Enzymatic Synthesis of Self-assembled Dicer Substrate RNA Nanostructures for Programmable Gene Silencing

Enzymatic synthesis
of RNA nanostructures is achieved by isothermal
rolling circle transcription (RCT). Each arm of RNA nanostructures
provides a functional role of Dicer substrate RNA inducing sequence
specific RNA interference (RNAi). Three different RNAi sequences (GFP,
RFP, and BFP) are incorporated within the three-arm junction RNA nanostructures
(Y-RNA). The template and helper DNA strands are designed for the
large-scale in vitro synthesis of RNA strands to prepare self-assembled
Y-RNA. Interestingly, Dicer processing of Y-RNA is highly influenced
by its physical structure and different gene silencing activity is
achieved depending on its arm length and overhang. In addition, enzymatic
synthesis allows the preparation of various Y-RNA structures using
a single DNA template offering on demand regulation of multiple target
genes.