| 總結: | <p>Fluorogenic oligonucleotide-based systems are important tools for sequence-specific DNA targeting. Fluorescence systems use the emission of a photon from an excited molecule (fluorophore) to produce a detectable signal. It is a highly sensitive process and its versatility has expanded its use to DNA targeting probes. The aim of the work herein is to investigate the design and synthesis of fluorogenic oligonucleotide-based systems specifically for targeting double-stranded DNA.</p>
<p>Triplex-forming oligonucleotides can bind in the major groove of dsDNA through Hoogsteen hydrogen bonding to form triplexes (triple helixes). While other methods of dsDNA detection rely on denaturation of the double-stranded target, triplexes do not, and, hence, they provide a versatile approach to DNA recognition. However, the instability of triplexes, especially at neutral pH, limits their applications. The incorporation of a fluorogenic intercalator, thiazole orange, covalently attached to the TFO provides a triplex-stabilising moiety. The stabilising effect of thiazole orange is additive; multiple incorporations can produce triplexes with increased stability of +50 °C even at neutral pH. The extreme stability provided from the modified fluorogenic TFOs allows increased duplex binding affinity and can compensate for the limited target range of TFOs.
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<p>CRISPR-Cas is a programmable biological research tool that is invaluable in gene- editing and genomic imaging technologies. The introduction of fluorogenic moieties both on the dCas9 enzyme and in guide RNA is crucial for imaging but often leads to high background fluorescence in biological systems. By modifying the gRNA to contain an ‘on/off’ fluorogenic system, genomic imaging in live cells of repetitive elements is improved and is cross-validated by GFP-tagged dCas9.</p>
<p>The efficient synthesis of long, modified RNA is fundamental, particularly in CRISPR research. The introduction of an azide moiety into RNA to provide a means of ‘clicking’ modified RNA strands together is an important potential approach. However, it is limited by the protecting groups used for RNA and, in turn, this hinders the length of efficient RNA synthesis. The synthesis of a clickable moiety compatible with 2'-O-TC RNA chemistry is investigated as it could provide a simple and cost- effective route to modified guide RNA and could be instrumental in applications such as modified guide RNA library generation.</p>
<p>Overall, the aims of this study were to synthesise and develop fluorogenic oligonucleotide-based systems and investigate their potential applications.</p>
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