Development of Conditionally Activated (“Caged”) Oligonucleotides for Gene Expression Regulation and Transcriptome in Vivo Analysis (Tiva)
Caged oligonucleotides are DNA or RNA molecules whose biological function is temporarily blocked until restored by programmed stimuli. Caging provides useful spatiotemporal control to study dynamic biological processes. Many oligo caging strategies have been explored in the literature: A circular structure incorporating a photocleavable linker is particularly elegant. Target binding is inhibited by the curvature of the caged oligo until photoactivation reveals the bioactive, linear form. However, circular oligo synthesis described in early studies is laborious and low yielding. We present an efficient route to cyclize oligonucleotides incorporating a nitrobenzyl photocleavable linker through intramolecular copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). Oligo cyclization was achieved for several sequences in nearly quantitative yield. A circular antisense DNA strand targeting gfap, a marker gene encoding glial fibrillary acidic protein (GFAP) in astrocytes, was successfully tested, where irradiation decreased GFAP protein expression 10-fold. The circular caging design was extended to Transcriptome In Vivo Analysis (TIVA), a technology developed by our laboratory to extract mRNA from living single cells while preserving contextual information. Caging stability was investigated while varying circular probe size and oligo backbone modification. A caged circular 14U 2′-OMe RNA was synthesized as a TIVA probe, which gave excellent caging stability as well as target binding upon light activation. Although commonly used, near-UV light limits uncaging to thin tissues and optically transparent model organisms. Alternatively, cellular stimuli such as enzyme reactivity can connect oligo function with biochemical conditions. We have developed the first protease-activated oligo, demonstrated here for caspase-3 in cells undergoing apoptosis. The caged probe was introduced into HeLa cells and successfully activated once apoptosis was triggered.
Yang, Linlin, "Development of Conditionally Activated (“Caged”) Oligonucleotides for Gene Expression Regulation and Transcriptome in Vivo Analysis (Tiva)" (2020). Dissertations available from ProQuest. AAI28087837.