Fluorogenic RNA Aptamers: A Nano-platform for Fabrication of Simple and Combinatorial Logic Gates

Abstract

RNA aptamers that bind non-fluorescent dyes and activate their fluorescence are highly sensitive, nonperturbing, and convenient probes in the field of synthetic biology. These RNA molecules, referred to as light-up aptamers, operate as molecular nanoswitches that alter folding and fluorescence function in response to ligand binding, which is important in biosensing and molecular computing. Herein, we demonstrate a conceptually new generation of smart RNA nano-devices based on malachite green (MG)-binding RNA aptamer, which fluorescence output controlled by addition of short DNA oligonucleotides inputs. Four types of RNA switches possessing AND, OR, NAND, and NOR Boolean logic functions were created in modular form, allowing MG dye binding affinity to be changed by altering 3D conformation of the RNA aptamer. It is essential to develop higher-level logic circuits for the production of multi-task nanodevices for data processing, typically requiring combinatorial logic gates. Therefore, we further designed and synthetized higher-level half adder logic circuit by “in parallel” integration of two logic gates XOR and AND within a single RNA nanoparticle. The design utilizes fluorescence emissions from two different RNA aptamers: MG-binding RNA aptamer (AND gate) and Broccoli RNA aptamer that binds DFHBI dye (XOR gate). All computationally designed RNA devices were synthesized and experimentally tested in vitro. The ability to design smart nanodevices based on RNA binding aptamers offers a new route to engineer “label-free” ligand-sensing regulatory circuits, nucleic acid detection systems, and gene control elements.

Broccoli Fluorets: Split Aptamers as a User-Friendly Fluorescent Toolkit for Dynamic RNA Nanotechnology

“…Logic gating and the development of programmable NANPs often utilize strand displacement for triggering dynamic interactions [53]. Likewise, the fluorets can act as “molecular switches” which are activated or de-activated by the addition of other oligonucleotides [54].…”

Smart-Responsive Nucleic Acid Nanoparticles (NANPs) with the Potential to Modulate Immune Behavior

“…However, when the halves of the split aptamer are brought together during the reassociation, the completed aptamer regains its function [40,63,64]. The development of split fluorescent aptamers, such as malachite green [65], Spinach [63], and Broccoli [66], has produced tools which are especially useful when applied as a validating output for dynamic RNA nanotechnology [63,64,66,67,68], in the visualization of NANP assemblies [45,69], and for logic gating [70].…”

Optimization of the Split-Spinach Aptamer for Monitoring Nanoparticle Assembly Involving Multiple Contiguous RNAs

“…Split-aptamer systems rely on the fact that the functional aptamer forms only when both non-functional halves combine in the presence of a small molecular fluorophore. Given the dynamic nature of aptamer assembly, such fluorogenic systems have opened up new applications that include high-throughput assays, controlled reporting of assembly and processing, the development of logic gates and molecular computation, and more [23,33,34,35]. While split-aptamers offer the ability to monitor the assembly of two unique RNA strands (or three if formed on a scaffold-strand), most RNA nanoparticles are composed of several unique strands of RNA.…”