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Mirror Image Nucleic Acid Probes for Microrna Detection and Live Cell Imaging
Abstract
Gene expression is regulated by a vast network of non-coding RNAs. In the past decades, many methods have been developed for measuring non-coding RNAs, especially microRNAs, in vitro and in vivo. In particular, DNA strand displacement reactions have enabled the development of an impressive repertoire of molecular devices with complex functionalities. By relying on DNA to function, dynamic strand displacement devices represent powerful tools for the interrogation and manipulation of non-coding RNAs. Yet, implementation in living systems has been a slow process due to several persistent challenges, including nuclease degradation.
To circumvent these issues, researchers are increasingly turning to chemically modified nucleotides as a means to increase device performance and reliability within harsh biological environments. L-DNA, the mirror image (i.e. enantiomer) of natural D-DNA, represents a critically underexplored modification for this application. L-DNAs have the same physical and chemical properties as their natural counterparts, but they are essentially ‘invisible’ to the stereospecific environment of biology. Therefore, the use of L-DNAs is of great promise for in vivo applications of strand displacement devices.
Here we describe the successful detection of endogenously expressed microRNA in living cells using a probe based on an L-RNA fluorogenic aptamer. Then we compare the operation of D- and L- DNA strand displacement reactions directly in living cells, showing the reliability and feasibility of adapting strand displacement reactions in L-. Lastly, we introduce a chimeric duplex that consists of L-/D-DNA that is capable of monitoring base excision repair activities in cells in real-time. Overall, work in this dissertation illustrates the advantages and potentials of using L-nucleic acids in the development of robust systems and devices that operate in living cells.
Citation
Zhong, Wenrui (2022). Mirror Image Nucleic Acid Probes for Microrna Detection and Live Cell Imaging. Doctoral dissertation, Texas A&M University. Available electronically from https : / /hdl .handle .net /1969 .1 /198508.