Nucleic acid amplification tests (NAATs) enable sensitive detection of low density infections that microscopy and rapid diagnostic test are not capable of detecting. They enable quantitative and qualitative nucleic acid detection, genotype analysis, and single nucleotide polymorphism (SNP) detection. Current state of the art molecular probes used with NAATs includes molecular beacon (MB), Taqman and its variations. This work presents novel molecular probe designed from Spinach and Dapoxyl aptamers. The aptamers are split into two parts (split aptamer), allowing greater sensitivity and selectivity towards fully complementary nucleic acid analyte. The major advantage of split aptamer probe over state-of-the-art fluorescent probes is its high selectivity: in the presence of a single base mismatched analyte, it produces only background fluorescence, even at room temperature. SSA is a promising tool for label-free analysis of nucleic acids at ambient temperatures. Split spinach aptamer (SSA) probes and split dapoxyl aptamer (SDA) for fluorescent analysis of nucleic acids were designed and tested. In both split aptamer design, two RNA or RNA/DNA or DNA strands hybridized to a specific nucleic acid analyte and formed a binding site for fluorescent dye, which was accompanied by up to 270-fold and 69-fold increase in fluorescence. SSAr consisted entirely of ribonucleotides which potentially be expressed in live cells and used for imaging of specific mRNAs. For in vitro RNA/DNA analysis, SDA consisting of entirely DNA are preferable due to greater chemical stability, lower synthetic cost and reduced ability to form intramolecular structures. Additionally, we designed two DNA strands that function as an adapter for SSA and demonstrated how a single universal spinach aptamer (USSA) probe can be used to detect multiple (potentially any) nucleic acid sequences. USSA can be used for cost-efficient and highly selective analysis of even folded DNA and RNA analytes, as well as for the readout of outputs of DNA logic circuits.
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Doctor of Philosophy (Ph.D.)
College of Sciences
Length of Campus-only Access
Doctoral Dissertation (Open Access)
Kikuchi, Nanami, "Split Aptameric Turn-On Fluorescence Sensor for Detection of Sequence Specific Nucleic Acid at Ambient Temperature" (2018). Electronic Theses and Dissertations, 2004-2019. 5768.