designed with the PB model. This showed a detectable signal, but needs to be optimized.
We describe a quantitative detection method for mutated microRNA in human plasma samples. Specific oligonucleotides designed from a Peyrard-Bishop model allowed accurate prediction of target:probe recognition affinity and specificity. The assay allowed identification of singlenucleotide polymorphism mismatch profiles in clinically relevant microRNA-128-2-3p, showing terminal mutations that correlate positively with inflammatory colitis and colorectal cancer.
Related to production of probes, we describe a new approach to make pools of microRNA targeting breast cancer cells. The microRNA pools were synthesized at once on the same solid-support using ‘Tandem Oligonucleotide Synthesis’ strategy. We make up to four consecutive microRNA using 2’/3’OAc nucleotide phosphoramidites. The developed phosphoramidites combined give a cleavable moiety that separates the microRNAs and are cleaved using standard cleavage conditions. Furthermore, we investigate making branched pools (microRNA dendrimers) versus linear pools as a strategy to further improve the product yields. Our approach provides microRNA pools in high yields, which is of relevance to the growing demand on synthetic RNA oligomers for nucleic acid research and technology.
Targeted sequencing has great importance in finding new variants of nucleic acid biomarkers. In this thesis, we optimize Oligonucleotide-Selective Sequencing, established in the Ji Research lab. We investigate the effect of primer spacing using synthesized branched oligonucleotide as primers, with the aim of improving yield and sensitivity. The branching primers were found to improve sensitivity by 2-fold compared to control primers which is promising.
Original languageEnglish
PublisherDTU Chemistry
Number of pages194
Publication statusPublished - 2023

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